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- Abteilung für Funktionswerkstoffe der Medizin und der Zahnheilkunde (37)
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- Klinik und Poliklinik für Mund-, Kiefer- und Plastische Gesichtschirurgie (2)
- Klinik und Polikliniken für Zahn-, Mund- und Kieferkrankheiten (2)
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Aim of this thesis was the development of functionalizable hydrogel coatings for melt electrowritten PCL scaffolds and of bioprintable hydrogels for biofabrication.
Hydrogel coatings of melt electrowritten scaffolds enabled to control the surface hydrophilicity, thereby allowing cell-material interaction studies of biofunctionalized scaffolds in minimal protein adhesive environments. For this purpose, a hydrophilic star- shaped crosslinkable polymer was used and the coating conditions were optimized. Moreover, newly developed photosensitive scaffolds facilitated a time and pH independent biofunctionalization.
Bioprintable hydrogels for biofabrication were based on the allyl-functionalization of gelatin (GelAGE) and modified hyaluronic acid-products, to enable hydrogel crosslinking by means of the thiol-ene click chemistry. Optimization of GelAGE hydrogel properties was achieved through an in-depth analysis of the synthesis parameters, varying Ene:SH ratios, different crosslinking molecules and photoinitiators. Homogeneity of thiol-ene crosslinked networks was compared to free radical polymerized hydrogels and the applicability of GelAGE as bioink for extrusion-based bioprinting was investigated. Purely hyaluronic acid-based bioinks were hypothesized to maintain mechanical- and rheological properties, cell viabilities and the processability, upon further decreasing the overall hydrogel polymer and thiol content.
Hydrogel coatings: Highly structured PCL scaffolds were fabricated with MEW and subjected to coatings with six-armed star-shaped crosslinkable polymers (sP(EO-stat-PO)). Crosslinking results from the aqueous induced hydrolysis of reactive isocyanate groups (NCO) of sP(EO-stat-PO) and increased the surface hydrophilicity and provided a platform for biofunctionalizations in minimal protein adhesive environments. Not only the coating procedure was optimized with respect to sP(EO-stat-PO) concentrations and coating durations, instead scaffold pre-treatments were developed, which were fundamental to enhance the final hydrophilicity to completely avoid unspecific protein adsorption on sP(EO-stat-PO) coated scaffolds. The sP(EO-stat-PO) layer thickness of around 100 nm generally allows in vitro studies not only in dependence on the scaffold biofunctionalization but also on the scaffold architecture. The hydrogel coating extent was assessed via an indirect quantification of the NCO-hydrolysis products. Knowledge of NCO-hydrolysis kinetics enabled to achieve a balance of sufficiently coated scaffolds while maintaining the presence of NCO-groups that were exploited for subsequent biofunctionalizations. However, this time and pH dependent biofunctionalization was restricted to small biomolecules. In order to overcome this limitation and to couple high molecular weight biomolecules another reaction route was developed. This route was based on the photolysis of diazirine moieties and enabled a time and pH independent scaffold biofunctionalization with streptavidin and collagen type I. The fibril formation ability of collagen was used to obtain different collagen conformations on the scaffolds and a preliminary in vitro study demonstrated the applicability to investigate cell-material interactions.
The herein developed scaffolds could be applied to gain deeper insights into the fundamentals of cellular sensing. Especially the complexity by which cells sense e.g. collagen remain to be further elucidated. Therefore, different hierarchies of collagen-like conformations could be coupled to the scaffolds, e.g. gelatin or collagen-derived peptide sequences, and the activation of DDR receptors in dependence on the complexity of the coupled substances could be determined. Due to the strong streptavidin-biotin bond, streptavidin functionalized scaffolds could be applied as a versatile platform to allow immobilization of any biotinylated molecules.
Gelatin-based bioinks: First the GelAGE products were synthesized with respect to molecular weight distributions and amino acid composition integrity. A detailed study was conducted with varying molar ratios of reactants and synthesis durations and implied that gelatin degradation was most dominant for high alkaline synthesis conditions with long reaction times. Gelatin possesses multiple functionalizable groups and the predominant functionalization of amine groups was confirmed via different model substances and analyses. Polymer network homogeneity was proven for the GelAGE system compared to free radical polymerized hydrogels with GelMA. A detailed analysis of hydrogel compositions with varying functional group ratios and UV- or Vis-light photoinitiators was executed. The UV-initiator concentration is restricted due to cytotoxicity and potential cellular DNA damages upon UV-irradiation, whereas the more cytocompatible Vis- initiator system enabled mechanical stiffness tuning over a wide range by controlling the photoinitiator concentration at constant Ene:SH ratios and polymer weight percentages. Versatility of the GelAGE bioink for different AM techniques was proved by exploiting the thermo-gelling behavior of differently degraded GelAGE products for stereolithography and extrusion-based printing. Moreover, the viability of cell-laden GelAGE constructs was demonstrated for extrusion-based bioprinting. By applying different multifunctional thiol-macromolecular crosslinkers the mechanical and rheological properties improved concurrently to the processability. Importantly, lower thiol-crosslinker concentrations were required to yield superior mechanical strengths and physico-chemical properties of the hydrogels as compared to the small bis-thiol-crosslinker. Extrusion-based bioprinting with distinct encapsulated cells underlined the need for individual optimization of cell-laden hydrogel formulations.
Not only the viability of encapsulated cells in extrusion-based bioprinted constructs should be assessed, instead other parameters such as cell morphology or production of collagen or glycosaminoglycans should be considered as these represent some of the crucial prerequisites for cartilage Tissue Engineering applications. Moreover, these studies should be expanded to the stereolithographic approach and ultimately the versatility and cytocompatibility of formulations with macromolecular crosslinkers would be of interest. Macromolecular crosslinkers allowed reducing polymer weight percentages and amounts of thiol groups and are thus expected to contribute to increased cytocompatibility, especially in combination with the more cytocompatible Vis-initiator system, which remains to be elucidated.
Hyaluronic acid-based bioinks: Different molecular weight hyaluronic acid (HA) products were synthesized to bear ene- (HAPA) or thiol-functionalities (LHASH) to enable pure HA thiol-ene crosslinked hydrogels. Depending on the molecular weight of modified HA products, polymer weight percentages and Ene:SH ratios, a wide range of mechanical stiffness was covered. However, the manageability of high molecular weight HA (HHAPA) product solutions (HHAPA + LHASH) was restricted to 5.0 wt.-% as a consequence of the high viscosity. Based on the same HA thiol component (LHASH), hybrid hydrogels of HA with GelAGE were compared to pure HA hydrogels. Although the overall polymer weight percentage of HHAPA + LHASH hydrogels was significantly lowered compared to hybrid hydrogels (GelAGE + LHASH), similar mechanical and physico-chemical properties of pure HA hydrogels were determined with maintained Ene:SH ratios. Low viscous low molecular weight HA precursor solutions (LHAPA + LHASH) prevented the applicability for extrusion-based bioprinting, whereas the non-thermoresponsive HHAPA + LHASH system could be bioprinted with only one-fourth of the polymer content of hybrid formulations. The high viscous behavior of HHAPA + LHASH solutions, lower polymer weight percentages, decreased printing pressures and consequently declined shear stress during printing, were hypothesized to contribute to high cell viabilities in extrusion-based bioprinted constructs compared to the hybrid bioink.
The low molecular weight HA precursor formulation (LHAPA + LHASH) was not applicable for extrusion-based printing, but this system has potential for other AM techniques such as stereolithography. Similar to the GelAGE system a more detailed study on the functions of encapsulated cells would be useful to further develop this system. Moreover, the initiation with the Vis-initiator should be conducted.
In this work, a toolbox was provided to create three-component polymer conjugates with a defined architecture, designed to bear different biocomponents that can interact with larger biological systems in biomacromolecular recognition experiments. The target architecture is the attachment of two biomolecule ‘arms’ to the alpha telechelic end point of a polymer and fixating the conjugate to the gold surface of SAW and SPR sensor chips with the polymer’s other omega chain end. This specific design of a conjugate will be implemented by using a strategy to yield novel double alpha as well as omega telechelic functionalized POx and the success of all cascade reaction steps leading to the final conjugation product will be proven through affinity measurements between covalently bound mannose and ConA. All reactions were performed on a low molecular model level first and then transferred to telechelic and also side chain functionalized polymer systems.
Tibiakopfimpressionsfrakturen (AO 41-B2.2 – Schatzker III), welche aufgrund der demographischen Entwicklung in ihrer klinischen Relevanz zunehmen, erfordern zur bestmöglichen Frakturstabilisierung eine Schraubenosteosynthese sowie eine stabile metaphysäre Defektauffüllung mittels Knochenersatzmaterial, da anderenfalls ein sekundärer Repositionsverlust mit konsekutiver Gonarthrose droht. Die hierbei eingesetzten Kalziumphosphatzemente bringen klinische Probleme wie geringe mechanische Stabilität, fehlende Bohrbarkeit, welche eine unvollständige Defektauffüllung bedingt, ungewisse Resorption und unüberprüfte Herstellerangaben mit sich.
Diese Studie hatte daher zum Ziel, einen bohrbaren Kalziumphosphatzement und einen Magnesiumphosphatzement, welche als vielversprechende Alternativen aufgrund der klinischen Schwierigkeiten erscheinen, gegen Graftys® Quickset und ChronOS™ Inject biomechanisch einzuordnen und somit langfristig zu einer verbesserten Frakturversorgung beizutragen.
Der erste Teil der Studie bestand aus einer reinen Materialprüfung, in der mittels Zementquader Druckversuche und mittels Ausrisskörper Zugversuche durchgeführt wurden. Im zweiten Teil wurde ein Frakturmodell für Impressionsfrakturen an Kunstknochen benutzt, um die Zemente hierbei zur Defektauffüllung zu verwenden und alleine sowie in Kombination mit einer Osteosynthese in der Jail-Technik zu testen. Es erfolgte eine zyklische Belastung mit 3000 Zyklen zu je 250 N sowie anschließend eine Maximalkrafttestung (Load-To-Failure) mit Hilfe einer Materialprüfmaschine.
Der Magnesiumphosphatzement zeigte die signifikant höchste Kompressionsfestigkeit von 100,50 MPa ± 15,97 MPa und Ausrisskraft sowie im Verbund mit Knochen das geringste Displacement, höchste Maximalkraft und Steifigkeit. Kalziumphosphat bohrbar wies aufgrund seines pseudoplastischen Verhaltens eine geringe biomechanische Stabilität und ein hohes Displacement auf, konnte aber durch seine Bohrbarkeit gegenüber Graftys® Quickset bei Einsatz mit Schrauben einen Vorteil im Displacement erreichen und somit die Vorzüge eines bohrbaren Knochenzements aufzeigen. ChronOS™ zeigte nach Aushärtung im Wasserbad mit einer Kompressionsfestigkeit von 0,58 MPa ± 0,14 MPa eine niedrige biomechanische Stabilität und wurde daher nicht weiter untersucht. Da die Viskosität eines Zements neben anderen Faktoren für die Interdigitation mit den Spongiosahohlräumen im Knochen verantwortlich ist, lässt sich, sofern diese angemessen ist, Rückschlüsse von der Materialprüfung auf das Verhalten im Knochen ziehen.
Magnesiumphosphatzemente erscheinen aufgrund ihrer hohen biomechanischen Stabilität und vermutlich guten Resorptionsrate als vielversprechende Alternative zu herkömmlichen Kalziumphosphatzementen und bedürfen daher einer weiteren Überprüfung im Tierversuch.
The main focus of this thesis was the processing of different calcium and magnesium phosphate cements together with an optimization of mechanical and biological properties. Therefore, different manufacturing techniques like 3D powder printing and centrifugally casting were employed for the fabrication of reinforced or biomedically improved implants.
One of the main problems during 3D powder printing is the low green strength of many materials, especially when they are only physically bonded and do not undergo a setting reaction. Such materials need post-treatments like sintering to exhibit their full mechanical performance. However, the green bodies have to be removed from the printer requiring a certain stability. With the help of fiber reinforcement, the green strength of printed gypsum samples could be increased by the addition of polymeric and glass fibers within the printing process. The results showed that fiber reinforcement during 3D powder printing is possible and opens up diverse opportunities to enhance the damage tolerance of green bodies as well as directly printed samples. The transfer to biomedically relevant materials like calcium and magnesium phosphate cements and biocompatible fibers would be the next step towards reinforced patient-specific implants.
In a second approach, centrifugally casting derived from construction industries was established for the fabrication of hollow bioceramic cylinders. The aim was the replacement of the diaphysis of long bones, which exhibit a tubular structure with a high density of cortical bone on the fringe. By centrifugation, cement slurries with and without additives could be fabricated to tubes. As a first establishment, the processing parameters regarding the material (e.g. cement composition) as well as the set-up (e.g. rotation times) had to be optimized for each system. In respect of mechanics, such tubes can keep up with 3D powder printed tubes, although the mechanical performance of 3D printed tubes is strongly dependent on printing directions. Additionally, some material compositions like dual setting systems cannot be fabricated by 3D powder printing. Therefore, a transfer of such techniques to centrifugally casting enabled the fabrication of tubular structures with an extremely high damage tolerance due to high deformation ability. A similar effect was achieved by fiber (mesh) addition, as already shown for 3D powder printing. Another possibility of centrifugally casting is the combination of different materials resulting in graded structures to adjust implant degradation or bone formation. This became especially apparent for the incorporation of the antibiotic vancomycin, which is used for the treatment of bacterial implant infections. A long-term release could be achieved by the entrapment of the drug between magnesium phosphate cement layers. Therefore, the release of the drug could be regulated by the degradation of the outer shell, which supports the release into an acidic bacterial environment. The centrifugally casting technique exhibited to be a versatile tool for numerous materials and applications including the fabrication of non-centrosymmetric patient-specific implants for the reconstruction of human long bones.
The third project aimed to manufacture strontium-substituted magnesium phosphate implants with improved biological behavior by 3D powder printing. As the promoting effect of strontium on bone formation and the inhibitory impact on bone resorption is already well investigated, the incorporation of strontium into a degradable magnesium phosphate cement promised a fast integration and replacement of the implant. Porous structures were obtained with a high pore interconnectivity that is favorable for cell invasion and bone ingrowth. Despite the porosity, the mechanical performance was comparable to pure magnesium phosphate cement with a high reliability of the printed samples as quantitatively determined by Weibull statistics. However, the biological testing was impeded by the high degradation rate and the relating ion release. The high release of phosphate ions into surrounding media and the detachment of cement particles from the surface inhibited osteoblast growth and activity. To distinguish those two effects, a direct and indirect cell seeding is always required for degradable materials. Furthermore, the high phosphate release compared to the strontium release has to be managed during degradation such that the adverse effect of phosphate ions does not overwhelm the bone promoting effect of the strontium ions.
The manufacturing techniques presented in this thesis together with the material property improvement offer a diverse tool box for the fabrication of patient-specific implants. This includes not just the individual implant shape but also the application like bone growth promotion, damage tolerance and local drug delivery. Therefore, this can act as the basis for further research on specific medical indications.
The limited intrinsic self-healing capability of articular cartilage requires treatment of
cartilage defects. Material assisted and cell based therapies are in clinical practice but
tend to result in formation of mechanical inferior fibro-cartilage in long term follow up. If
a lesion has not been properly restored degenerative diseases are diagnosed as late sequela
causing pain and loss in morbidity. Complex three dimensional tissue models mimicking
physiological situation allow investigation of cartilage metabolism and mechanisms involved
in repair. A standardized and reproducible model cultured under controllable conditions
ex vivo to maintain tissue properties is of relevance for comparable studies.
Topic of this thesis was the establishment of an cartilage defect model that allows for
testing novel biomaterials and investigate the effect of defined defect depths on formation
of repair tissue.
In part I an ex vivo osteochondral defect model was established based on isolation of
porcine osteochondral explants (OCE) from medial condyles, 8 mm in diameter and 5 mm
in height. Full thickness cartilage defects with 1 mm to 4 mm in diameter were created
to define ex vivo cartilage critical size after 28 days culture with custom developed static
culture device. In part II of this thesis hydrogel materials, namely collagen I isolated from
rat tail, commercially available fibrin glue, matrix-metalloproteinase clevable poly(ethylene
glycol) polymerized with heparin (starPEGh), methacrylated poly(N-(2-hydroxypropyl)
methacrylamide mono-dilactate-poly(ethylene glycol) triblock copolymer/methacrylated
hyaluronic acid (MP/HA), thiol functionalized HA/allyl functionalized poly(glycidol)
(P(AGE/G)-HA-SH), were tested cell free and chondrocyte loaded (20 mio/ml) as implant
in 4 mm cartilage defects to investigate cartilage regeneration. Reproducible chondral
defects, 8 mm in diameter and 1 mm in height, were generated with an artificial tissue
cutter (ARTcut®) to investigate effect of defect depth on defect regeneration in part III.
In all approaches OCE were analyzed by Safranin-O staining to visualize proteoglycans
in cartilage and/or hydrogels. Immuno-histological and -fluorescent stainings (aggrecan,
collagen II, VI and X, proCollagen I, SOX9, RUNX2), gene expression analysis (aggrecan,
collagen II and X, SOX9, RUNX2) of chondrocyte loaded hydrogels (part II) and proteoglycan
and DNA content (Part I & II) were performed for detailed analysis of cartilage
regeneration.
Part I: The development of custom made static culture device, consisting of inserts in which OCE is fixed and deep well plate, allowed tissue specific media supply without
supplementation of TGF . Critical size diameter was defined to be 4 mm.
Part II: Biomaterials revealed differences in cartilage regeneration. Collagen I and fibrin
glue showed presence of cells migrated from OCE into cell free hydrogels with indication
of fibrous tissue formation by presence of proCollagen I. In chondrocyte loaded study
cartilage matrix proteins aggrecan, collagen II and VI and transcription factor SOX9 were
detected after ex vivo culture throughout the two natural hydrogels collagen I and fibrin
glue whereas markers were localized in pericellular matrix in starPEGh. Weak stainings resulted
for MP/HA and P(AGE/G)-HA-SH in some cell clusters. Gene expression data and
proteoglycan quantification supported histological findings with tendency of hypertrophy
indicated by upregulation of collagen X and RunX2 in MP/HA and P(AGE/G)-HA-SH.
Part III: In life-dead stainings recruitment of cells from OCE into empty or cell free
collagen I treated chondral defects was seen.
Separated and tissue specific media supply is critical to maintain ECM composition in
cartilage. Presence of OCE stimulates cartilage matrix synthesis in chondrocyte loaded
collagen I hydrogel and reduces hypertrophy compared to free swelling conditions and
pellet cultures. Differences in cartilage repair tissue formation resulted in preference of
natural derived polymers compared to synthetic based materials. The ex vivo cartilage
defect model represents a platform for testing novel hydrogels as cartilage materials, but
also to investigate the effect of cell seeding densities, cell gradients, cell co-cultures on
defect regeneration dependent on defect depth. The separated media compartments allow
for systematic analysis of pharmaceutics, media components or inflammatory cytokines on
bone and cartilage metabolism and matrix stability.
Ziel dieser Arbeit war die Herstellung fluoreszent markierter Präpolymere sowie deren Optimierung, die kontrollierte und reproduzierbare Synthese von redox-sensitiven und nicht redox-sensitiven NG mit und ohne Fluoreszenzmarkierung in einem durchschnittlichen Partikelgrößenbereich von 150 – 300 nm und mit einer Konzentration > 10*10 Partikel/ml, die Charakterisierung der NG, ihre Untersuchung bezüglich ihrer Stabilität und des Assoziationsverhaltens zu BSA sowie die Erlangung von Erkenntnissen bezüglich des Aufnahmemechanismus der NG in Abhängigkeit vom Transportpeptid Tat.
Abschließend kann zusammenfassend gesagt werden:
1. Das große Potential von PG-basierten NG für biologische bzw. medizinische Einsatzgebiete konnte weiter untermauert werden.
2. Das mit Cy5-Alkin markierte PG PG-SH-Cy5 erscheint aufgrund des relativ hohen erreichten Markierungsgrades bei der Herstellung als aussichtsreichster Kandidat für weitere Untersuchungen. Diese Umsetzung besitzt noch Optimierungspotentiale bezüglich einer Verringerung des Polymerverlusts bei der Aufarbeitung, des erreichbaren Markierungsgrades und der Markierungsausbeute. Möglichkeiten, dies zu erreichen, wurden diskutiert.
3. Klare Aussagen über den Einfluss des esterhaltigen bzw. esterfreien Ausgangspolymers PG-SH auf die Konzentration und die Partikelgröße konnten aufgrund einer nicht ausreichenden Datenlage nicht getroffen werden.
4. Die esterhaltigen PG-SH-Moleküle erscheinen aufgrund ihrer Labilität gegenüber Hydrolyse für die NP-Synthese weniger geeignet (geringere Stabilität).
5. Die Charakterisierung der aus den markierten und unmarkierten Ausgangspolymeren hergestellten NG, welche teilweise zusätzlich mit dem Transportpeptid Tat funktionalisiert wurden, erfolgte mittels NTA und zeigt für die meisten Spezies relativ schmale, gut definierte, monomodale Größenverteilungen mit einem Maximum um 100-200 nm im Bereich von ca. 40 – max. 400 nm mit Partikelkonzentrationen im Bereich von 1010 - 1011 Partikeln/ml.
6. Insgesamt konnte gezeigt werden, dass der untersuchte, von PG-SH abgeleitete NP-Typ (z. B. NG_3, redox-sensitiv unmarkiert) aufgrund seiner Einheitlichkeit, Partikelgröße und der Reproduzierbarkeit der Herstellung als gut geeignet für den geplanten Einsatz in biologischen Systemen erscheint. Von den weiter derivatisierten NG erscheinen die folgenden aufgrund der oben geschilderten Kriterien als besonders geeignet für den geplanten Einsatz in biologischen Systemen und weiterer Untersuchungen wert: NG680_(TAT)_1-4 (redox-sensitiv, markiert), NGCy5_(TAT)_1 (redox-sensitiv, markiert), NG_MA_2 (nicht redox-sensitiv, unmarkiert), NGCy7_MA_1 (nicht redox-sensitiv, markiert). Aufgrund des relativ hohen erreichbaren Markierungsgrades bei der Markierung der Ausgangspolymere erscheinen die mit Cy5-markierten Verbindungen als besonders vorteilhaft.
7. Die esterfreien, redox-sensitiven NP erwiesen sich bei 14-tägiger Lagerung unter physiologischen Bedingungen als stabil. Ihre Konzentration nahm über 14 Tage um ca. 60 % vom Ausgangswert ab. Gleichzeitig nahm der Teilchendurchmesser während des Beobachtungszeitraums um ca. 25 % zu. Die Abnahme der Teilchenzahl ist - zumindest teilweise - durch eine Vergrößerung des mittleren Teilchendurchmessers und mögliche Adsorptionseffekte an die Gefäßwände des Versuchsaufbaus zu erklären.
8. Die Konzentration der esterfreien, nicht redox-sensitiven NP verringert sich bei 14-tägiger Inkubation unter physiologischen Bedingungen deutlich auf ca. 10 % des Ausgangswerts. Der mittlere Durchmesser der Partikel bleibt innerhalb des Untersuchungszeitraums innerhalb der Fehlergrenzen konstant. Die starke Abnahme der Partikelkonzentration ist wahrscheinlich auf die Hydrolyse des verwendeten esterhaltigen Crosslinkers PEGDA zurückzuführen. Desweiteren sind Adsorptionsphänomene an Oberflächen des Versuchsaufbaus nicht auszuschließen. Insgesamt hervorzuheben ist die wesentlich höhere Stabiliät der redox-sensitiven NP unter den Versuchsbedingungen. Diese Substanzklasse sollte daher weiter verfolgt werden.
9. Es wurde gezeigt, dass sowohl die NG, die das Aufnahmeprotein Tat enthalten, als auch die NG ohne Tat mit Fluoreszenz-markiertem BSA (8,3 µg/ml) wechselwirken und zusammen mit diesem bei der Zentrifugation abgeschieden werden. Über die Art der Wechselwirkung kann keine Aussage getroffen werden.
10. Durch in vitro Zellaufnahmeuntersuchungen an Hela-Zellen konnte gezeigt werden, dass die mit Tat funktionalisierten, redox-sensitiven, Fluoreszenz-markierten NP von den Zellen aufgenommen werden. Die Aufnahme erfolgt über eine deutlich erkennbare Vesikelbildung, die an der Plasmamembran verstärkt beobachtet werden kann. Im Gegensatz hierzu konnte bei den nicht mit Tat funktionalisierten NP keine vergleichbare in vitro Zellaufnahme beobachtet werden.
Die Ergebnisse dieser Arbeit bestätigen insgesamt das große Potential der von Thiol-funktionalisierten PG abgeleiteten NG für die medizinische Forschung und zukünftige Anwendungen in der Diagnostik und Therapie. Es wird eine Reihe von Ansatzpunkten aufgezeigt, auf deren Basis weitere vertiefende Untersuchungen zur Charakterisierung und Optimierung sowie zu zukünftigen nutzbringenden Anwendungen vorgenommen werden sollten.
In order to mimic the extracellular matrix for tissue engineering, recent research approaches often involve 3D printing or electrospinning of fibres to scaffolds as cell carrier material. Within this thesis, a micron fibre printing process, called melt electrospinning writing (MEW), combining both additive manufacturing and electrospinning, has been investigated and improved. Thus, a unique device was developed for accurate process control and manufacturing of high quality constructs. Thereby, different studies could be conducted in order to understand the electrohydrodynamic printing behaviour of different medically relevant thermoplastics as well as to characterise the influence of MEW on the resulting scaffold performance.
For reproducible scaffold printing, a commonly occurring processing instability was investigated and defined as pulsing, or in extreme cases as long beading. Here, processing analysis could be performed with the aim to overcome those instabilities and prevent the resulting manufacturing issues. Two different biocompatible polymers were utilised for this study: poly(ε-caprolactone) (PCL) as the only material available for MEW until then and poly(2-ethyl-2-oxazoline) for the first time. A hypothesis including the dependency of pulsing regarding involved mass flows regulated by the feeding pressure and the electrical field strength could be presented. Further, a guide via fibre diameter quantification was established to assess and accomplish high quality printing of scaffolds for subsequent research tasks.
By following a combined approach including small sized spinnerets, small flow rates and high field strengths, PCL fibres with submicron-sized fibre diameters (fØ = 817 ± 165 nm) were deposited to defined scaffolds. The resulting material characteristics could be investigated regarding molecular orientation and morphological aspects. Thereby, an alignment and isotropic crystallinity was observed that can be attributed to the distinct acceleration of the solidifying jet in the electrical field and by the collector uptake. Resulting submicron fibres formed accurate but mechanically sensitive structures requiring further preparation for a suitable use in cell biology. To overcome this handling issue, a coating procedure, by using hydrophilic and cross-linkable star-shaped molecules for preparing fibre adhesive but cell repellent collector surfaces, was used.
Printing PCL fibre patterns below the critical translation speed (CTS) revealed the opportunity to manufacture sinusoidal shaped fibres analogously to those observed using purely viscous fluids falling on a moving belt. No significant influence of the high voltage field during MEW processing could be observed on the buckling phenomenon. A study on the sinusoidal geometry revealed increasing peak-to-peak values and decreasing wavelengths as a function of decreasing collector speeds sc between CTS > sc ≥ 2/3 CTS independent of feeding pressures. Resulting scaffolds printed at 100 %, 90 %, 80 % and 70 % of CTS exhibited significantly different tensile properties, foremost regarding Young’s moduli (E = 42 ± 7 MPa to 173 ± 22 MPa at 1 – 3 % strain). As known from literature, a changed morphology and mechanical environment can impact cell performance substantially leading to a new opportunity of tailoring TE scaffolds.
Further, poly(L-lactide-co-ε-caprolactone-co-acryloyl carbonate) as well as poly(ε-caprolactone-co-acryloyl carbonate) (PCLAC) copolymers could be used for MEW printing. Those exhibit the opportunity for UV-initiated radical cross-linking in a post-processing step leading to significantly increased mechanical characteristics. Here, single fibres of the polymer composed of 90 mol.% CL and 10 mol.% AC showed a considerable maximum tensile strength of σmax = 53 ± 16 MPa. Furthermore, sinusoidal meanders made of PCLAC yielded a specific tensile stress-strain characteristic mimicking the qualitative behaviour of tendons or ligaments. Cell viability by L929 murine fibroblasts and live/dead staining with human mesenchymal stem cells revealed a promising biomaterial behaviour pointing out MEW printed PCLAC scaffolds as promising choice for medical repair of load-bearing soft tissue.
Indeed, one apparent drawback, the small throughput similar to other AM methods, may still prevent MEW’s industrial application yet. However, ongoing research focusses on enlargement of manufacturing speed with the clear perspective of relevant improvement. Thereby, the utilisation of large spinneret sizes may enable printing of high volume rates, while downsizing the resulting fibre diameter via electrical field and mechanical stretching by the collector uptake. Using this approach, limitations of FDM by small nozzle sizes could be overcome. Thinking visionary, such printing devices could be placed in hospitals for patient-specific printing-on-demand therapies one day. Taking the evolved high deposition precision combined with the unique small fibre diameter sizes into account, technical processing of high performance membranes, filters or functional surface finishes also stands to reason.
Metals are the most used materials for implant devices, especially in orthopedics, but despite their long history of application issues such as material failure through wear and corrosion remain unsolved leading to a certain number of revision surgeries. Apart from the problems associated with insufficient material properties, another serious issue is an implant associated infection due to the formation of a biofilm on the surface of the material after implantation. Thus, improvements in implant technology are demanded, especially since there is a projected rise of implants needed in the future. Surface modification methods such as physical vapour deposition (PVD), oxygen diffusion hardening and electrochemical anodization have shown to be efficient methods to improve the surfaces of metallic bulk materials regarding biomedical issues. This thesis was focused on the development of functional PVD coatings that are suitable for further treatment with surface modification techniques originally developed for bulk metals. The aim was to precisely adjust the surface properties of the implant according to the targeted application to prevent possible failure mechanisms such as coating delamination, wear or the occurrence of post-operative infections.
Initially, tantalum layers with approx 5 µm thickness were deposited at elevated substrate temperatures on cp Ti by RF magnetron sputtering. Due to the high affinity of tantalum to oxygen, these coatings are known to provide a self healing capacity since the rapid oxide formation is known to close surface cracks. Here, the work aimed to reduce the abrupt change of mechanical properties between the hard and brittle coating and the ductile substrate by creating an oxygen diffusion zone. It was found that the hardness and adhesion could be significantly increased when the coatings were treated afterwards by oxygen diffusion hardening in a two step process. Firstly, the surface was oxidized at a pressure of 6.7•10-3 mbar at 350 450 °C, followed by 1-2 h annealing in oxygen-free atmosphere at the same temperature leading to a diffusion of oxygen atoms into deeper parts of the substrate as proved by X-ray diffraction (XRD) analysis. The hereby caused mechanical stress in the crystal lattice led to an increase in Vickers hardness of the Ta layers from 570 HV to over 900 HV. Investigations into the adhesion of oxygen diffusion treated samples by Rockwell measurements demonstrated an increase of critical force for coating delamination from 12 N for untreated samples up to 25 N for diffusion treated samples.
In a second approach, the development of modular targets aimed to produce functional coatings by metallic doping of titanium with biologically active agents. This was demonstrated by the fabrication of antimicrobial Ti(Ag) coatings using a single magnetron sputtering source equipped with a titanium target containing implemented silver modules under variation of bias voltage and substrate temperature. The deposition of both Ti and Ag was confirmed by X-ray diffraction and a clear correlation between the applied sputtering parameters and the silver content of the coatings was demonstrated by ICP-MS and EDX. Surface-sensitive XPS measurements revealed that higher substrate temperatures led to an accumulation of Ag in the near-surface region, while the application of a bias voltage had the opposite effect. SEM and AFM microscopy revealed that substrate heating during film deposition supported the formation of even and dense surface layers with small roughness values, which could even be enforced by applying a substrate bias voltage. Additional elution measurements using ICP-MS showed that the release kinetics depended on the amount of silver located at the film surface and hence could be tailored by variation of the sputter parameters.
In a final step, the applied Ti and Ti(Ag) coatings deposited on cp Ti, stainless steel (316L) and glass substrates were subsequently nanostructured using a self-ordering process induced by electrochemical anodization in aqueous fluoride containing electrolytes. SEM analysis showed that nanotube arrays could be grown from the Ti and Ti(Ag) coatings deposited at elevated temperatures on any substrate, whereby no influence of the substrate on nanotube morphology could be observed. EDX measurements indicated that the anodization process led to the selective etching of Ti from Ti(Ag) coating. Further experiments on coatings deposited on glass surfaces revealed that moderate substrate temperatures during deposition resulting in smooth Ti layers as determined by AFM measurements, are favorable for the generation of highly ordered nanotube arrays. Such arrays exhibited superhydrophilic behavior as proved by contact angle measurements. XRD analysis revealed that the nanostructured coatings were amorphous after anodization but could be crystallized to anatase structure by thermal treatment at temperatures of 450°C.
Ziel der Arbeit ist es, verschiedene Knochenersatzmaterialien der Tympanoplastik Typ 3
(autogenes Gewebe, Titan, Ionomerzement) bezüglich ihres Langzeitverhaltens im Mittelohr zu vergleichen. Es werden zwischen dem 21.12.1995 und dem 30.04.2011 in der Hals-Nasen-Ohrenklinik des Städtischen Klinikums Solingen operierte Patienten nachuntersucht. Insgesamt handelt es sich um 957 mit einer Tympanoplastik Typ III versorgte Patienten, die in diesem Zeitraum insgesamt 1093mal operiert worden sind. 676mal ist die Kette mit einer Titanprothese rekonstruiert worden, davon 301mal mit einer PORP und 375mal mit einer TORP (davon 21 bei intakter Stapessuprastruktur). Zu Beginn des Beobachtungszeitraums sind 56 Ionomerzement-prothesen eingesetzt worden, so dass 40 Ionomerzement-PORP und 16 Ionomerzement-TORP mit berücksichtigt worden sind. In 19 Fällen sind „sonstige“ Methoden (z.B. Knorpelüberhöhung des Steigbügels) zur Gehörknöchelchenkettenrekonstruktion gewählt worden.
Die Untersuchung zeigt, dass zur Kettenrekonstruktion eine Transposition autogener Ossikel angestrebt werden sollte. Stehen diese nicht zur Verfügung, empfiehlt sich die Verwendung von Titan-Prothesen. Aufgrund ihres hervorragenden In-Situ-Verhaltens sowie der nachgewiesen guten audiologischen Resultate sind sie derzeit das Mittel der Wahl.
Induced pluripotent stem cells (iPSCs) have been recognised as a virtually unlimited source of stem cells that can be generated in a patient-specific manner. Due to these cells’ potential to give rise to all differentiated cell types of the human body, they have been widely used to derive differentiated cells for drug screening and disease modelling purposes. iPSCs also garner much interest as they can potentially serve as a source for cell replacement therapy. Towards the realisation of these biomedical applications, this thesis aims to address challenges that are associated with scale-up, safety and biofabrication.
Firstly, the manufacture of a high number of human iPSCs (hiPSCs) will require standardised procedures for scale-up and the development of a flexible bioprocessing method, since standard adherent hiPSC culture exhibits limited scalability and is labour-intensive. While the quantity of cells that are required for cell therapy depends largely on the tissue and defect that these replacing cells are meant to correct, an estimate of 1 × 10^9 has been suggested to be sufficient for several indications, including myocardial infarction and islet replacement for diabetes. Here, the development of an integrated, microcarrier-free workflow to transition standard adherent hiPSC culture (6-well plates) to scalable stirred suspension culture in bioreactors (1 L working volume, 2.4 L maximum working volume) is presented. The two-phase bioprocess lasts 14 days and generates hiPSC aggregates measuring 198 ± 58 μm in diameter on the harvesting day, yielding close to 2 × 10^9 cells. hiPSCs can be maintained in stirred suspension for at least 7 weeks with weekly passaging, while exhibiting pluripotency-associated markers TRA-1-60, TRA-1-81, SSEA-4, OCT4, and SOX2. These cells retain their ability to differentiate into cells of all the three germ layers in vitro, exemplified by cells positive for AFP, SMA, or TUBB3. Additionally, they maintain a stable karyotype and continue to respond to specification cues, demonstrated by directed differentiation into beating cardiomyocyte-like cells. Therefore, the aim of manufacturing high hiPSC quantities was met using a state-of-the-art scalable suspension bioreactor platform.
Secondly, multipotent stem cells such as induced neural stem cells (iNSCs) may represent a safer source of renewable cells compared to pluripotent stem cells. However, pre-conditioning of stem cells prior to transplantation is a delicate issue to ensure not only proper function in the host but also safety. Here, iNSCs which are normally maintained in the presence of factors such as hLIF, CHIR99021, and SB431542 were cultured in basal medium for distinct periods of time. This wash-out procedure results in lower proliferation while maintaining key neural stem cell marker PAX6, suggesting a transient pre-differentiated state. Such pre-treatment may aid transplantation studies to suppress tumourigenesis through transplanted cells, an approach that is being evaluated using a mouse model of experimental focal demyelination and autoimmune encephalomyelitis.
Thirdly, biomedical applications of stem cells can benefit from recent advancements in biofabrication, where cells can be arranged in customisable topographical layouts. Employing a 3DDiscovery bioprinter, a bioink consisting of hiPSCs in gelatin-alginate was extruded into disc-shaped moulds or printed in a cross-hatch infill pattern and cross-linked with calcium ions. In both discs and printed patterns, hiPSCs recovered from these bioprints showed viability of around 70% even after 4 days of culture when loaded into gelatin-alginate solution in aggregate form. They maintained pluripotency-associated markers TRA-1-60 and SSEA-4 and continued to proliferate after re-plating. As further proof-of-principle, printed hiPSC 3D constructs were subjected to targeted neuronal differentiation, developing typical neurite outgrowth and resulting in a widespread network of cells throughout and within the topology of the printed matrix. Staining against TUBB3 confirmed neuronal identity of the differentiated cellular progeny. In conclusion, these data demonstrate that hiPSCs not only survive the 3D-printing process but were able to differentiate along the printed topology in cellular networks.
In Tissue Engineering, scaffolds composed of natural polymers often show a distinct lack in stability. The natural polymer gelatin is highly fragile under physiological conditions, nevertheless displaying a broad variety of favorable properties. The aim of this study was to fabricate electrospun gelatin nanofibers, in situ functionalized and stabilized during the spinning process with highly reactive star polymer NCO-sP(EO-stat-PO) (“sPEG”). A spinning protocol for homogenous, non-beaded, 500 to 1000 nm thick nanofibers from different ratios of gelatin and sPEG was successfully established. Fibers were subsequently characterized and tested with SEM imaging, tensile tests, water incubation, FTIR, EDX, and cell culture. It was shown that adding sPEG during the spinning process leads to an increase in visible fiber crosslinking, mechanical stability, and stability in water. The nanofibers were further shown to be biocompatible in cell culture with RAW 264.7 macrophages.
The aim of this thesis was the application of the functional prepolymer NCO-sP(EO-stat-PO) for the development of new biomaterials. First, the influence of the star-shaped polymers on the mechanical properties of biocements and bone adhesives was investigated. 3-armed star-shaped macromers were used as an additive for a mineral bone cement, and the influence on the mechanical properties was studied. Additionally, a previously developed bone adhesive was examined regarding cytocompatibility. The second topic was the examination of novel functionalization steps which were performed on the surface of electrospun fibers modified with NCO-sP(EO-stat-PO). This established method of functionalizing electrospun meshes was advanced regarding the modification with proteins which was then demonstrated in a biological application. Two different kinds of antibodies were immobilized on the fiber surface in a consecutive manner and the influence of these proteins on the cell behavior was investigated. The final topic involved the quantification of surface-bound peptide sequences. By functionalization of the peptides with the UV-reactive molecule 2-mercaptopyridine it was possible to quantify this compound via UV measurements by cleavage of disulfide bridges and indirectly draw conclusions about the number of immobilized peptides.
In the field of mineral biocements and bone adhesives, NCO-sP(EO-stat-PO) was able to influence the setting behavior and mechanical performance of mineral bone cements based on calcium phosphate chemistry. The addition of NCO-sP(EO-stat-PO) resulted in a pseudo-ductile fracture behavior due to the formation of a hydrogel network in the cement, which was then mineralized by nanosized hydroxyapatite crystals following cement setting. Accordingly, a commercially available aluminum silicate cement from civil engineering could be modified.
In addition, it could be shown that the use of NCO-sP(EO-stat-PO) is beneficial for adjusting specific material properties of bone adhesives. Here, the crosslinking behavior of the prepolymer in an aqueous medium was exploited to form an interpenetrating network (IPN) together with a photochemically curing poly(ethylene glycol) dimethacrylate (PEGDMA) matrix. This could be used for the development of a bone adhesive with an improved adhesion to bone in a wet environment. The developed bone adhesive was further investigated in terms of possible influences of the initiator systems. In addition, the material system was tested for cytocompatibility by using different cell lines.
Moreover, the preparation of electrospun fiber meshes via solution electrospinning consisting of poly(lactide-co-glycolide) (PLGA) as a backbone polymer and NCO-sP(EO-stat-PO) as functional additive is an established method for the application of the meshes as a replacement of the native extracellular matrix (ECM). In general, these fibers reveal diameters in the nanometer range, are protein and cell repellent due to the hydrophilic properties of the prepolymer and show a specific biofunctionalization by immobilization of peptide sequences. Here, the isocyanate groups presented on the fiber surface after electrospinning were used to carry out various functionalization steps, while retaining the properties of protein and cell repellency. The modification of the electrospun fibers involved the immobilization of analogs or antagonists of tumor necrosis factor (TNF) and the indirect detection of these by interaction with a light-producing enzyme. Here, a multimodal modification of the fiber surface with RGD to mediate cell adhesion and two different antibodies could be achieved. After culturing the cell line HT1080, the pro- or anti-inflammatory response of cells could be detected by IL-8 specific ELISA measurements.
Furthermore, the quantification of molecules on the surface of electrospun fibers was investigated. It was tested whether the detection by means of super-resolution microscopy would be possible. Therefore, experiments were performed with short amino acid sequences such as RGD for quantification by fluorescence microscopy. Based on earlier results, in which a UV-spectrometrically active molecule was used to detect the quantification of RGD, it was shown that short peptides can also be quantified in a small scale on flat functional substrates (2D) such as NCO-sP(EO-stat-PO) hydrogel coatings, and modified electrospun fibers produced from PLGA and NCO-sP(EO-stat-PO) (3D). In addition, a collagen sequence was used to prove that a successful quantification can be carried out as well for longer peptide chains.
These studies have revealed that NCO-sP(EO-stat-PO) can serve as a functional additive for many applications and should be considered for further studies on the development of novel biomaterials. The rapid crosslinking reaction, the resulting hydrogel formation and the biocompatibility are to be mentioned as positive properties, which makes the prepolymer interesting for future applications.
The aim of the work was the development of thiol-ene cross-linked hydrogels based on functionalized poly(glycidol)s (PG) and hyaluronic acid (HA) for extrusion based 3D bioprinting. Additionally, the functionalization of the synthesized PG with peptides and the suitability of these polymers for physically cross-linked gels were investigated, in a proof of principle study in order to demonstrate the versatile use of PG polymers in hydrogel development.
First, the precursor polymers of the different hydrogel systems were synthesized. For thiol-ene cross-linked hydogels, linear allyl-functionalized PG (P(AGE-co-G)) and three different thiol-(SH-)functionalized polymers, ester-containing PG-SH (PG SHec), ester-free PG-SH (PG-SHef) and HA-SH were synthesized and analysed, The degree of functionalization of these polymers was adjustable.
For physically cross-linked hydrogels, peptide-functionalized PG (P(peptide-co-G)), was synthesized through polymer analogue thiol-ene modification of P(AGE-co-G).
Subsequently, thiol-ene cross-linked hydrogels were prepared with the synthesized thiol- and allyl-functionalized polymers. Depending on the origin of the used polymers, two different systems were obtained: on the one hand synthetic hydrogels consisting of PG-SHec/ef and P(AGE-co-G) and on the other hand hybrid gels, consisting of HA-SH and P(AGE-co-G). In synthetic gels, the degradability of the gels was determined by the applied PG-SH. The use of PG-SHec resulted in hydrolytically degradable hydrogels, whereas the cross-linking with PG-SHef resulted in non-degradable gels.
The physical properties of these different hydrogel systems were determined by swelling, mechanical and diffusion studies and subsequently compared among each other. In swelling studies the differences of degradable and non-degradable synthetic hydrogels as well as the differences of synthetic compared to hybrid hydrogels were demonstrated.
Next, the stiffness and the swelling ratios (SR) of the established hydrogel systems were examined in dependency of different parameters, such as incubation time, polymer concentration and UV irradiation. In general, these measurements revealed the same trends for synthetic and hybrid hydrogels: an increased polymer concentration as well as prolonged UV irradiation led to an increased network density. Moreover, it was demonstrated that the incorporation of additional non-bound HMW HA hampered the hydrogel cross-linking resulting in gels with decreased stiffness and increased SR. This effect was strongly dependent on the amount of additional HMW HA.
The diffusion of different molecular weight fluorescein isothiocyanate-dextran (FITC-dextran) through hybrid hydrogels (with/without HMW HA) gave information about the mesh size of these gels. The smallest FITC-dextran (4 kDa) completely diffused through both hydrogel systems within the first week, whereas only 55 % of 40 kDa and 5-10 % HMW FITC-dextrans (500 kDa and 2 MDa) could diffuse through the networks.
The applicability of synthetic and hybrid hydrogels for cartilage regeneration purpose was investigated through by biological examinations. It was proven that both gels support the survival of embedded human mesenchymal stromal cells (hMSCs) (21/28 d in vitro culture), however, the chondrogenic differentiation was significantly improved in hybrid hydrogels compared to synthetic gels. The addition of non-bound HMW HA resulted in a slightly less distinct chondrogenesis.
Lastly the printability of the established hydrogel systems was examined. Therefore, the viscoelastic properties of the hydrogel solutions were adjusted by incorporation of non-bound HMW HA. Both systems could be successfully printed with high resolution and high shape fidelity.
The introduction of the double printing approach with reinforcing PCL allowed printing of hydrogel solutions with lower viscosities. As a consequence, the amount of additional HMW HA necessary for printing could be reduced allowing successful printing of hybrid hydrogel solutions with embedded cells. It was demonstrated that the integrated cells survived the printing process with high viability measured after 21 d. Moreover, by this reinforcing technique, robust hydrogel-containing constructs were fabricated.
In addition to thiol-ene cross-linked hydrogels, hydrogel cross-linking via ionic interactions was investigated with a hybrid hydrogel based on HMW HA and peptide-functionalized PG. Rheological measurements revealed an increase in the viscosity of a 2 wt.% HMW HA solution by the addition of peptide-functionalized PG. The increase in viscosity could be attributed to the ionic interactions between the positively charge PG and the negatively charge HMW HA.
In conclusion, throughout this thesis thiol-ene chemistry and PG were introduced as promising cross-linking reaction and polymer precursor for the field of biofabrication. Furthermore, the differences of hybrid and synthetic hydrogels as well as chemically and physically cross-linked hydrogels were demonstrated.
Moreover, the double printing approach was demonstrated to be a promising tool for the fabrication of robust hydrogel-containing constructs. It opens the possibility of printing hydrogels that were not printable yet, due to too low viscosities.
Biofabrication is an advancing new research field that might, one day, lead to complex products like tissue replacements or tissue analogues for drug testing. Although great progress was made during the last years, there are still major hurdles like new types of materials and advanced processing techniques. The main focus of this thesis was to help overcoming this hurdles by challenging and improving existing fabrication processes like extrusion-based bioprinting but also by developing new techniques. Furthermore, this thesis assisted in designing and processing materials from novel building blocks like recombinant spider silk proteins or inks loaded with charged nanoparticles.
A novel 3D printing technique called Melt Electrospinning Writing (MEW) was used in Chapter 3 to create tubular constructs from thin polymer fibers (roughly 12 μm in diameter) by collecting the fibers onto rotating and translating cylinders. The main focus was put on the influence of the collector diameter and its rotation and translation on the morphology of the constructs generated by this approach. In a first step, the collector was not moving and the pattern generated by these settings was analyzed. It could be shown that the diameter of the stationary collectors had a big impact on the morphology of the constructs. The bigger the diameter of the mandrel (smallest collector diameters 0.5 mm, biggest 4.8 mm) got, the more the shape of the generated footprint converged into a circular one known from flat collectors. In a second set of experiments the mandrels were only rotated. Increasing the rotational velocity from 4.2 to 42.0 rpm transformed the morphology of the constructs from a figure-of-eight pattern to a sinusoidal and ultimately to a straight fiber morphology. It was possible to prove that the transformation of the pattern was comparable to what was known from increasing the speed using flat collectors and that at a critical speed, the so called critical translation speed, straight fibers would appear that were precisely stacking on top of each other. By combining rotation and translation of the mandrel, it was possible to print tubular constructs with defined winding angles. Using collections speeds close to the critical translation speed enabled higher control of fiber positioning and it was possible to generate precisely stacked constructs with winding angles between 5 and 60°.
In Chapter 4 a different approach was followed. It was based on extrusion-based bioprinting in combination with a hydrogel ink system. The ink was loaded with nanoparticles and the nanoparticle release was analyzed. In other words, two systems, a printable polyglycidol/hyaluronic acid ink and mesoporous silica nanoparticles (MSN), were combined to analyze charge driven release mechanism that could be fine-tuned using bioprinting. Thorough rheological evaluations proved that the charged nanoparticles, both negatively charged MSN-COOH and positively charged MSN-NH2, did not alter the shear thinning properties of the ink that revealed a negative base charge due to hyaluronic acid as one of its main components. Furthermore, it could be shown that the particles did also not have a negative effect on the recovery properties of the material after exposure to high shear. During printing, the observations made via rheological testing were supported by the fact that all materials could be printed at the same settings of the bioprinter. Using theses inks, it was possible to make constructs as big as 12x12x3 mm3 composed of 16 layers. The fiber diameters produced were about 627±31 μm and two-component constructs could be realized utilizing the two hydrogel print heads of the printer to fabricate one hybrid construct. The particle distribution within those constructs was homogeneous, both from a microscopic and a macroscopic point of view. Particle release from printed constructs was tracked over 6 weeks and revealed that the print geometry had an influence on the particle release. Printed in a geometry with direct contact between the strands containing different MSN, the positively charged particles quickly migrated into the strand previously containing only negatively charged MSN-COOH. The MSN-COOH seemed to be rather released into the surrounding liquid and also after 6 weeks no MSN-COOH signal could be detected in the strand previously only containing MSN-NH2. In case of a geometry without direct contact between the strands, the migration of the positively charged nanoparticles into the MSN-COOH containing strand was strongly delayed. This proved that the architecture of the printed construct can be used to fine-tune the particle release from nanoparticle containing printable hydrogel ink systems.
Chapter 5 discusses an approach using hydrogel inks based on recombinant spider silk proteins processed via extrusion-based bioprinting. The ink could be applied for printing at protein concentrations of 3 % w/v without the addition of thickeners or any post process crosslinking. Both, the recombinant protein eADF4(C16) and a modification introducing a RGD-sequence to the protein (eADF4(C16)-RGD), could be printed revealing a very good print fidelity. The RGD modification had positive effect on the adhesion of cells seeded onto printed constructs. Furthermore, human fibroblasts encapsulated in the ink at concentrations of 1.2 million cells per mL did not alter the print fidelity and did not interfere with the crosslinking mechanism of the ink. This enabled printing cell laden constructs with a cell survival rate of 70.1±7.6 %. Although the cell survival rate needs to be improved in further trials, the approach shown is one of the first leading towards the shift of the window of biofabrication because it is based on a new material that does not need potentially harmful post-process crosslinking and allows the direct encapsulation of cells staying viable throughout the print process.
The aim of this thesis was the development of a multifunctional coating system for AuNPs based on thioether polymers, providing both excellent colloidal stability and a variable possibility to introduce functionalities for biological applications.
First, two thioether-polymer systems were synthesised as a systematic investigation into colloidal stabilisation efficacy. Besides commonly used monovalent poly(ethylene glycol) (PEG-SR), its structural analogue linear poly(glycidol) (PG-SR) bearing multiple statistically distributed thioether moieties along the backbone was synthesised. Additionally, respective thiol analogues (PEG-SH and PG-SH) were produced and applied as reference.
Successive modification of varyingly large AuNPs with aforementioned thiol- and thioether-polymers was performed via ligand exchange reaction on citrate stabilised AuNPs. An increased stabilisation efficacy of both thioether-polymers against biological and physiological conditions, as well as against freeze-drying compared to thiol analogues was determined.
Based on the excellent colloidal stabilisation efficacy and multi-functionalisability of thioether-PG, a plethora of functional groups, such as charged groups, hydrophilic/hydrophobic chains, as well as bio-active moieties namely diazirine and biotin was introduced to the AuNP surface. Moreover, the generic and covalent binding of diazirine-modified PG-SR with biomolecules including peptides and proteins was thoroughly demonstrated.
Lastly, diverse applicability and bioactivity of aforementioned modified particles in various studies was displayed, once more verifying the introduction of functionalities. On the one hand the electrostatic interaction of charged AuNPs with hydrogels based on hyaluronic acid was applied to tune the release kinetics of particles from three-dimensional scaffolds. On the other hand the strong complexation of siRNA onto two positively charged AuNPs was proven. The amount of siRNA payload was tuneable by varying the surface charge, ionic strength of the surrounding medium and the N/P ratio. Moreover, the biological activity and selectivity of the biotin-streptavidin conjugation was verified with respectively functionalised particles in controlled agglomeration test and in laser-triggered cell elimination experiments. In the latter, streptavidin-functionalised AuNPs resulted in excellent depletion of biotinylated cells whereas unfunctionalised control particles failed, excluding unspecific binding of these particles to the cell surface.
The key hypothesis of this work represented the question, if mimicking the zonal composition and structural porosity of musculoskeletal tissues influences invading cells positively and leads to advantageous results for tissue engineering. Conventional approaches in tissue engineering are limited in producing monolithic “scaffolds” that provide locally variating biological key signals and pore architectures, imitating the alignment of collagenous fibres in bone and cartilage tissues, respectively. In order to fill this gap in available tissue engineering strategies, a new fabrication technique was evolved for the production of scaffolds to validate the hypothesis.
Therefore, a new solidification based platform procedure was developed. This process comprises the directional solidification of multiple flowable precursors that are “cryostructured” to prepare a controlled anisotropic pore structure. Porous scaffolds are attained through ice crystal removal by lyophilisation. Optionally, electrostatic spinning of polymers may be applied to provide an external mesh on top or around the scaffolds. A consolidation step generates monolithic matrices from multi zonal structures. To serve as matrix for tissue engineering approaches or direct implantation as medical device, the scaffold is sterilized.
An Adjustable Cryostructuring Device (ACD) was successively developed; individual parts were conceptualized by computer aided design (CAD) and assembled. During optimisation, a significant performance improvement of the ACDs accessible external temperature gradient was achieved, from (1.3 ± 0.1) K/mm to (9.0 ± 0.1) K/mm. Additionally, four different configurations of the device were made available that enabled the directional solidification of collagenous precursors in a highly controlled manner with various sample sizes and shapes.
By using alginate as a model substance the process was systematically evaluated. Cryostructuring diagraphs were analysed yielding solidification parameters, which were associated to pore sizes and alignments that were determined by image processing. Thereby, a precise control over pore size and alignment through electrical regulation of the ACD could be demonstrated.
To obtain tissue mimetic scaffolds for the musculoskeletal system, collagens and calcium phosphates had to be prepared to serve as raw materials. Extraction and purification protocols were established to generate collagen I and collagen II, while the calcium phosphates brushite and hydroxyapatite were produced by precipitation reactions.
Besides the successive augmentation of the ACD also an optimization of the processing steps was crucial. Firstly, the concentrations and the individual behaviour of respective precursor components had to be screened. Together with the insights gained by videographic examination of solidifying collagen solutions, essential knowledge was gained that facilitated the production of more complex scaffolds. Phenomena of ice crystal growth during cryostructuring were discussed. By evolutionary steps, a cryostructuring of multi-layered precursors with consecutive anisotropic pores could be achieved and successfully transferred from alginate to collagenous precursors. Finally, very smooth interfaces that were hardly detectable by scanning electron microscopy (SEM) could be attained. For the used collagenous systems, a dependency relation between adjustable processing parameters and different resulting solidification morphologies was created.
Dehydrothermal-, diisocyanate-, and carbodiimide- based cross linking methods were evaluated, whereby the “zero length” cross linking by carbodiimide was found to be most suitable. Afterwards, a formulation for the cross linking solution was elaborated, which generated favourable outcomes by application inside a reduced pressure apparatus. As a consequence, a pore collapse during wet chemical cross linking could be avoided.
Complex monolithic scaffolds featuring continuous pores were fabricated that mimicked structure and respective composition of different areas of native tissues by the presence of biochemical key stimulants. At first, three types of bone scaffolds were produced from collagen I and hydroxyapatite with appropriate sizes to fit critical sized defects in rat femurs. They either featured an isotropic or anisotropic porosity and partly also contained glycosaminoglycans (GAGs). Furthermore, meniscus scaffolds were prepared by processing two precursors with biomimetic contents of collagen I, collagen II and GAGs. Here, the pore structures were created under boundary conditions, which allowed an ice crystal growth that was nearly orthogonal to the external temperature gradient. Thereby, the preferential alignment of collagen fibres in the natural meniscus tissue could be mimicked. Those scaffolds owned appropriate sizes for cell culture in well plates or even an authentic meniscus shape and size. Finally, osteochondral scaffolds, sized to either fit well plates or perfusion reactors for cell culture, were fabricated to mimic the composition of subchondral bone and different cartilage zones. Collagen I and the resorbable calcium phosphate brushite were used for the subchondral zone, whereas the cartilage zones were composed out of collagen I, collagen II and tissue mimetic contents of GAGs. The pore structure corresponded to the one that is dominating the volume of natural osteochondral tissue.
Energy dispersive X-ray spectroscopy (EDX) and SEM were used to analyse the composition and pore structure of the individual scaffold zones, respectively. The cross section pore diameters were determined to (65 ± 25) µm, (88 ± 35) µm and(93 ± 42) µm for the anisotropic, the isotropic and GAG containing isotropic bone scaffolds. Furthermore, the meniscus scaffolds showed pore diameters of (93 ± 21) µm in the inner meniscus zone and (248 ± 63) µm inside the outer meniscus zone. Pore sizes of (82 ± 25) µm, (83 ± 29) µm and (85 ± 39) µm were present inside the subchondral, the lower chondral and the upper chondral zone of osteochondral scaffolds. Depending on the fabrication parameters, the respective scaffold zones were also found to feature a specific micro- and nanostructure at their inner surfaces.
Degradation studies were carried out under physiological conditions and resulted in a mean mass loss of (0.52 ± 0.13) %, (1.56 ± 0.10) % and (0.80 ± 0.10) % per day for bone, meniscus and osteochondral scaffolds, respectively. Rheological measurements were used to determine the viscosity changes upon cooling of different precursors. Micro computer tomography (µ-CT) investigations were applied to characterize the 3D microstructure of osteochondral scaffolds. To obtain an osteochondral scaffold with four zones of tissue mimetic microstructure alignment, a poly (D, L-lactide-co-glycolide) mesh was deposited on the upper chondral zone by electrostatic spinning. In case of the bone scaffolds, the retention / release capacity of bone morphogenetic protein 2 (BMP-2) was evaluated by an enzyme linked immunosorbent assay (ELISA). Due to the high presence of attractive BMP binding sites, only less than 0.1 % of the initially loaded cytokine was released. The suitability of combining the cryostructuring process with 3D powder printed calcium phosphate substrates was evaluated with osteochondral scaffolds, but did not appear to yield more preferable results than the non-combined approach.
A new custom build confined compression setup was elaborated together with a suitable evaluation procedure for the mechanical characterisation under physiological conditions. For bone and cartilage scaffolds, apparent elastic moduli of (37.6 ± 6.9) kPa and (3.14 ± 0.85) kPa were measured. A similar behaviour of the scaffolds to natural cartilage and bone tissue was demonstrated in terms of elastic energy storage. Under physiological frequencies, less than 1.0 % and 0.8 % of the exerted energy was lost for bone and cartilage scaffolds, respectively. With average relaxation times of (0.613 ± 0.040) sec and (0.815 ± 0.077) sec, measured for the cartilage and bone scaffolds, they respond four orders of magnitude faster than the native tissues. Additionally, all kinds of produced scaffolds were able to withstand cyclic compression at un-physiological frequencies as high as 20 Hz without a loss in structural integrity.
With the presented new method, scaffolds could be fabricated whose extent in mimicking of native tissues exceeded the one of scaffolds producible by state of the art methods. This allowed a testing of the key hypothesis: The biological evaluation of an anisotropic pore structure in vivo revealed a higher functionality of immigrated cells and led finally to advantageous healing outcomes. Moreover, the mimicking of local compositions in combination with a consecutive anisotropic porosity that approaches native tissue structures could be demonstrated to induce zone specific matrix remodelling in stem cells in vitro. Additionally, clues for a zone specific chondrogenic stem cell differentiation were attained without the supplementation of growth factors.
Thereby, the hypothesis that an increased approximation of the hierarchically compositional and structurally anisotropic properties of musculoskeletal tissues would lead to an improved cellular response and a better healing quality, could be confirmed. With a special focus on cell free in situ tissue engineering approaches, the insights gained within this thesis may be directly transferred to clinical regenerative therapies.
Chemoselective poly(oxazolines) (POx) and poly[(oligo ethylene glycol) acrylates] were synthesized. An initiator was produced for the preparation of poly(oxazoline)s capable of participating in click chemistry reactions which allows the functionalization of the polymer at the α terminus which was confirmed by 1H NMR spectroscopy. The initiator was used for the polymerization of hydrophilic 2 methyl 2 oxazoline (MeOx), whereby chemoselective, alkyne functionalized polymers could be prepared for Cu-catalyzed azide–alkyne cycloaddition. The desired molecular weight could be achieved through the living, ring opening cationic polymerization and was confirmed by 1H NMR, SEC and MALDI ToF measurements. Polymers were terminated with piperidine if no further functionalization was needed, or with an ester derivate for enabling amine attachment in a subsequent step. In addition, polymers were functionalized by termination with NaN3 in order to provide the counterpart to the azide–alkyne reaction. IR spectroscopy was suitable for the azide detection. The coupling of polymers showed the reactivity and could be confirmed by SEC, 1H NMR and IR spectroscopy.
The composition of cysteine functionalized POx was completed by thiol–ene chemistry. Since the commercially available iso 2 propyl 2 oxazoline is not available for the cationic polymerization, 2 butenyl and 2 decenyl 2 oxazoline (ButenOx and DecenOx) were first prepared. The synthesis of both copolymers, based on MeOx could be confirmed by 1H NMR as well as with SEC, whereby narrow distributions with dispersities of 1.06 could be achieved. The cysteine functionalization of the copolymers was enabled by the creation of a thiazolidine component which could be synthesized by acetal and formyl protection of cysteine and subsequent functionalization with a thiol. The component enabled the reaction with a polymer by thiol–ene reaction which was started by the addition of dimethoxyphenyl-acetophenone and was catalyzed by irradiation with UV light. Both copolymers, with a shorter (polymers with BuenOx) and longer (polymers with DecenOx) hydrophobic sidechain could be functionalized. 1H NMR spectroscopic analysis showed a quantitative reaction with the thiazolidine derivate. After deprotection by acidic workup the desired, cysteine functionalized polymer could be isolated. Quantification of cysteine functions was ensured by a modified TNBSA assay, whereby the thiols were first oxidized in order to confirm an independent measurement of amine functions. Both, the TNBSA assay as well as the NMR measurement showed the desired number of cysteine residues.
The cytotoxicity of functionalized polymers with different compositions was tested by a luminescent cell viability assay (LCVA). Both, the amount of cysteine functions (5–10%) in the copolymers as well as the length of the hydrophobic side chain were varied. All polymers did not show cytotoxicity up to concentrations of 10 mg∙mL-1. The cell activity and cell numbers only decreased below 50% and 20% respectively, when copolymers with 5% cysteine and longer sidechains were measured, which was attributed to a contamination of the sample itself. The cooperation partner performed Native Chemical Ligation (NCL) with model peptides and purified the products by HPLC. A sterically non demanding peptide was synthesized, consisting of an aromatic amino acid and four glycine units. The aromatic unit was used for the quantification of the polymer–peptide conjugate in the 1H NMR spectroscopy. A polymer having five cysteine side chains has been fully implemented by NCL to a conjugate of one polymer with five peptides. A sterically more demanding peptide was additionally used and MALDI ToF measurements confirmed the successful conjugation.
Furthermore the cysteine functionalized polymer was used for nanogel synthesis. The thiol of the cysteine function was oxidized in an inverse mini-emulsion by H2O2, resulting in nanogels (~500 nm) which could be confirmed by SEM, AFM, DLS and NTA measurements.
Besides POx, oligo (ethylene glycol)acrylates (OEGA) were polymerized; by copolymerization with the reactive pentafluorophenyl acrylate (PFPA) reactive and amphiphilic polymers were obtained. The synthesis of PFPA could be confirmed spectroscopically by 1H , 19F NMR, and by FT IR. Copolymers were synthesized by RAFT polymerization with narrow dispersities. Functionalization with an amine functionalized thiazolidine led to a hydrophilic cysteine functionalized polymer after acidic deprotection. Apart from this polymer, a thioester functionalization was successfully performed by reaction of the active polymer with a cyclic amine functionalized thioester which does not release a toxic by product (such as the resulting thiol) during NCL and thus features a very high potential to replace former thioester.
The aim of the thesis was to develop water soluble poly(2-oxazoline) (POx) copolymers with new side group functionalities, which can be used for the formation of hydrogels in biomedical applications and for the development of peptide-polymer conjugates.
First, random copolymers of the monomer MeOx or EtOx with ButEnOx and EtOx with DecEnOx were synthesized and characterized. The vinyl functionality brought into the copolymer by the monomers ButEnOx and DecEnOx would later serve for post-polymerization functionalization. The synthesized copolymers were further functionalized with thiols via post-polymerization functionalization using a newly developed synthesis protocol or with a protected catechol molecule for hydrogel formation. For the formation of peptide-polymer conjugates, a cyclic thioester, namely thiolactone acrylamide and an azlactone precursor, whose synthesis was newly developed, were attached to the side chain of P(EtOx-co-ButEnOx) copolymers.
The application of the functionalized thiol copolymers as hydrogels using thiol-ene chemistry for cross-linking was demonstrated. The swelling behavior and mechanical properties were characterized. The hydrophilicity of the network as well as the cross-linking density strongly influenced the swelling behavior and the mechanical strength of the hydrogels. All hydrogels showed good cell viability results.
The hydrogel networks based on MeOx and EtOx were loaded with two dyes, fluorescein and methylene blue. It was observed that the uptake of the more hydrophilic dye fluorescein depended more on the ability of the hydrogel to swell. In contrast, the uptake of the more hydrophobic dye methylene blue was less dependent on the swelling degree, but much more on the hydrophilicity of the network.
For the potential application as cartilage glue, (biohybrid) hydrogels were synthesized based on the catechol-functionalized copolymers, with and without additional fibrinogen, using sodium periodate as the oxidizing agent. The system allowed for degradation due to the incorporated ester linkages at the cross-linking points. The swelling behavior as well as the mechanical properties were characterized. As expected, hydrogels with higher degrees of cross-linking showed less swelling and higher elastic modulus. The addition of fibrinogen however increased the elasticity of the network, which can be favorable for the intended application as a cartilage glue. Biological evaluation clearly demonstrated the advantage of degradable ester links in the hydrogel network, where chondrocytes were able to bridge the artificial gap in contrast to hydrogels without any ester motifs.
Lastly, different ways to form peptide-polymer conjugates were presented. Peptides were attached with the thiol of the terminal cysteine group to the vinyl side chain of P(EtOx-co-ButEnOx) copolymers by radical thiol-ene chemistry. Another approach was to use a cyclic thioester, thiolactone, or an azlactone functionality to bind a model peptide via native chemical ligation. The two latter named strategies to bind peptides to POx side chains are especially interesting as one and in the case of thiolactone two free thiols are still present at the binding site after the reaction, which can, for example, be used for further thiol-ene cross-linking to form POx hydrogels.
In summary, side functional poly(oxazoline) copolymers show great potential for numerous biomedical applications. The various side chain functionalities can be introduced by an appropriate monomer or by post-polymerization functionalization, as demonstrated. By their multi-functionality, hydrogel characteristics, such as cross-linking degree and mechanical strength, can be fine-tuned and adjusted depending on the application in the human body. In addition, the presented chemoselective and orthogonal reaction strategies can be used in the future to synthesize polymer conjugates, which can, for example, be used in drug delivery or in tissue regeneration.
The outcome of the innate immune response to biomaterials mainly determines whether the material will be incorporated in the body to fulfill its desired function or, when it gets encapsulated, will be rejected in the worst case. Macrophages are key players in this process, and their polarization state with either pro- (M1), anti-inflammatory (M2), or intermediate characteristics is crucial for deciding on the biomaterial’s fate. While a transient initial pro-inflammatory state is helpful, a prolonged inflammation deteriorates the proper healing and subsequent regeneration. Therefore, biomaterial-based polarization may aid in driving macrophages in the desired direction. However, the in vivo process is highly complex, and a mono-culture of macrophages in vitro displays only one part of the cellular system, but, to this date, there is a lack of established co-cultures to assess the immune response to biomaterials. Thus, this thesis aimed to establish a functional co-culture system of human macrophages and human mesenchymal stromal cells (hMSCs) to improve the assessment of the immune response to biomaterials in vitro. Together with macrophages, hMSCs are involved in tissue regeneration and inflammatory reactions and can modulate the immune response. In particular, endogenously derived hMSCs considerably contribute to the successful engrafting of biomaterials. This thesis focused on poly(ε-caprolactone) (PCL) fiber-based scaffolds produced by the technique of melt electrowriting (MEW) as biomaterial constructs. Via this fabrication technique, uniform, precisely ordered scaffolds varying in geometry and pore size have been created in-house.
To determine the impact of scaffold geometries and pore sizes on macrophages, mono-cultures incubated on scaffolds were conducted. As a pre-requisite to achieve a functional co-culture system on scaffolds, setups for direct and indirect systems in 2D have initially been established. These setups were analyzed for the capability of cell-cell communication. In parallel, a co-culture medium suitable for both cell types was defined, prior to the establishment of a step-by-step procedure for the co-cultivation of human macrophages and hMSCs on fiber-based scaffolds.
Regarding the scaffold morphologies tested within this thesis to improve M2-like polarization, box-shaped scaffolds outperformed triangular-, round- or disordered-shaped ones. Upon further investigation of scaffolds with box-shaped pores and precise inter-fiber spacing from 100 µm down to only 40 µm, decreasing pore sizes facilitated primary human macrophage elongation accompanied by their differentiation towards the M2 type, which was most pronounced for the smallest pore size of 40 µm. To the best of my knowledge, this was the first time that the elongation of human macrophages in a 3D environment has been correlated to their M2-like polarization. Thus, these results may set the stage for the design, the assessment, and the selection of new biomaterials, which can positively affect the tissue regeneration.
The cell communication of both cell types, detected via mitochondria exchange in direct and indirect co-cultures systems, took place in both directions, i.e., from hMSCs to macrophages and vice versa. Thereby, in direct co-culture, tunneling nanotubes enabled the transfer from one cell type to the respective other, while in indirect co-culture, a non-directional transfer through extracellular vesicles (EVs) released into the medium seemed likely. Moreover, the phagocytic activity of macrophages after 2D co-cultivation and hence immunomodulation by hMSCs increased with the highest phagocytic rate after 48 h being most pronounced in direct co-cultivation.
As the commonly used serum supplements for macrophages and hMSCs, i.e., human serum (hS) and fetal calf serum (FCS), respectively, failed to support the respective other cell type during prolonged cultivation, these sera were replaced by human platelet lysate (hPL), which has been proven to be the optimal supplement for the co-cultivation of human macrophages with hMSCs within this thesis. Thereby, the phenotype of both cell types, the distribution of both cell populations, the phagocytic activity of macrophages, and the gene expression profiles were maintained and comparable to the respective standard mono-culture conditions. This was even true when hPL was applied without the anticoagulant heparin in all cultures with macrophages, and therefore, heparin was omitted for further experiments comprising hPL and macrophages.
Accordingly, a step-by-step operating procedure for the co-cultivation on fiber-based scaffolds has been established comprising the setup for 3D cultivation as well as the description of methods for the analysis of phenotypical and molecular changes upon contact with the biomaterial. The evaluation of the macrophage response depending on the cultivation with or without hMSCs and either on scaffolds or on plastic surfaces has been successfully achieved and confirmed the functionality of the suggested procedures.
In conclusion, the functional co-culture system of human macrophages and hMSCs established here can now be employed to assess biomaterials in terms of the immune response in a more in vivo-related way. Moreover, specifically designed scaffolds used within the present thesis showed auspicious design criteria positively influencing the macrophage polarization towards the anti-inflammatory, pro-healing type and might be adaptable to other biomaterials in future approaches.
Hence, follow-up experiments should focus on the evaluation of the co-culture outcome on promising scaffolds, and the suggested operating procedures should be adjusted to further kinds of biomaterials, such as cements or hydrogels.
The implantation of any foreign material into the body automatically starts an immune reaction that serves as the first, mandatory step to regenerate tissue. The course of this initial immune reaction decides on the fate of the implant: either the biomaterial will be integrated into the host tissue to subsequently fulfill its intended function (e.g., tissue regeneration), or it will be repelled by fibrous encapsulation that determines the implant failure. Especially neutrophils and macrophages play major roles during this inflammatory response and hence mainly decide on the biomaterial's fate. For clinically relevant tissue engineering approaches, biomaterials may be designed in shape and morphology as well as in their surface functionality to improve the healing outcome, but also to trigger stem cell responses during the subsequent tissue regeneration phase.
The main focus of this thesis was to unravel the influence of scaffold characteristics, including scaffold morphology and surface functionality, on primary human innate immune cells (neutrophils and macrophages) and human mesenchymal stromal cells (hMSCs) to assess their in vitro immune response and tissue regeneration capacity, respectively. The fiber-based constructs were produced either via melt electrowriting (MEW), when the precise control over scaffold morphology was required, or via solution electrospinning (ES), when the scaffold design could be neglected. All the fiber-based scaffolds used throughout this thesis were composed of the polymer poly(ε caprolactone) (PCL).
A novel strategy to model and alleviate the first direct cell contact of the immune system with a peptide-bioactived fibrous material was presented in chapter 3 by treating the material with human neutrophil elastase (HNE) to imitate the neutrophil attack. The main focus of this study was put on the effect of HNE towards an RGDS-based peptide that was immobilized on the surface of a fibrous material to improve subsequent L929 cell adhesion. The elastase efficiently degraded the peptide-functionality, as evidenced by a decreased L929 cell adhesion, since the peptide integrated a specific HNE-cleavage site (AAPV-motif). A sacrificial hydrogel coating based on primary oxidized hyaluronic acid (proxHA), which dissolved within a few days after the neutrophil attack, provided an optimal protection of the peptide-bioactivated fibrous mesh, i.e, the hydrogel alleviated the neutrophil attack and largely ensured the biomaterial's integrity. Thus, according to these results, a means to protect the biomaterial is required to overcome the neutrophil attack.
Chapter 4 was based on the advancement of melt electrowriting (MEW) to improve the printing resolution of MEW scaffolds in terms of minimal inter-fiber distances and a concomitant high stacking precision. Initially, to gain a better MEW understanding, the influence of several parameters, including spinneret diameter, applied pressure, and collector velocity on mechanical properties, crystallinity, fiber diameter and fiber surface morphology was analyzed. Afterward, innovative MEW designs (e.g., box-, triangle-, round , and wall-shaped scaffolds) have been established by pushing the printing parameters to their physical limits. Further, the inter-fiber distance within a standardized box-structured scaffold was successfully reduced to 40 µm, while simultaneously a high stacking precision was maintained. In collaboration with a co-worker of my department (Tina Tylek, who performed all cell-based experiments in this study), these novel MEW scaffolds have been proven to facilitate human monocyte-derived macrophage polarization towards the regenerative M2 type in an elongation-driven manner with a more pronounced effect with decreasing pore sizes.
Finally, a pro-adipogenic platform for hMSCs was developed in chapter 5 using MEW scaffolds with immobilized, complex ECM proteins (e.g., human decellularized adipose tissue (DAT), laminin (LN), and fibronectin (FN)) to test for the adipogenic differentiation potential in vitro. Within this thesis, a special short-term adipogenic induction regime enabled to more thoroughly assess the intrinsic pro-adipogenic capacity of the composite biomaterials and prevented any possible masking by the commonly used long-term application of adipogenic differentiation reagents. The scaffolds with incorporated DAT consistently showed the highest adipogenic outcome and hence provided an adipo-inductive microenvironment for hMSCs, which holds great promise for applications in soft tissue regeneration.
Future studies should combine all three addressed projects in a more in vivo-related manner, comprising a co-cultivation setup of neutrophils, macrophages, and MSCs. The MEW-scaffold, particularly due to its ability to combine surface functionality and adjustable morphology, has been proven to be a successful approach for wound healing and paves the way for subsequent tissue regeneration.
Synthetic bone replacement materials have their application in non-load bearing defects with the function of (re-)construction or substitution of bone. This tissue itself represents a biological composite material based on mineralized collagen fibrils and combines the mechanical strength of the mineral with the ductility of the organic matrix. By mimicking these outstanding properties with polymer-cement-composites, an imitation of bone is feasible. A promising approach for such replacement materials are dual setting systems, which are generated by dissolution-precipitation reaction with cement setting in parallel to polymerization and gelation of the organic phase forming a coherent hydrogel network. Hereby, the high brittleness of the pure inorganic network was shifted to a more ductile and elastic behavior.
The aim of this thesis was focused on the development of different dual setting systems to modify pure calcium phosphate cements’ (CPCs’) mechanical performance by incorporation of a hydrogel matrix.
A dual setting system based on hydroxyapatite (HA) and cross-linked 2-hydroxyethyl methacrylate (HEMA) via radical polymerization was advanced by homogenous incorporation of a degradable cross-linker composed of poly(ethylene glycol) (PEG) as well as poly(lactic acid) (PLA) with reactive terminal methacrylate functionalities (PEG-PLLA-DMA). By integration of this high molecular weight structure in the HEMA-hydrogel network, a significant increase in energy absorption (toughness) under 4-point bending testing was observed. An addition of only 10 wt% hydrogel precursor (referred to the liquid phase) resulted in a duplication of stress over a period of 8 days. Additionally, the calculated elasticity was positively affected and up to six times higher compared to pure HA. With a constantly applied force during compressive strength testing, a deformation and thus strain levels of about 10 % were reached immediately after preparation.
For higher degradability, the system was modified in a second approach regarding organic as well as inorganic phase. The latter component was changed by brushite forming cement that is resorbable in vivo due to solubility processes. This CPC was combined with a hydrogel based on PEG-PLLA-DMA and other dimethacrylated PEGs with different molecular weights and concentrations. Hereby, new reaction conditions were created including a shift to acidic conditions. On this ground, the challenge was to find a new radical initiator system. Suitable candidates were ascorbic acid and hydrogen peroxide. that started the polymerization and successful gelation in this environment. These highly flexible dual set composites showed a very high ductility with an overall low strength compared to HA-based models. After removal of the applied force during compressive strength testing, a complete shape recovery was observed for the samples containing the highest polymeric amount (50 wt%) of PEG-PLLA-DMA.
Regarding phase distribution in the constructs, a homogenously incorporated hydrogel network was demonstrated in a decalcifying study with ethylenediaminetetraacetic acid. Intact, coherent hydrogels remained after dissolution of the inorganic phase via calcium ion complexation.
In a third approach, the synthetic hydrogel matrix of the previously described system was replaced by the natural biopolymer gelatin. Simultaneously to brushite formation, physical as well as chemical cross-linking by the compound genipin was performed in the dual setting materials. Thanks to the incorporation of gelatin, elasticity increased significantly, in which concentrations up to 10.0 w/v% resulted in a certain cohesion of samples after compressive strength testing. They did not dissociate in little pieces but remained intact cuboid specimens though having cracks or fissures. Furthermore, the drug release of two active pharmaceutical ingredients (vancomycin and rifampicin) was investigated over a time frame of 5 weeks. The release exponent was determined according to Korsmeyer-Peppas with n = 0.5 which corresponds to the drug liberation model of Higuchi. A sustained release was observed for the antibiotic vancomycin encapsulated in composites with a gelatin concentration of 10.0 w/v% and a powder-to-liquid ratio of 2.5 g/mL.
With respect to these developments of different dual setting systems, three novel approaches were successfully established by polymerization of monomers and cross-linking of precursors forming an incorporated, homogenous hydrogel matrix in a calcium phosphate network. All studies showed an essential transfer of mechanical performance in direction of flexibility and bendability.
Adipose tissue defects and related pathologies still represent major challenges in reconstructive surgery. Based on to the paradigm ‘replace with alike’, adipose tissue is considered the ideal substitute material for damaged soft tissue [1-3]. Yet the transfer of autologous fat, particularly larger volumes, is confined by deficient and unpredictable long term results, as well as considerable operative morbidity at the donor and recipient site [4-6], calling for innovative treatment options to improve patient care.
With the aim to achieve complete regeneration of soft tissue defects, adipose tissue engineering holds great promise to provide functional, biologically active adipose tissue equivalents. Here, especially long-term maintenance of volume and shape, as well as sufficient vascularization of engineered adipose tissue represent critical and unresolved challenges [7-9]. For adipose tissue engineering approaches to be successful, it is thus essential to generate constructs that retain their initial volume in vivo, as well as to ensure their rapid vascularization to support cell survival and differentiation for full tissue regeneration [9,10]. Therefore, it was the ultimate goal of this thesis to develop volume-stable 3D adipose tissue constructs and to identify applicable strategies for sufficient vascularization of engineered constructs. The feasibility of the investigated approaches was verified by translation from in vitro to in vivo as a critical step for the advancement of potential regenerative therapies.
For the development of volume-stable constructs, the combination of two biomaterials with complementary properties was successfully implemented. In contrast to previous approaches in the field using mainly non-degradable solid structures for mechanical protection of developing adipose tissue [11-13], the combination of a cell-instructive hydrogel component with a biodegradable porous support structure of adequate texture was shown advantageous for the generation of volume-stable adipose tissue. Specifically, stable fibrin hydrogels previously developed in our group [14] served as cell carrier and supported the adipogenic development of adipose-derived stem cells (ASCs) as reflected by lipid accumulation and leptin secretion. Stable fibrin gels were thereby shown to be equally supportive of adipogenesis compared to commercial TissuCol hydrogels in vitro. Using ASCs as a safe source of autologous cells [15,16] added substantial practicability to the approach. To enhance the mechanical strength of the engineered constructs, porous biodegradable poly(ε caprolactone)-based polyurethane (PU) scaffolds were introduced as support structures and shown to exhibit adequately sized pores to host adipocytes as well as interconnectivity to allow coherent tissue formation and vascularization. Low wettability and impaired cell attachment indicated that PU scaffolds alone were insufficient in retaining cells within the pores, yet cytocompatibility and differentiation of ASCs were adequately demonstrated, rendering the PU scaffolds suitable as support structures for the generation of stable fibrin/PU composite constructs (Chapter 3).
Volume-stable adipose tissue constructs were generated by seeding the pre-established stable fibrin/PU composites with ASCs. Investigation of size and weight in vitro revealed that composite constructs featured enhanced stability relative to stable fibrin gels alone. Comparing stable fibrin gels and TissuCol as hydrogel components, it was found that TissuCol gels were less resilient to degradation and contraction. Composite constructs were fully characterized, showing good cell viability of ASCs and strong adipogenic development as indicated by functional analysis via histological Oil Red O staining of lipid vacuoles, qRT-PCR analysis of prominent adipogenic markers (PPARγ, C/EBPα, GLUT4, aP2) and quantification of leptin secretion. In a pilot study in vivo, investigating the suitability of the constructs for transplantation, stable fibrin/PU composites provided with a vascular pedicle gave rise to areas of well-vascularized adipose tissue, contrasted by insufficient capillary formation and adipogenesis in constructs implanted without pedicle. The biomaterial combination of stable fibrin gels and porous biodegradable PU scaffolds was thereby shown highly suitable for the generation of volume-stable adipose tissue constructs in vivo, and in addition, the effectiveness of immediate vascularization upon implantation to support adipose tissue formation was demonstrated (Chapter 4).
Further pursuing the objective to investigate adequate vascularization strategies for engineered adipose tissue, hypoxic preconditioning was conducted as a possible approach for in vitro prevascularization. In 2D culture experiments, analysis on the cellular level illustrated that the adipogenic potential of ASCs was reduced under hypoxic conditions when applied in the differentiation phase, irrespective of the oxygen tension encountered by the cells during expansion. Hypoxic treatment of ASCs in 3D constructs prepared from stable fibrin gels similarly resulted in reduced adipogenesis, whereas endothelial CD31 expression as well as enhanced leptin and vascular endothelial growth factor (VEGF) secretion indicated that hypoxic treatment indeed resulted in a pro-angiogenic response of ASCs. Especially the observed profound regulation of leptin production by hypoxia and the dual role of leptin as adipokine and angiogenic modulator were considered an interesting connection advocating further study. Having confirmed the hypothesis that hypoxia may generate a pro-angiogenic milieu inside ASC-seeded constructs, faster vessel ingrowth and improved vascularization as well as an enhanced tolerance of hypoxia-treated ASCs towards ischemic conditions upon implanatation may be expected, but remain to be verified in rodent models in vivo (Chapter 5).
Having previously been utilized for bone and cartilage engineering [17-19], as well as for revascularization and wound healing applications [20-22], stromal-vascular fraction (SVF) cells were investigated as a novel cell source for adipose tissue engineering. Providing cells with adipogenic differentiation as well as vascularization potential, the SVF was applied with the specific aim to promote adipogenesis and vascularization in engineered constructs in vivo. With only basic in vitro investigations by Lin et al. addressing the SVF for adipose repair to date [23], the present work thoroughly investigated SVF cells for adipose tissue construct generation in vitro, and in particular, pioneered the application of these cells for adipose tissue engineering in vivo.
Initial in vitro experiments compared SVF- and ASC-seeded stable fibrin constructs in different medium compositions employing preadipocyte (PGM-2) and endothelial cell culture medium (EGM-2). It was found that a 1:1 mixture of PGM-2 and EGM-2, as previously established for co-culture models of adipogenesis [24], efficiently maintained cells with adipogenic and endothelial potential in SVF-seeded constructs in short and long-term culture setups. Observations on the cellular level were supported by analysis of mRNA expression of characteristic adipogenic and endothelial markers. In preparation of the evaluation of SVF-seeded constructs under in vivo conditions, a whole mount staining (WMS) method, facilitating the 3D visualization of adipocytes and blood vessels, was successfully established and optimized using native adipose tissue as template (Chapter 6).
In a subcutaneous nude mouse model, SVF cells were, for the first time in vivo, elucidated for their potential to support the functional assembly of vascularized adipose tissue. Investigating the effect of adipogenic precultivation of SVF-seeded stable fibrin constructs in vitro prior to implantation on the in vivo outcome, hormonal induction was shown beneficial in terms of adipocyte development, whereas a strong vascularization potential was observed when no adipogenic inducers were added. Via histological analysis, it was proven that the developed structures were of human origin and derived from the implanted cells. Applying SVF cells without precultivation in vitro but comparing two different fibrin carriers, namely stable fibrin and TissuCol gels, revealed that TissuCol profoundly supported adipose formation by SVF cells in vivo. This was contrasted by only minor SVF cell development and a strong reduction of cell numbers in stable fibrin gels implanted without precultivation. Histomorphometric analysis of adipocytes and capillary structures was conducted to verify the qualitative results, concluding that particularly SVF cells in TissuCol were highly suited for adipose regeneration in vivo. Employing the established WMS technique, the close interaction of mature adipocytes and blood vessels in TissuCol constructs was impressively shown and via species-specific human vimentin staining, the expected strong involvement of implanted SVF cells in the formation of coherent adipose tissue was confirmed (Chapter 7).
With the development of biodegradable volume-stable adipose tissue constructs, the application of ASCs and SVF cells as two promising cell sources for functional adipose regeneration, as well as the thorough evaluation of strategies for construct vascularization in vitro and in vivo, this thesis provides valuable solutions to current challenges in adipose tissue engineering. The presented findings further open up new perspectives for innovative treatments to cure soft tissue defects and serve as a basis for directed approaches towards the generation of clinically applicable soft tissue substitutes.
Zur Erhöhung der mechanischen Stabilität mineralischer Knochenzemente aus Calciumorthophosphaten (CPC) wurde in einem TTCP/DCPA-System das Zementedukt TTCP mit verschiedenen biokompatiblen Oxiden (SiO2, TiO2, ZrO2) während des Herstellungsprozesses dotiert. Dies führte zur Bildung von Calciummetallaten und einer Herabsetzung der Löslichkeit der TTCP-Komponente des Zements. Gegenüber einem oxidfreien Zement konnte die Druckfestigkeit von 65 MPa auf 80 MPa (SiO2) bzw. 100 MPa (TiO2) gesteigert werden.
In einem zweiten Ansatz zur Verbesserung der Injizierbarkeit wurden die Wechselwirkungen der Partikeloberflächen mit der flüssigen Zementphase betrachtet. Durch biokompatible Additive sollte eine repulsive elektrostatische Wechselwirkung eingestellt werden, um Partikelagglomerate effektiv zu dispergieren und eine verflüssigende Wirkung zu erreichen. Die Injizierbarkeit eines TTCP/DCPA-Zements durch eine Kanüle mit 800 µm Durchmesser konnte durch die Verwendung von 500 mM tri-Natriumzitrat-Lösung aufgrund einer deutlichen Herabsetzung der Viskosität der Zementpaste signifikant gesteigert werden (>95%, P/L 3,3/1, Kraftaufwand 20 N).
Abschließend wurde der Einfluss der Partikelgrößenverteilung auf die Festigkeit und Injizierbarkeit einer auf monomodaler Partikelgrößenverteilung basierten Zementmatrix untersucht. Hierzu wurden einem mechanisch aktivierten a-TCP-System unreaktive, feinkörnige Füllstoffpopulationen (TiO2, CaHPO4, CaCO3) zugesetzt und systematisch deren Effekt in Verbindung mit einer Partikelaufladung durch tri-Natriumzitrat auf die rheologischen und mechanischen Eigenschaften untersucht.
Erst die Kombination einer bimodalen Partikelgrößenverteilung mit tri-Natriumzitrat-Lösung führte zu einer starken Erniedrigung der Viskosität, damit zur nahezu vollständigen Injizierbarkeit der Zemente und einer teilweise signifikanten Steigerung der mechanischen Festigkeiten (z.B. 72 MPa reiner a-TCP-Zement auf 142 MPa mit Zusatz von CaHPO4).
Aim of this thesis was to combine the versatility of sulfur-chemistry, regarding redox-sensitivity as well as chemo- and site-specific conjugation, with multifunctionality of poly(glycidol)s as an alternative to poly(ethylene glycol).
First the homo- and copolymerizations of EEGE and AGE were performed with respect to molar-mass distribution and reaction kinetics. A detailed study was given, varying the polymerization parameters such as DP, counter ion, solvent and monomer influence. It can be concluded that in general the rates for all polymerizations are higher using K+, in contrast to Cs+, as counter ion for the active alkoxide species. Unfortunately, K+ as counter ion commonly leads to a reduced control over polymer dispersity. In this thesis it was shown that the broad molar-mass distributions might be reduced by adding the monomer in a step-wise manner. In experiments with a syringe pump, for continuously adding the monomer, a significant reduction of the dispersities could be found using K+ as counter ion.
In analogy to the oxyanionic polymerization of epoxides, the polymerization of episulfides via a thioanionic mechanism with various DPs was successful with thiols/DBU as initiator. In most experiments bimodality could be observed due to the dimerization, caused by oxidation processes by introduced oxygen during synthesis. Reducing this was successful by modifying the degassing procedure, e.g. repeated degassing cycles after each step, i.e. initiation, monomer addition and quenching. Unfortunately, it was not always possible to completely avoid the dimerization due to oxidation. Thiophenol, butanethiol, mercaptoethanol and dithiothreitol were used as thiol initiators, all being capable to initiate the polymerization. With the prediction and the narrow molar-mass distributions, the living character of the polymerization is therefore indicated.
Homo- and copolymers of poly(glycidol) were used to functionalize these polymers with side-chains bearing amines, thiols, carboxylic acids and cysteines. The cysteine side-chains were obtained using a newly synthesized thiol-functional thiazolidine. For this, cysteine was protected using a condensation reaction with acetone yielding a dimethyl-substituted thiazolidine. Protection of the ring-amine was obtained via a mixed-anhydride route using formic acid and acetic anhydride. The carboxylic acid of 2,2-dimethylthiazolidine-4-carboxylic acid was activated with CDI and cysteamine attached. The obtained crystalline mercaptothiazolidine was subjected to thiol-ene click chemistry with allyl-functional poly(glycidol). A systematic comparison of thermal- versus photo-initiation showed a much higher yield and reaction rate for the UV-light mediated thiol-ene synthesis with DMPA as photo-initiator. Hydrolysis of the protected thiazolidine-functionalities was obtained upon heating the samples for 5 d at 70 °C in 0.1 M HCl. Dialysis against acetic acid lead to cysteine-functional poly(glycidol)s, storable as the acetate salt even under non-inert atmosphere. An oxidative TNBSA assay was developed to quantify the cysteine-content without the influence of the thiol-functionality. A cooperation partner coupled C-terminal thioester peptides with the cysteine-functional poly(glycidol)s and showed the good accessibility and reactivity of the cysteines along the backbone. SDS-PAGE, HPLC and MALDI-ToF measurements confirmed the successful coupling.
Tumorzellen, Stromazellen, Extrazellulärmatrix (EZM) und lösliche Faktoren in der Tumormikroumgebung beeinflussen und verstärken sich gegenseitig in der Ausbildung eines malignen Phänotyps. Sowohl die fibrotische EZM als auch eine kleine Subpopulation von pluripotenten Tumorstammzellen sind bekanntermaßen für die Steigerung der Tumoraggressivität verantwortlich. Inwiefern diese beiden unabhängigen Faktoren im Kontext von Brustkrebs miteinander in Beziehung stehen, ist jedoch bis heute unklar.
Um untersuchen zu können, welchen Beitrag Tumorzellen, Stromazellen, EZM und lösliche Faktoren einzeln und im Zusammenspiel zur Malignität eines Tumors leisten, ist die Entwicklung geeigneter in-vitro-Modelle unabdingbar. Daher war es das Ziel dieser Arbeit, ein 3D-Mikrotumormodell zu generieren, in dem eine Analyse dieser genannten Faktoren stattfinden könnte. An diesem Modell wurden darüber hinaus erste Untersuchungen von im Tumorkontext bekannten EZM-Proteinen durchgeführt. Um die dreidimensionale Anordnung von Tumorzellen und ihrer Gewebeumgebung adäquat wiedergeben zu können, beinhalteten die 3D-Tumorsphäroide sowohl Brustkrebszellen (MDA-MB-231) als auch Stromazellen (hASCs).
Die EZM als wichtiger Bestandteil der (Tumor-) Mikroumgebung sollte übersichtshalber durch Hämatoxylin-Eosin-Färbung und detaillierter durch immunhistochemische Analyse nach zwei verschiedenen Kulturzeitpunkten charakterisiert werden, um EZM-Veränderungen im zeitlichen Verlauf darzustellen. Im Fokus der Analyse standen die beiden wichtigsten profibrotischen EZM-Proteine Fibronektin und Kollagen I, die maßgeblich an der Pathogenese von Brustkrebs beteiligt sind. Zudem wurde das Vorkommen des Myofibroblastenmarkers α-SMA untersucht.
An den Sphäroiden einer Kontrollgruppe, die lediglich hASCs beinhaltete, sollte vergleichend eine Analyse der genannten EZM-Proteine sowie α-SMA durchgeführt werden. Um schließlich den Einfluss der von Tumorzellen sezernierten löslichen Faktoren in der Tumormikroumgebung herauszustellen, wurden Sphäroide aus hASCs in tumorkonditioniertem Medium gezüchtet und darin ebenfalls Matrixproteine und α-SMA untersucht.
Abschließend erfolgte eine Korrelation der EZM-Analyse mit dem Vorhandensein von Tumorstammzellen in den 3D-Tumorsphäroiden. Dafür wurden die Tumorstammzellen mithilfe eines GFP-basierten Reporters für den Stammzellmarker NANOG (NANOG-GFP-Reporterzelllinie) in mikroskopischen Aufnahmen der 3D-Tumorsphäroide nachgewiesen und im Kontext mit der EZM lokalisiert.
In this thesis, non-modified POx, namely PnPrOx and PcycloPrOx, with an LCST in the physiological range between 20 and 37°C have been utilized as materials for three different biofabrication approaches. Their thermoresponsive behavior and processability were exploited to establish an easy-to-apply coating for cell sheet engineering, a novel method to create biomimetic scaffolds based on aligned fibrils via Melt Electrowriting (MEW) and the application of melt electrowritten sacrificial scaffolds for microchannel creation for hydrogels.
Chapter 3 describes the establishment of a thermoresponsive coating for tissue culture plates. Here, PnPrOx was simply dissolved in water and dried in well plates and petri dishes in an oven. PnPrOx adsorbed to the surface, and the addition of warm media generated a cell culture compatible coating. It was shown that different cell types were able to attach and proliferate. After confluency, temperature reduction led to the detachment of cell sheets. Compared to standard procedures for surface coating, the thermoresponsive polymer is not bound covalently to the surface and therefore does not require specialized equipment and chemical knowledge. However, it should be noted that the detachment of the cell layer requires the dissolution of the PnPrOx-coating, leading to possible polymer contamination. Although it is only a small amount of polymer dissolved in the media, the detached cell sheets need to be washed by media exchange for further processing if required. ...
Aufgrund der sich umkehrenden Alterspyramide in Deutschland leiden bereits jetzt immer mehr Menschen an Gelenkknorpelschäden. Doch nicht nur das Alter, sondern auch Unfälle und Sportverletzungen und Übergewicht können zu irreversiblen Knorpeldefekten führen. Obwohl es diverse Behandlungsmöglichkeiten gibt, können die bisherige Methoden nicht als dauerhafte Heilung betrachtet werden. Im Rahmen des internationalen Forschungsprojektes BIO-CHIP sollte eine vielsprechende Behandlungsmethode mit neuartigen Arzneimitteln untersucht werden.
Als Ausgangsmaterial des Arzneimittels, ein hergestelltes Knorpelimplantat, dienen patienteneigene Knorpelzellen aus der Nase. Diese werden isoliert, vermehrt und letztlich auf einer Matrix zu einem Knorpelimplantat kultiviert. Wesentliche Voraussetzung für die Implantatfreigabe stellt neben toxikologischen und biologischen Unbedenklichkeitstests die Beurteilung der Viabilität dar. Diese wurde bisher anhand von Histologieschnitten von der Pathologie durchgeführt.
Ziel der vorliegenden Arbeit war die Entwicklung und Validierung eines standardisierten und objektiven Viabilitätstests für die Chondrozyten innerhalb der Knorpelmatrix. Hierfür wurde die LDH als Marker für irreversibel geschädigte Zellen verwendet. Die LDH Konzentration konnte mit dem CyQuant LDH-Assay durch die Messung der Absorption gemessen werden. Es konnte nachgewiesen werden, dass LDH die erforderliche Stabilität und Nachweisbarkeit im Medium besitzt. Mithilfe der Lyse, analog zum Herstellungsprozess, gezüchteter Mini-Knorpelimplantate, konnten die maximal erreichbaren LDH Konzentrationen ermittelt werden. Mithilfe dieser Konzentrationen wurde eine Eichkurve generiert. Diese dient als Beurteilung der Viabilität zukünftig gemessener Absorptionen des Überstandmediums.
Das entwickelte Verfahren erfordert keine invasiven Eingriffe am Implantat und zeichnet sich durch eine einfache Durchführung aus, da nur der Überstand gemessen werden muss. Die durchgeführte Validierung der Methode bescheinigte eine hohe Robustheit, Linearität, Genauigkeit und Präzision.
In einigen Punkten konnte diese Arbeit die Ergebnisse früherer Studien bestätigen.
Einstellungs- und Nadelmodi (wenn auch mit unterschiedlicher Ausprägung)
haben einen Einfluss auf die verbesserte Sichtbarkeit der Regionalanästhesienadeln
im Ultraschall. Damit diese sonographisch besser erkannt werden, sollten sie
in einem flacheren Winkel (30-45°) eingestochen werden. Größere Nadellumina
haben einen Vorteil in der Sichtbarkeit im Ultraschall. Auch konnte gezeigt werden,
dass der Nadelschliff einen Einfluss darauf hat, wie gut die Anästhesiekanülenspitze
sonographisch gesehen wird. Allein die (in der Arbeit untersuchten) Nadelbeschichtungs-
Arten brachten keinen wesentlichen Effekt für die verbesserte
Erkennbarkeit.
Es war jedoch auffallend, dass die subjektiven und objektiven Resultate in einem
relativ hohen Maße nicht übereinstimmten. Gründe dafür sehe ich v.a. im gewählten
Verfahren der objektiven Untersuchung (mit der Bildbearbeitungssoftware
muss die region of interest zielgenauer erfasst werden, um störende Umgebungseinflüsse
auszuschließen).
Echogene Nadeloberflächen (auch wenn diese nicht Gegenstand der vorliegenden
Untersuchung waren), weisen eine verbesserte Ultraschall-Reflexion auf. Sie
werden dazu beitragen, dass die sichere Anwendung der USRA weiter zunimmt.
Verbesserte Sonographiegeräte, zusätzliche Hilfsmittel (GPS-Unterstützung) und
neueste Nadelentwicklungen versprechen einen wichtigen Fortschritt auf dem
Gebiet der ultraschallunterstützten Regionalanästhesie (USRA). Diese Weiterentwicklungen
werden der USRA den Stellenwert als Gold-Standard in der
Regionalanästhesie sichern.
The basement membrane separates the epithelium from the stroma of any given barrier tissue and is essential in regulating cellular behavior, as mechanical barrier and as structural support. It further plays an important role for new tissue formation, homeostasis, and pathological processes, such as diabetes or cancer. Breakdown of the basement membrane is believed to be essential for tumor invasion and metastasization. Since the basement membrane is crucial for many body functions, the development of artificial basement membranes is indispensable for the ultimate formation of engineered functional tissue, however, challenging due to their complex structure.
Electrospinning enables the production of fibers in the nano- or microscale range with morphological similarities to the randomly orientated collagen and elastic fibers in the basement membrane. However, electrospun fibers often lack the functional similarity to guide cells and maintain tissue-specific functions. Hence, their possible applications as matrix structure for tissue engineering are limited.
Herein, the potential of polyester meshes, modified with six armed star-shaped pre-polymers and cell-adhesion-mediating peptides, was evaluated to act as functional isotropic and bipolar artificial basement membranes. Thereby, the meshes were shown to be biocompatible and stable including under dynamic conditions, and the degradation profile to correlate with the rate of new tissue formation. The different peptide sequences did not influence the morphology and integrity of the fibers. The modified membranes exhibited protein-repellent properties over 12 months, indicating the long-term stability of the cross-linked star-polymer surfaces.
Cell culture experiments with primary fibroblasts and a human keratinocyte cell line (HaCaT) revealed that cell adhesion and growth strongly depends on the peptide sequences and their combinations employed. HaCaT cells grew to confluence on membranes modified with a combination of laminin/collagen type IV derived binding sequences and with a combination of fibronectin/laminin/collagen type IV derived peptide sequences. Fibroblasts strongly adhered to the fibronectin derived binding sequence and to membranes containing a combination of fibronectin/laminin/collagen type IV derived peptide sequences. The adhesion and growth of fibroblasts and HaCaT cells were significantly reduced on membranes modified with laminin, as well as collagen IV derived peptide sequences. HaCaT cells and fibroblasts barely adhered onto meshes without peptide sequences.
Co-culture experiments at the air-liquid interface with fibroblasts and HaCaT cells confirmed the possibility of creating biocompatible, biofunctional and biomimetic isotropic and bipolar basement membranes, based on the functionalized fibers. HaCaT cells grew in several layers, differentiating towards the surface and expressing cytokeratin 10 in the suprabasal and cytokeratin 14 in the basal layers. Migration of fibroblasts into the electrospun membrane was shown by vimentin staining. Moreover, specific staining against laminin type V, collagen type I, III, IV and fibronectin illustrated that cells started to remodel the electrospun membrane and produced new extracellular matrix proteins following the adhesion to the synthetic surface structures.
The culturing of primary human skin keratinocytes proved to be difficult on electrospun fibers. Cells attached to the membrane, but failed to form a multilayered, well-stratified, and keratinized epidermal layer. Changing the fiber composition and fixation methods did not promote tissue development. Further investigations of the membrane demonstrated the tremendous influence of the pore size of the membrane on epithelial formation. Furthermore, primary keratinocytes reacted more sensitive to pH changes in the medium than HaCaT cells did.
Since primary keratinocytes did not adequately develop on the functionalized meshes, polycarbonate membranes were used instead of electrospun meshes to establish oral mucosa models. The tissue-engineered models represented important features of native human oral mucosa. They consisted of a multilayered epithelium with stratum basale, stratum spinosum, stratum granulosum, and stratum corneum. The models formed a physical barrier and the expression of characteristic cell markers was comparable with that in native human oral mucosa. The results from the ET-50 assay and the irritation study reflected the reproducibility of the tissue equivalents.
Altogether, electrospinning enables the production of fibers with structural similarity to the basement membrane. Incorporating extracellular matrix components to mimic the functional composition offers a safe and promising way to modify the fibers so that they can be used for different tissue engineering applications. The resultant biomimetic membranes that can be functionalized with binding sequences derived from widely varying proteins can be used as a toolbox to study the influence of isotropic and bipolar basement membranes on tissue formation and matrix remodeling systematically, with regards to the biochemical composition and the influence and importance of mono- and co-culture. The oral mucosa models may be useful for toxicity and permeation studies, to monitor the irritation potential of oral health care products and biomaterials or as a disease model.
Intraperitoneal adhesions are fibrous bands that connect tissues in the peritoneal cavity that are usually separated. These adhesions form as a consequence of trauma, inflammation or surgical interventions and often result in severe consequences such as chronic pain, small bowel obstructions or female infertility.
The aim of this thesis was to develop a synthetic barrier device for adhesion prevention made of modified poly(lactide) [PLA]. Solid PLA films (SurgiWrap®) are already successfully in clinical use due to the good biocompatibility and the biodegradability of the material resulting in non-toxic degradation products since lactic acid is naturally part of the metabolic circles of the human body. Considering the brittleness and stiffness of the films, the long degradation time of several months as well as the need for suturing, there is potential for optimization. Through a copolymerization with the hydrophilic poly(ethylene glycol) [PEG], a reduction of the degradation time was intendend. Moreover, the copolymerization should also lead to an improvement of the mechanical properties of the films since PEG acts as plasticizer for PLA. Linear PLA-PEG-PLA triblock copolymers as well as star-shaped PEG-PLA copolymers were synthesized via standard ring opening polymerization to tailor the barrier properties. Besides solid films, solution electrospun meshes from PLA and the synthesized PEG-PLA copolymers were investigated for a potential application as well. Since suturing of a barrier additionally induces adhesion formation, alginate coated membranes were prepared in order to achieve self-adhesiveness. With the intention to reduce infections and consequently inflammation, electrospun meshes and solvent cast films were loaded with the antibacterial drug triclosan and drug release as well as antibacterial efficacy was investigated.
Mechanical tests confirmed that through the variation of the PEG content and branching the mechanical properties can be tailored and are in good accordance with the glass transition temperatures [Tg] of the polymers. Consequently, potentially adequate mechanical properties for surgical handling as well as for the performance within the patient’s body were successfully achieved. Degradation studies revealed that the degradation time was significantly shorter for PEG-PLA membranes than for PLA films and with an appropriate PEG content could be adjusted to the intended time frame. Cell adhesion and viability tests confirmed the non-toxicity of the clinically used PLA films as well as of PEG-PLA films and meshes. With a bioadhesion test the benefit of an alginate coated side towards the pure PLA film concerning self-adhesiveness was successfully demonstrated. Moreover, optical evaluations and a T-peel test of different alginate coated PLA films showed that the cohesion between the chemically different layers was distinctly enhanced by the use of an appropriate PEG-PLA mesh as intermediate cohesion promoting layer. In in vitro release studies with triclosan loaded films a higher release was determined for PEG-PLA than for PLA films. In agar diffusion tests a higher and longer inhibition of staphylococcus aureus growth was observed confirming the release results. Moreover, drug loaded meshes (especially drug loaded after electrospinning) showed enhanced and elongated bacterial inhibition in comparison to films.
Die vorliegende Arbeit hatte zum Ziel, die Zytokompatibilität von im 3D-Pulverdruckverfahren hergestellten Zellkulturträgern aus Dicalciumphosphat Anhydrat (CaHPO4, Monetit) in vitro zu untersuchen. Dieses Material lässt sich der Substanzklasse der Calciumphosphate zuordnen, welche aufgrund ihrer chemischen Ähnlichkeit zur mineralischen Phase des Knochens einen hohen Stellenwert als Knochenersatzmaterial besitzen.
Die Trägerstrukturen wurden mittels CAD-CAM Technologie im 3D-Pulverdruckverfahren fabriziert. Dabei wurde auf ein entsprechend adaptiertes Zementsystem zurückgegriffen, bestehend aus Tricalciumphosphatpulver und Phosphorsäure. Die primär aus Dicalciumphosphat Dihydrat (Bruschit) bestehenden Konstrukte wurden anschließend durch Autoklavieren hydrothermal in Monetit umgewandelt.
Die Kombination einer bei Raumtemperatur ablaufenden Zementabbindereaktion mit einem generativen Fertigungsverfahren wie dem Pulverdruck ermöglichte die Herstellung monolithischer Formkörper ohne thermische Verfestigung (Sinterung). Daher kann eine im Vergleich zu gesinterten Formkörpern gute thermodynamische Löslichkeit und somit gute Biodegradierbarkeit erwartet werden.
Zur Evaluierung der Zytokompatibilität des pulvergedruckten Materials wurde nach Besiedlung mit osteoblastären Zellen deren Proliferations- und Differenzierungsverhalten in vitro untersucht. Die Zellviabilität, die Aktivität der Alkalischen Phosphatase sowie die Konzentration von Osteocalcin dienten als Parameter. Weiterhin wurden die Konzentration freier Elektrolyte und der pH-Wert im Nährmedium zur Evaluierung der Löslichkeit der Träger in vitro herangezogen. Anhand licht- und rasterelektronenmikroskopischer Aufnahmen erfolgte eine qualitativ-morphologische Einschätzung des Zellwachstums.
Die Untersuchungen zeigen eine gute Zytokompatibilität des Trägermaterials aus Monetit. Die im Vergleich zu den Positiv-Kontrollen etwas erniedrigten Werte lassen sich durch die im Nährmedium festgestellten Elektrolytverschiebungen erklären, welche durch die thermodynamische Löslichkeit von Monetit zustande kommen. Diese Problematik der Zellkultur als geschlossenem System sollte jedoch in vivo bei stetigem Flüssigkeits- und Metabolitenaustausch keine Rolle spielen. Die Ergebnisse liefern einen Beitrag zur Erarbeitung neuartiger Knochenzemente, insbesondere aus Monetit. Klinisch interessant erscheint die verfahrensbedingte Möglichkeit, die Anforderungen nach guter Degradierbarkeit, präoperativer Fabrizierung und individueller Formgebung (z.B. passend zu einem individuellen Defekt) miteinander kombinieren zu können.
Die Therapie von Patienten mit fortgeschrittener sensorineuraler Schwerhörigkeit oder Taubheit mit auditorischen Implantaten ist heute Standard in der medizinischen Versorgung. Durch direkte elektrische Stimulation der noch vorhanden auditorischen Neurone wird versucht, die physiologische Informationsübertragung entlang der Hörbahn künstlich nachzubilden. Strukturierte Halbleiterelektrodenträger aus Silizium stellen dabei eine mögliche Alternative zu den herkömmlichen Kabelbündel-Elektrodenträgern heutiger Implantate dar. Durch die Möglichkeit der Abstandsverringerung zwischen Stimulationselektroden und Nervenfasern sowie durch die Möglichkeit der Erhöhung der Anzahl an Stimulationselektroden könnte die Leistungsfähigkeit heutiger Implantate verbessert werden.
Ziel der vorliegenden Arbeit war es, das Wachstumsverhalten corticaler neuronaler Vorläuferzellen der Maus auf strukturierten Siliziumhalbleitern zu untersuchen und das Wachstumsverhalten näher zu charakterisieren. Zwei unterschiedliche Oberflä-chenstrukturen wurden durch den Einsatz der Elektronenstrahllithografie auf Silizi-umwafern erzeugt. Zylinder auf der Oberfläche der Halbleiter wurden als Modellstruktur gewählt, um eine erhöhte Anzahl an Elektrodenkontakten zu simulieren und die neuronale Interaktion mit diesen zu untersuchen. Daneben wurden Furchen auf Siliziumoberflächen verwendet, um die Wachstumsrichtung der neuronalen Zellen zu beeinflussen.
Die durchgeführten Untersuchungen konnten zeigen, dass strukturierte Halbleiter-elektrodenträger in zukünftigen auditorischen Implantaten grundsätzlich eine Alternative zu den Kabelbündel-Elektrodenträgern heutiger Implantate sein könnten. Durch den Einsatz der Elektronenstrahllithografie konnten Siliziumwafer mit präziser Oberflächenstrukturierung hergestellt werden und deren Biokompatibilität durch Kultivierung neuronaler Zellen gezeigt werden. Die Geometrie der eingeätzten Oberflächenstruktur hatte dabei entscheidenden Einfluss auf das Wachstumsverhalten der Zellen. Während durch Furchen die Orientierung der Neurone gezielt beeinflusst werden konnte und die Neuritenlängen mit zunehmender Ätztiefe abnahmen, konnten derartige Effekte bei den untersuchten Zylindern nicht beobachtet werden.
Die durchgeführten rasterelektronenmikroskopischen Untersuchungen gaben Auf-schluss über die Interaktion der Neurite mit der Oberflächenstruktur. Auf vertikalen Wachstumsstress reagierten die kortikalen neuronalen Vorläuferzellen unabhängig von der Oberflächenstruktur mit der Ausbildung von neuronalen Brücken. Der Modus der Brückenbildung war bei beiden Strukturen dabei gleich, jedoch wurden unterschiedliche Verankerungspunkte an der Siliziumoberfläche beobachtet. Darüber hinaus konnte durch Echtzeituntersuchungen an lebenden Zellen gezeigt werden, dass diese während des Wachstums ihre Lage verändern konnten und somit nicht dauerhaft mit der Oberfläche verbunden waren.
Die Ergebnisse zeigen, dass die beiden untersuchten Oberflächenstrukturen grundsätzlich für den Einsatz auf zukünftigen Halbleiterelektrodenträgern geeignet sind, jedoch noch weiterführende Untersuchungen nötig sind, um diese weiter zu optimieren.
Die vorliegende Arbeit befasste sich mit der bisher relativ unbekannten Polymerklasse der Polypeptoide, die hinsichtlich ihrer Verwendung als Biomaterial näher untersucht werden sollte. Hierbei war die Untersuchung des Polymerisationssystems ein wesentlicher Schwerpunkt. Dies beinhaltete zum einen die Synthesen verschiedener Monomere sowie deren Polymerisationskinetiken und zum anderen Studien über die Stabilität des aktiven Kettenendes. Um mehr über die Polypeptoide zu erfahren, wurden die erhaltenen Homopolymere nach der Strukturanalyse hinsichtlich ihrer physikochemischen Eigen-schaften untersucht. Im Anschluss erfolgte die Synthese von (amphiphilen) Blockco-polypeptoiden, die sich in wässrigen Lösungen zu definierten Morphologien zusammen-lagern. Die resultierenden Morphologien, sowohl mizellare als auch vesikuläre Strukturen, wurden mit verschiedenen Methoden, wie z. B. der Pyren-Fluoreszenz-Spektroskpie und der dynamischen Lichtstreuung, untersucht. Erste Erkenntnisse über die Biokompatibilität der Polypeptoide sollte die Bestimmung der Zellviabilität in verschiedenen Polymerlösungen liefern.
Die verschiedenen Studien über die Polypeptoide zeigten, dass diese Polymerklasse über eine besonders lebende Polymerisation synthetisiert werden kann. Dabei resultieren Produkte, die sich durch eine Poisson-Verteilung und eine hohe Endgruppengenauigkeit auszeichnen. Zusätzlich bestehen Polypeptoide aus einem abbaubaren Rückgrat und, im Vergleich zu den Polypeptiden, besitzen sie eine erhöhte proteolytische Stabilität. Amphiphile Blockcopolypeptoide sind zudem in der Lage, sich in Lösung zu verschiedenen Morphologien anzuordnen. Durch die Variierung der Seitenkette und des f kann sowohl die Selbstorganisation als auch das Mikroumfeld der Aggregate abgestimmt werden. Darüber hinaus können die amphiphile Blockcopolymere, die sich zu Mizellen anordnen, hydrophobe Substanzen solubilisieren.
Polypeptoide liefern all die nötige chemische Vielseitigkeit und potentielle Biokompatibilität, um bestehende sowie neuartige Probleme in biomedizinischen Anwendungen zu bewältigen. Zukünftige in vivo und in vitro Test werden das Potential, aber auch die Grenzen dieser neuen Polymerklasse als Biomaterial zeigen.
Hintergrund: Narbenhernien stellen nach Operationen ein unerwünschtes Ereignis dar. Dabei werden in der Literatur verschiedene Theorien zur Entstehung diskutiert. Sowohl beim Aortenaneurysma als auch bei der Narbenhernie soll das Kollagen ein entscheidender Faktor in der Entstehung. Historisch wird von ausgegangen das Patienten mit einem Aortenaneurysma auch ein erhöhtes Risiko für die Entwicklung einer Narbenhernie haben. Aus diesem Grund vergleichen wir die Inzidenzrate zwischen Patienten mit Aortenaneurysmen und Patienten mit einem kolorektalen Eingriff um Risikofaktoren zu identifizieren.
Methoden: Diese Studie ist eine retrospektive „Matched Control“ Kohorten-Studie. Es wurden alle Patienten eingeschlossen die sich zwischen dem 01.01.2006 und dem 31.12.2008 an der chirurgischen Universitätsklinik Würzburg an einem Aortenaneurysma oder einem kolorektalen Eingriff unterzogen haben.
Ergebnisse: In unserer Studie konnten wir eine Gesamtinzidenzrate von 17,2% nachweisen. In der Gruppe der Aortenaneurysmen bestand eine Inzidenzrate von 13,9%, in der Gruppe der Kolorektalen Eingriffe von 25,9%. Es bestand kein signifikanter Unterschied zwischen den beiden Gruppen bezüglich der Inzidenzrate von Narbenhernien
Schlussfolgerung: Es besteht kein signifikanter Unterschied zwischen den beiden Vergleichsgruppen. In der Gruppe der Aortenaneurysmen war die Inzidenzrate sogar geringer.
Modifikation von Titanoberflächen mittels elektrochemischer Abscheidung von Magnesiumphosphaten
(2013)
In der vorliegenden Arbeit ist es gelungen, die experimentellen Parameter für eine erfolgreiche elektrochemische Abscheidung sowohl von Struvit (MgNH4PO4 • 6H2O) als auch Newberyit (MgHPO4 • 3H2O) auf durch Sandstrahlen aufgeraute Titanproben zu ermitteln. Welche der beiden Phasen auf den Titanoberflächen abgeschieden wurde, hing dabei hauptsächlich von der jeweiligen Elektrolytzusammensetzung ab. Bei der Elektrodeposition selbst erwiesen sich eine Elektrolyttemperatur von 50 °C und Stromdichten von etwa 79 – 105 mA/cm2 als optimal, um geschlossene Schichten von hinreichender Dicke reproduzierbar herzustellen. Es zeigte sich, dass die für die jeweiligen Abscheidungsprodukte optimierten Parameter (79 mA/cm2 für Struvit und 105,3 mA/cm2 für Newberyit) zu deutlich unterschiedlichen Massenabscheidungen (4,4 mg/cm2 für Struvit und 0,6 mg/cm2 für Newberyit bei einer Beschichtungsdauer von 15 min) führten. Das Monohydrat Dittmarit (MgNH4PO4 • H2O) ließ sich nicht direkt abscheiden, konnte aber durch Dampfsterilisation von zuvor erzeugten Struvitschichten in einem Autoklaven erzeugt werden.
Um das Verhalten der Oberflächenmodifikationen in einer in-vivo-Umgebung zu simulieren, wurden die Beschichtungen für eine maximale Dauer von 14 Tagen in Simulated Body Fluid (SBF), Dulbecco's Modified Eagle Medium (DMEM) und in fötalem Kälberserum (FCS) eingelagert. In bestimmten Zeitabständen wurden eingelagerte Proben ihrem Medium entnommen, getrocknet und die Schichten mit Hilfe der Röntgendiffraktometrie und der Rasterelektronen-mikroskopie hinsichtlich ihrer kristallographischen und morphologischen Eigenschaften charakterisiert. Dabei zeigten die drei Magnesiumphosphate jeweils unterschiedliches Degradationsverhalten in den verschiedenen Einlagerungsmedien. Struvit wandelte sich nach 14 Tagen in DMEM teilweise, in FCS größtenteils und in SBF vollständig zu Bobierrit (Mg3(PO4)2 • 8H2O) um. Ein ähnliches Verhalten zeigte sich bei Dittmarit, allerdings kam es hier in allen Medien zur Bildung einer weiteren Phase (Tri-Magnesium-Di-Phosphat-5-Hydrat, Mg3(PO4)2 • 5H2O), in FCS bildete sich zusätzlich noch Di-Magnesiumphosphathydroxid-4-Hydrat (Mg2PO4OH • 4H2O). Die Newberyit-Schichten hingegen zeigten keinerlei Phasenumwandlungen, lösten sich aber in den Einlagerungsversuchen teilweise auf.
Diese Ergebnisse zeigen, dass elektrochemisch erzeugte Beschichtungen auf Magnesiumphosphatbasis durchaus vielversprechend im Hinblick auf die funktionelle Modifikation metallischer Implantatoberflächen sind. Neben den literaturbekannten positiven Eigenschaften der Magnesiumphosphate (gute Zytokompatibilität, hohe Löslichkeit und mechanische Festigkeit) ist für zukünftige Forschungen vor allem das in dieser Arbeit untersuchte Degradationsverhalten von Interesse. Die in fast allen untersuchten Kombinationen aus Schichtmodifikation und Einlagerungsmedium auftretenden Phasenumwandlungen weisen auf durch die physiologische Umgebung hervorgerufene Resorptionsprozesse hin, die wiederum in vivo die Osteointegration des Implantats unterstützen könnten.
Ein weiterer Aspekt zukünftiger Untersuchungen ist die mögliche Beladung der biokompatiblen Schichten mit bioaktiven Substanzen (antibakterielle oder osteointegrative Wirkstoffe sowie Metallionen zur Unterstützung bzw. Steuerung biologischer Prozesse im implantatnahen Bereich). Hier könnten die unterschiedlichen Degradationsmechanismen der verschiedenen untersuchten Magnesiumphosphat-Modifikationen die Grundlage für kontrollierte und maßgeschneiderte Freisetzungskinetiken liefern.
In modern medicine hip and knee joint replacement are common surgical procedures. However, about 11 % of hip implants and about 7 % of knee implants need re-operations. The comparison of implant registers revealed two major indications for re-operations: aseptic loosening and implant infections, that both severely impact the patients’ health and are an economic burden for the health care system. To address these problems, a calcium hydroxide coating on titanium was investigated in this thesis. Calcium hydroxide is a well-known antibacterial agent and used with success in dentistry. The coatings were applied with electrochemically assisted deposition, a versatile tool that combines easiness of process with the ability to coat complex geometries homogeneously. The pH-gradient during coating was investigated and showed the surface confinement of the coating process. Surface pre-treatment altered the surface morphology and chemistry of the titanium substrates and was shown to affect the morphology of the calcium hydroxide coatings. The influence of the coating parameters stirring speed and current pulsing were examined in various configurations and combinations and could also affect the surface morphology. A change in surface morphology results in a changed adhesion and behavior of cells and bacteria. Thus, the parameters surface pre-treatment, stirring speed and current pulsing presented a toolset for tailoring cellular response and antibacterial properties. Microbiological tests with S. aureus and S. epidermidis were performed to test the time-dependent antibacterial activity of the calcium hydroxide coatings. A reduction of both strains could be achieved for 13 h, which makes calcium hydroxide a promising antibacterial coating. To give insight into biofilm growth, a protocol for biofilm staining was investigated on titanium disks with S. aureus and S. epidermidis. Biofilm growth could be detected after 5 days of bacterial incubation, which was much earlier than the 3 weeks that are currently assumed in medical treatment. Thus, it should be considered to treat infections as if a biofilm were present from day 5 on. The ephemeral antibacterial properties of calcium hydroxide were further enhanced and prolonged with the addition of silver and copper ions. Both ionic modifications significantly enhanced the bactericidal potential. The copper modification showed higher antibacterial effects than the silver modification and had a higher cytocompatibility which was comparable to the pure calcium hydroxide coating. Thus, copper ions are an auspicious option to enhance the antibacterial properties. Calcium hydroxide coatings presented in this thesis have promising antibacterial properties and can easily be applied to complex geometries, thus they are a step in fighting aseptic loosening and implant infections.
Entzündliche Prozesse stellen einen zentralen Aspekt der Karzinogenese dar und können sowohl zur Induktion als auch zum Progress von Tumoren beitragen. Der NF-kB-Signalweg ist einer der wichtigsten Signaltransduktionswege der In- flammation und Tumorpromotion, was ihn zur plausiblen Zielstruktur für die pros- pektive klinische Tumortherapie machen könnte. In der vorliegenden Arbeit wur- den die Eigenschaften von vier unterschiedlich targetierenden NF-kB-pathway- Inhibitoren – Cortisol, MLN4924, QNZ und TPCA1 – auf die Inflammation, Zell- proliferation und proapoptotische Sensitivierung am in vitro Modell des HNSCC untersucht. Es konnte gezeigt werden, dass die spezifische Auswahl des Inhi- bitors bzw. seines targets entscheidend für den wirkungsvollen Einsatz dieser Wirkstoffgruppe in der antiproliferativen Therapie des HNSCC zu sein scheint. Beispielsweise vermittelte MLN4924 die Freisetzung von IL-8. Cortisol bewirkte die Resistenz der FasL-induzierten Apoptose von HNSCC-Zellen. QNZ wirkte in einigen Zelllinien antiproliferativ und sensitivierend für den FasL-induzierten Zell- tod, beeinflusste jedoch in diesem Zusammenhang kontraproduktiv die IL-8-Sek- retion. Dies disqualifizierte diese Wirkstoffe für die Anwendung in der Therapie von Kopf-Hals-Tumoren. Dahingegen qualifizierte sich TPCA-1 aufgrund folgen- der Eigenschaften als geeigneter Wirkstoff für den prospektiven klinischen Ein- satz:
1) TPCA-1 wirkte antiproliferativ,
2) hemmte die TNF-a-induzierte Inflammation,
3) regulierte die IL-8-Expression herab,
4) wirkte sensitivierend für den TNF-a-induzierten Zelltod,
5) interferierte kaum mit der FasL-vermittelten Apoptose und 6) induzierte Apoptose.
In the field of biofabrication, biopolymer-based hydrogels are often used as bulk materials with defined structures or as bioinks. Despite their excellent biocompatibility, biopolymers need chemical modification to fulfill mechanical stability.
In this thesis, the primary alcohol of hyaluronic acid was oxidized using TEMPO/TCC oxidation to generate aldehyde groups without ring-opening mechanism of glycol cleavage using sodium periodate. For crosslinking reaction of the aldehyde groups, adipic acid dihydrazide was used as bivalent crosslinker for Schiff Base chemistry. This hydrogel system with fast and reversible crosslinking mechanism was used successfully as bulk hydrogel for chondrogenic differentiation with human mesenchymal stem cells (hMSC).
Gelatin was modified with pentenoic acid for crosslinking reaction via light controllable thiol-ene reaction, using thiolated 4-arm sPEG as multivalent crosslinker. Due to preservation of the thermo responsive property of gelatin by avoiding chain degradation during modification reaction, this gelatin-based hydrogel system was successfully processed via 3D printing with low polymer concentration. Good cell viability was achieved using hMSC in various concentrations after 3D bioprinting and chondrogenic differentiation showed promising results.
The focus of this thesis was to investigate how PCL and PLGA react to the heat exposure that comes with the MEW process over a defined timespan.
To assess the thermal stability of PCL during MEW over 25 d, an automated collection of fibers has been used to determine the CTS on each day of heating for three different temperatures. PCL is exceptionally stable over 25 d at 75 °C, whereas for 85 °C and 95 °C a slight upward trend during the last 10 d could be observed, which is an indication for thermal degradation. Same trend could be observed for diameter of fibers produced at a fixed collector speed. For all temperatures, CTS during the first 5 d decreased due to inhomogeneities of the melt. Physical analysis of the fibers by XRD and mechanical testing showed no significant changes.
To investigate the chemical details of the thermal durability, PCL was artificially aged over 25 d at 75 °C, 85 °C and 95 °C. Data from GPC analysis and rheology revealed that PCL is degrading steadily at all three temperatures. Combined with GC-MS analysis, two different mechanisms for degradation could be observed: random chain scission and unzipping. Additional GPC experiment using a mixture of PCL and a fluorescence labelled PCL showed that PCL was undergoing ester interchange reactions, which could explain its thermal stability.
PLGA was established successfully as material for MEW. GPC results revealed that PLGA degraded heavily in the one-hour preheating period. To reduce the processing temperature, ATEC was blended with PLGA in three mixtures. This slowed down degradation and a processing window of 6 h could be established. Mechanical testing with fibers produced with PLGA and all three blends was performed. PLGA was very brittle, whereas the blends showed an elastic behavior. This could be explained by ester interchange reactions that formed a loosely crosslinked network with ATEC.
The objective of this thesis was the synthesis and characterisation of two linear multifunctional PEG-alternatives for bioconjugation and hydrogel formation: i) Hydrophilic acrylate based copolymers containing peptide binding units and ii) hydrophilic polyether based copolymers containing different functional groups for a physical crosslinking.
In section 3.1 the successful synthesis of water soluble and linear acrylate based polymers containing oligo(ethylene glycol) methyl ether acrylate with either linear thioester functional 2-hydroxyethyl acrylate, thiolactone acrylamide, or vinyl azlactone via the living radical polymerisation technique Reversible Addition Fragmentation Chain Transfer (RAFT) and via free-radical polymerisation is described. The obtained polymers were characterized via GPC, 1H NMR, IR and RAMAN spectroscopy.
The RAFT end group was found to be difficult to remove from these short polymer chains and accordingly underwent the undesired side reaction aminolysis with the peptide during the conjugation studies. Besides that, polymers without RAFT end groups did not show any binding of the peptide at the thioester groups, which can be improved in future by using higher reactant concentrations and higher amount of binding units at the polymer. Polymers containing the highly reactive azlactone group showed a peptide binding of 19 %, but unfortunately this function also underwent spontaneous hydrolysis before the peptide could even be bound. In all cases, oligo(ethylene glycol) methyl ether acrylate was used with a relatively high molecular weight (Mn = 480 Da) was used, which eventually was efficiently shielding the introduced binding units from the added peptide. In future, a shorter monomer with Mn = 300 Da or less or hydrophilic N,N’-dialkyl acrylamide based polymers with less steric hindrance could be used to improve this bioconjugation system. Additionally, the amount of monomers containing peptide binding units in the polymer can be increased and have an additional spacer to achieve higher loading efficiency.
The water soluble, linear and short polyether based polymers, so called polyglycidols, were successfully synthesized and modified as described in section 3.2. The obtained polymers were characterized using GPC, 1H NMR, 31P{1H} NMR, IR, and RAMAN spectroscopy. The allyl groups which were present up to 20 % were used for radical induced thiol-ene chemistry for the introduction of functional groups intended for the formation of the physically crosslinking hydrogels. For the positively charged polymers, first a chloride group had to be introduced for the subsequent nucleophilic substitution with the imidazolium compound. There, degrees of modifications were found in the range 40-97 % due to the repulsion forces of the charges, decreased concentration of active chloride groups, and limiting solution concentrations of the polymer for this reaction. For the negatively charged polymers, first a protected phosphonamide moiety was introduced with a deprotection step afterwards showing 100 % conversion for all reactions. Preliminary hydrogel tests did not show a formation of a three-dimensional network of the polymer chains which was attributed to the short backbone length of the used polymers, but the gained knowledge about the synthetic routes for the modification of the polymer was successfully transferred to longer linear polyglycidols. The same applies to the introduction of electron rich and electron poor compounds showing π-π stacking interactions by UV-vis spectroscopy.
Finally, long linear polyglycidyl ethers were synthesised successfully up to molecular weights of Mn ~ 30 kDa in section 3.3, which was also proven by GPC, 1H NMR, IR and RAMAN spectroscopy. This applies to the homopolymerisation of ethoxyethyl glycidyl ether, allyl glycidyl ether and their copolymerisation with an amount of the allyl compound ~ 10 %. Attempts for higher molecular weights up to 100 kDa showed an uncontrolled polymerisation behaviour and eventually can be improved in future by choosing a lower initiation temperature. Also, the allyl side groups were modified via radical induced thiol-ene chemistry to obtain positively charged functionalities via imidazolium moieties (85 %) and negatively charged functionalities via phosphonamide moieties (100 %) with quantitative degree of modifications. Hydrogel tests have still shown a remaining solution by using long linear polyglycidols carrying negative charges with long/short linear polyglycidols carrying positive charges. The addition of calcium chloride led to a precipitate of the polymer instead of a three-dimensional network formation representing a too high concentration of ions and therefore shielding water molecules with prevention from dissolving the polymer. These systems can be improved by tuning the polymers structure like longer polymer chains, longer spacer between polymer backbone and charge, and higher amount of functional groups.
The objective of the thesis was partly reached containing detailed investigated synthetic routes for the design and characterisation of functional polymers which could be used in future with improvements for bioconjugation and hydrogel formation tests.
This thesis aimed the development of a correlated device which combines FluidFM® with Fluorescence Microscopy (FL) (FL-FluidFM®) and enables the simultaneous quantification of adhesion forces and fluorescent visualization of mature cells. The implementation of a PIFOC was crucial to achieve a high-resolution as well as a stable but dynamic focus level. The functionality of SCFS after hardware modification was verified by comparing two force-curves, both showing the typical force progression and measured with the optimized and conventional hardware, respectively. Then, the integration of FL was examined by detaching fluorescently labeled REF52 cells. The fluorescence illumination of the cytoskeleton showed the expected characteristic force profile and no evidence of interference effects. Afterwards a corresponding correlative data analysis was addressed including manual force step fitting, the identification of visualized cellular unbinding, and a time-dependent correlation. This procedure revealed a link between the area of cytoskeletal unbinding and force-jumps. This was followed by a comparison of the detachment characteristics of intercellular connected HUVECs and individual REF52 cells. HUVECs showed maximum detachment forces in the same order of magnitude as the ones of single REF52 cells. This contrasted with the expected strong cohesiveness of endothelial cells and indicated a lack of cell-cell contact formation. The latter was confirmed by a comparison of HUVECs, primary HBMVECs, and immortalized EA.hy926 cells fluorescently labeled for two marker proteins of intercellular junctions. This unveiled that both the previous cultivation duration and the cell type have a major impact on the development of intercellular junctions. In summary, the correlative FL FluidFM® represents a powerful novel approach, which enables a truly contemporaneous performance and, thus, has the potential to reveal new insights into the mechanobiological properties of cell adhesion.
A multitude of human tissues, such as bones, tendons, or muscles, are characterized by a hierarchical and highly ordered structure. In many cases, the loss of these tissues requires reconstruction using biocompatible replacement materials. In the field of bone replacement, the pore structure of the material has a crucial influence. Anisotropic porosity would have the advantage of facilitating the ingrowth of cells and newly formed blood vessels as well as the transport of nutrients.
In this thesis, scaffolds with a highly ordered and anisotropic pore structure were fabricated using unidirectional freezing.
Systematic investigations were carried out on biopolymer solutions (alginate and chitosan) to gain a deeper understanding of the freeze-structuring process. The knowledge gained was then applied to the development of anisotropically structured bone substitute materials. Here, the previously existing material platform for anisotropically structured calcium phosphates was extended to low-temperature phases such as calcium deficient hydroxyapatite (CDHA) or the secondary phosphates monetite and brushite.
After the implantation of a biomaterial, the inevitably triggered initial immune response plays a key role in the success of a graft, with immune cells such as neutrophils or macrophages being of particular importance. In this thesis, the influence of anisotropically structured alpha-TCP and CDHA scaffolds as well as their unstructured references on human monocytes/macrophages was investigated. Macrophages produced extracellular traps (ETs) due to mineral nanoparticles formed by the binding of phosphate and calcium ions to human platelet lysate. In particular, incubation of alpha-TCP samples in lysate containing cell culture medium resulted in pronounced particle formation and enhanced release of ETs.
Knorpeldefekte gelten in der Medizin als besonders schwierig zu beheben, da das avaskuläre und aneurale hyaline Knorpelgewebe nur über sehr begrenzte Selbstheilungskräfte verfügt. Die Entwicklung neuer klinischer Therapien für eine erfolgreiche Regeneration bis hin zum vollständigen Ersatz von beschädigtem oder erkranktem Knorpel stellt daher das Ziel umfangreicher Forschung dar. Darüber hinaus zeichnet sich Knorpel durch eine organisierte, zonale Zell-Matrix-Verteilung und -Dichte aus, die möglichst naturgetreu nachgebildet werden muss, um einen adäquaten Gelenkknorpelersatz zu schaffen. Das dreidimensionale Bioprinting von humanen mesenchymalen Stromazellen (hMSCs) in Hydrogelen ist hierbei ein vielversprechender Ansatz. Es sind jedoch umfangreiche Studien erforderlich, um herauszufinden, wie 3D-Stammzellkonstrukte mit unterschiedlichen Zelldichten und Zell-Zell-Wechselwirkungen in einer gedruckten Hydrogel Matrix interagieren. Deshalb wurde in dieser Arbeit untersucht, ob die mesenchymalen Stromazellen in Form von Einzelzellen oder Sphäroiden durch das Extrusionsdruckverfahren in ihrer Proliferationsfähigkeit und ihrem chondrogenen Differenzierungspotential beeinträchtigt werden.
Hierfür wurden in dieser Arbeit sowohl das Zellüberleben als auch Proliferations- und Differenzierungsmarker in gedruckten und nicht gedruckten Proben mit Einzelzellkonzentrationen von 2-20 Millionen Zellen sowie bei Sphäroiden mit ca 4000 Zellen/Sphäroid untersucht. Es konnte gezeigt werden, dass das extrusionsbasierte Druckverfahren keine negativen Auswirkungen auf die Überlebensfähigkeit und die Proliferation der hMSCs hat. Zum Nachweis der chondrogenen Differenzierung wurden mehrere Experimente durchgeführt. Durch die Expression von Typ-II-Kollagen und Aggrecan sowie durch die Quantifizierung von GAG welches zu einem großen Teil in der ECM von Knorpelgewebe zu finden ist, konnte bestätigt werden, dass die mesenchymalen Stromazellen durch den Druckprozess ihr chondrogenes Differenzierungspotential nicht einbüßen. Die beim 3D-Bioprinting auftretenden Scherkräfte scheinen die in-vitro Chondrogenese sogar ohne chemische Stimulation durch TGF-β1 anzustoßen. Außerdem zeigten die Sphäroidgruppen ein höheres chondrogenes Differenzierungspotential als die Einzelzellgruppen.
Um dies im Zusammenhang mit dem 3D Extrusionsdruckverfahren zu bestätigen, erscheint es sinnvoll, weitere Versuche mit noch höheren Zellkonzentrationen in Form von Sphäroiden durchzuführen. Zusammenfassend zeigte sich in dieser Arbeit, dass das extrusionsbasierte Druckverfahren in Alginat/Gelatine Hydrogelen keine Zellschädigung verursacht und weder die chondrogene Differenzierung von Einzelzellen noch von Sphäroiden beeinträchtigt.
Calcium phosphate cements (CPC) represent valuable synthetic bone grafts, as they are self-setting, biocompatible, osteoconductive and in their composition similar to the inorganic phase of human bone. Due to their long shelf-life, neutral setting and since water is sufficient for setting, hydroxyapatite (HA) forming cements are processed in different paste formulations. Those comprise dual setting, Ca2+ binding and premixed cement systems. With dual setting formulations, both dissolution and precipitation of the cement raw powder occur simultaneously to the polymerization of water-soluble monomers to form a hydrogel. Chelating agents are able to form complexes with Ca2+ released from the raw powder. Premixed systems mostly contain the raw powder of the cement and a non-aqueous binder liquid which delays the setting reaction until application in the moist physiological environment. In the present work, two of those reaction mechanisms allowed the development of HA based cement applications.
Drillable cements are of high clinical interest, as the quality of screw and plate osteosynthesis techniques can be improved by cement augmentation. A drillable, dual setting composite from HA and a poly(2-hydroxyethyl methacrylate) hydrogel was analyzed with respect to the influence of monomer content and powder-to-liquid ratio on setting kinetics and mechanical outcome. While the conversion to HA and crystal growth were constantly confined with increased monomer amount, a minimum concentration of 50 % was required to see impressive ameliorations including a low bending modulus and high fracture energy at improved bending strength. Increasing the liquid amount enabled injection of the paste as well as drilling after 10 min of pre-setting.
While classic bone wax formulations have drawbacks such as infection, inflammation, hindered osteogenesis and a lack of biodegradability, the as-presented premixed formulation is believed to exhibit outmatching properties. It consisted of HA raw powders and a non-aqueous, but water-miscible carrier liquid from poly(ethylene glycol) (PEG). The bone wax was proved to be cohesive and malleable, it withstood blood pressure conditions and among deposition in an aqueous environment, PEG was exchanged such that porous, nanocrystalline HA was formed. Incorporation of a model antibiotic proved the suitability of the novel bone wax formulation for drug release purposes.
Prefabricated laminates from premixed carbonated apatite forming cement and poly(ε-caprolactone) fiber mats with defined pore architecture were presented as a potential approach for the treatment of 2-dimensional, curved cranial defects. They are flexible until application and were produced in a layer-by-layer approach from both components such that the polymer scaffold prevents the cement from flowing. It was demonstrated that solution electrospinning with a patterned collector for the fabrication of perforated fiber mats was suitable, as high fiber volume contents in combination with an appropriate interface enabled the successful fabrication of mechanically reinforced laminates. Mild immersion of the scaffolds under alkaline conditions additionally improved the interphase followed by an increase in bending-strength.
Since few years, magnesium phosphate cements (MPC) have attracted increasing attention for bone replacement. Compared to CPC, MPC exhibit a higher degradation potential and high early strength and they release biologically valuable Mg2+. However, common systems offer some challenges while using them in non-classic cement formulations such as the need for foreign ion supply, the potential acidity of the reaction or the fast setting kinetics. Here, it was possible to develop a chelate-setting MPC paste with a broad spectrum of potential applications.
The general mechanism of the novel setting principle was tested in a proof-of-principle manner. The cement paste consisted of farringtonite with differently concentrated phytic acid solution for chelate formation with Mg2+ from the raw powder. Adjusting the phytic acid content and adding a magnesium oxide as setting regulator to compensate its retarding effect resulted in drillable formulations. Additionally, there is a strong clinical demand for well working bone adhesives especially in a moist environment. Mostly the existing formulations are non-biodegradable. Ex vivo adhesion of the above presented MPC under wet conditions on bone demonstrated over a course of 7 d shear strengths of 0.8 MPa. Further, the hardened cement specimens showed a mass loss of 2 wt.% within 24 d in an aqueous environment and released about 0.17 mg/g of osteogenic Mg2+ per day. Together with the demonstrated cytocompatibility towards human fetal osteoblasts, this cement system showed promising characteristics in terms of degradable biocements with special application purposes.
Erstellung eines genregulatorischen Netzwerkes zur Simulation der Entstehung von Zahnhartsubstanz
(2020)
In dieser Dissertation beschreibt der Autor die Erstellung eines grundlegenden bioinformatischen Modelles der menschlichen Zahnschmelzreifung. Mithilfe der KEGG Pathway-Datenbank wurde ein genregulatorisches Netzwerk (GRN) erstellt, welches maßgeblich auf den Signaltransduktionswegen Apoptose, Zellzyklus, Hedgehog-Signalweg, MAP-Kinase-Weg, mTOR-Signalweg Notch-Signalweg Signalweg, TGF-β-Signalweg und Wnt-Signalweg basiert. Im Weiteren wurde dieses Netzwerk durch zahlreiche verifizierte Wechselwirkungen erweitert und die zahnspezifischen Gene AMELX, AMELY, AMBN, ENAM und DSPP implementiert. In der anschließenden Simulation des Netzwerks mit dem Simulations-Tool Jimena konnten sechs stabile Zustände identifiziert werden. Diese wurden genauer untersucht und den Erkenntnissen eines GEO-Datensatzes gegenübergestellt. Langfristiges Ziel ist es, durch konsequente Optimierung des bioinformatischen Netzwerks Rückschlüsse auf die Odontogenese des Menschen zu ziehen.
Die Implantation eines Medizinprodukts in den menschlichen Körper ruft eine Immunreaktion hervor, die zur fibrösen Einkapselung führen kann. Makrophagen in direktem Kontakt mit der Oberfläche des Implantats erfassen sensorisch den Fremdkörper und übersetzten das Signal in die Freisetzung zahlreicher löslicher Mediatoren. Das generierte Entzündungsmilieu moduliert die Heilungsreaktion und kann zur Anreicherung von Fibroblasten sowie zur Erhöhung der Matrixsyntheserate in der Wundumgebung führen. Eine dichte fibröse Kapsel um ein Medizinprodukt beeinträchtigt den Ersatz von Körperstrukturen, das Unterstützen physiologischer Körperfunktionen sowie die Effizienz einer medizinischen Therapie. Zur Identifizierung potenzieller Biomaterialkandidaten mit optimalen Eigenschaften ist jedoch eine evidenzbasierte Entscheidungsfindung notwendig und diese wiederum muss durch geeignete Testmethoden unterstützt werden.
Zur Erfassung lokaler Effekte nach Implantation eines Biomaterials begründet die Komplexi-tät der ablaufenden Fremdkörperreaktion die Anwendung von Tiermodellen als Goldstandard. Die Eingliederung von in vitro Modellsystemen in standardisierte Testverfahren scheitert oft an der Verfügbarkeit validierter, verlässlicher und reproduzierbarer Methoden. Demnach ist kein standardisiertes in vitro Testverfahren beschrieben, das die komplexen dreidimensionalen Gewebsstrukturen während einer Fremdkörperreaktion abbildet und sich zur Testung über längere Kontaktphasen zwischen Blutkomponenten und Biomaterialien eignet. Jedoch können in vitro Testungen kosten- und zeiteffizienter sein und durch die Anwendung humaner Zellen eine höhere Übertragbarkeit auf den Menschen aufweisen. Zusätzlich adressiert die Präferenz zu in vitro Testmethoden den Aspekt „Reduzierung“ der 3R-Prinzipien „Replacement, Reduction, Refinement“ (Ersatz, Reduzierung, Verbesserung) von Russel und Burch (1959) zu einer bewussten und begründeten Anwendung von Tiermodellen in der Wissenschaft. Ziel von diesem Forschungsvorhaben war die Entwicklung von humanen in vitro Modellsystemen, die den Kontakt zu Blutkomponenten sowie die Reaktion des umliegenden Bindegewebes bei lokaler Implantation eines Biomaterials abbilden. Referenzmaterialien, deren Gewebsantwort nach Implantation in Tiere oder den Menschen bekannt ist, dienten als Validierungskriterium für die entwickelten Modellsysteme. Die Anreicherung von Zellen sowie die Bildung extrazellulärer Matrix in der Wundumgebung stellen wichtige Teilprozesse während einer Fremdkörperreaktion dar. Für beide Teilprozesse konnte in einem indirekten zellbasierten Modellsystem der Einfluss einer zellvermittelten Konditionierung wie die Freisetzung von löslichen Mediatoren durch materialadhärente Makrophagen auf die gerichtete Wanderung von Fibroblasten sowie den Umbau eines dreidimensionalen Bindegewebsmodells aufgezeigt werden.
Des Weiteren ließ sich das Freisetzungsprofil von Zytokinen durch materialständige Makrophagen unter verschiedenen Testbedingungen wie der Kontamination mit LPS, der Oberflächenbehandlung mit humanem Blutplasma und der Gegenwart von IL-4 bestimmen. Die anschließende vergleichende statistische Modellierung der generierten komplexen multifaktoriellen Datenmatrix ermöglichte die Übersetzung in eine Biomaterialbewertung. Dieses entwickelte Testverfahren eignete sich einerseits zur Validierung von in vitro Testbedingungen sowie andererseits zur Bewertung von Biomaterialien. Darüber hinaus konnte in einem dreidimensionalen Fremdkörpermodell die komplexe dreidimensionale Struktur der extrazellulären Matrix in einer Wunde durch die Kombination unterschiedlicher Zell- und Matrixkomponenten biomimetisch nachgebaut werden. Diese neuartigen dreidimensionalen Fremdkörpermodelle ermöglichten die Testung von Biomaterialien über längere Testphasen und können in anschließenden Studien angewandt werden, um dynamische Prozesse zu untersuchen. Zusammenfassend konnten in dieser Arbeit drei unterschiedliche Teststrategien entwickelt werden, die (I) die Bewertung von Teilprozessen ermöglichen, (II) die Identifizierung verlässlicher Testbedingungen unterstützen und (III) biomimetisch ein Wundgewebe abbilden. Wesentlich ist, dass biomimetisch ein dreidimensionales Gewebemodell entwickelt werden konnte, das eine verlässliche Unterscheidungskapazität zwischen Biomaterialien aufweist.
Herzschrittmachersysteme sind eine weitverbreitete Möglichkeit Herz-Kreislauf-Erkrankungen zu behandeln. Wegen der natürlichen Reaktion des Immunsystems auf Fremdkörper, erfolgt aber eine fortschreitende Verkapselung der Herzschrittmacherelektrode. Die Folge ist eine ansteigende Verminderung der Stimulationseffizienz durch Erhöhung der Anregungsschwelle. Die Integration der Elektrode in das Gewebe ist dabei mangelhaft und wird bestimmt durch Implantateigenschaften wie Größe, Flexibilität und Dimensionalität. Um die Integration zu verbessern, stellen dreidimensionale (3D) bzw. gewebeartige Elektroden eine Alternative zu den derzeit verwendeten planaren Metallelektroden dar. Zur Entwicklung einer leitfähigen, 3D und faserförmigen Elektrode wurden in dieser Arbeit Kohlenstoff-Nanofaser-Scaffolds über Elektrospinnen hergestellt. Durch die Modifikation des Fasergerüstes mit Natriumchlorid (NaCl) während der Scaffoldherstellung, konnte das Fasernetzwerk aufgelockert und Poren generiert werden. Die Kohlenstofffaser-Elektroden zeigten einen effizienten Energieübertrag, welcher vergleichbar mit heutigen Titannitrid (TiN) -Elektroden ist. Die Auflockerung des Fasergewebes hatte eine verbesserte Flexibilität des Faserscaffolds zu Folge. Neben der Flexibilität, konnte auch die Infiltration von Zellen in das poröse Faserscaffold erheblich verbessert werden. Dabei konnten Fibroblasten durch das gesamte Scaffold migrieren. Die Kompatibilität mit kardialen Zellen, die Grundvoraussetzung von Herzschrittmacherelektroden, wurde in vitro nachgewiesen. Durch die Kombination aus dem 3D-Elektrodengerüst mit einer Co-Kultur aus humanen Kardiomyozyten, mesenchymalen Stammzellen und Fibroblasten, erfolgte eine Einbettung der Elektrode in funktionelles kardiales Gewebe. Dadurch konnte ein lebender Gewebe-Elektroden-Hybrid generiert werden, welcher möglicherweise die Elektrode vor Immunzellen in vivo abschirmen kann. Eine Zusammenführung der hybriden Elektrode mit einen Tissue-Engineerten humanen kardialen Patch in vitro, führte zu Bildung einer nahtlosen Elektronik-Gewebe-Schnittstelle. Die fusionierte Einheit wurde abschließend auf ihre mechanische Belastbarkeit getestet und konnte über einen Elektroden-Anschluss elektrisch stimuliert werden.
Es wurde die Expression der osteogenen Markerproteine Alkalische Phosphatase, Bone Sialoprotein, Kollagen Typ I und Osteopontin von humanen mesenchymalen Stromazellen nach Kultur auf elektrogesponnenen Scaffolds analysiert. Die Scaffolds wurden mittels der Melt electrospinning writing Methode erstellt und unterschieden sich in ihrer Maschenweite. Anhand weiterer Kontrollversuche auf Monolayern wurde ein möglicher Einfluss der Geometrie auf die Proteinexpression untersucht.
Mesenchymal stem/stromal cells (MSCs) are a rare subpopulation of cells first identified in bone marrow with the potential to proliferate in plastic-adherent colonies and to generate de novo bone marrow stroma and its environment upon serial transplantation to heterotopic anatomical sites. Given their multipotency and self renewal competence, MSCs are prime prospective candidates for most modern musculoskeletal-tissue engineering and regenerative medicine approaches. Still, their envisioned therapeutic use is being questioned with concerns regarding their definition, characterization and integrative functions in vivo. It is well established that microenvironmental cues such as the extracellular matrix (ECM)-chemistry, the mechanical environment and local cellular and/or paracrine interactions critically control MSCs behavior. Yet, most of the scientific knowledge regarding the biology and therapeutic effect of MSCs originates from mechanistic in vitro studies where microenvironmental cues are hardly addressed. Therefore, manifestable changes in cell proliferation behavior and multilineage differentiation potential might be triggered that eventually compromise the translation of results to clinics. This thesis aims to address the complexity of MSCs interactions within the skeletal niche microenvironment in order to provide alternative methods to bypass the current MSCs in vitro culture limitations. Firstly, the influence of ECM-chemistry on MSCs behavior in vitro was explored by means of decellularized human bone models here established. Basal or osteogenic tailored cell-derived decellularized 2D matrices (dECM), proved to be suitable culture substrates for MSCs expansion by providing close-to-native cell-ECM interactions. Moreover, quantified morphological shape changes suggested a material osteo supportive potential, further functionally validated by observable spontaneous mineralization of MSCs. Aiming to identify novel intrinsic ECM regulatory features specific to the skeletal niche, 3D decellularized human trabecular bone scaffolds (dBone) were additionally developed and comprehensively characterized. Remarkably, the MSCs cultured on dBone scaffolds exhibit upregulation of genes associated with stemness as well as niche-related protein expression advocating for the conservation of the naïve MSCs phenotype. vi On the other hand, the effect of biomimetic mineralization on MSCs osteogenic lineage differentiation potential was further addressed by hydroxyapatite functionalization of type-I collagen in presence of magnesium. Mineralized scaffolds exhibited higher cell viability and a clear trend of osteogenic genes upregulation comparing with non-mineralized scaffolds. Lastly, in order to mimic the complexity of the native MSCs environment, a dynamic culture system was applied to the 3D decellularized bone constructs, previously studied in single static conditions. Mechanical stimuli generated by (1) continuous perfusion of cell culture medium at 1.7 mL/min and (2) compressive stress from 10% uniaxial load at 1 Hz, resulted in an improved cell repopulation within the scaffold and boosting of de novo ECM production. The stress-induced gene expression pattern suggested early MSCs commitment towards the osteogenic lineage mediated by integrin matrix adhesion, therefore further corroborating the recapitulation of a reliable in vitro bone niche model in dBone scaffolds. To conclude, the here developed in vitro models provide a progressive increased biomimicking complexity through which significant insights regarding MSC interactions with microenvironmental features in the skeletal niche can be obtained, thus surely paving the way for a better understanding of the role of MSCs in bone homeostasis and regeneration.
This thesis aimed to evaluate the possibility to use nanoparticles as antifungal drug carriers as well as their potential application in screening and diagnostics of invasive aspergillosis. The interaction of nanogels, superparamagnetic iron oxide nanoparticles (SPIOs) and gold nanoparticles (GNP) with fungal-specific polysaccharides, cells and biofilms was investigated.
Firstly, it was evaluated how the charge of nanogels influence their interaction with fungal cells. Linear poly(glycidol)s (pG) and poly(2-methyl-2-oxazoline) (pMOx) polymers were synthesized and further functionalized with thiol groups for preparation of redox responsive nanogels. Results showed that negatively charged nanogels were internalized by the fungi to a much greater extent than positively charged ones.
Furthermore, it was investigated how amphiphilicity of polymers used for preparation of nanogels influences nanogel-fungi interaction. It was concluded that nanogels prepared from polymers with degree of functionalization of 10% had the strongest interaction, regardless the length of the alkyl chain. Moreover, amphotericin B-loaded nanogels had a higher antifungal effect and lower toxicity towards mammalian cells than the free drug. In addition, inverse nanoprecipitation of thiol functionalized pGs was shown to be successful for preparation of nanogels with narrow size distribution.
It was also demonstrated that crosslinking of the polymeric coating in hydrogel-like network with thiol functionalized pGs improved the SPIOs imaging performance.
Finally, it was investigated whether GNPs could be used as model particles for the assessment of targeting to fungi. Fc dectin-1 was conjugated covalently to GNPs decorated with pGs, and binding affinity towards β-glucans was tested by surface plasmon resonance.
In summary, this thesis demonstrated evidence for the potential of pG nanogels and pG coated nanoparticles for antifungal therapy and diagnostics of fungal infections caused by A. fumigatus.