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Institute
- Abteilung für Funktionswerkstoffe der Medizin und der Zahnheilkunde (152) (remove)
Sonstige beteiligte Institutionen
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.
Magnesiumphosphatschäume nehmen auf Grund ihrer guten Resorbierbarkeit, unter physiologischen Bedingungen, einen immer größeren Stellenwert als Knochenersatzmaterial ein. Ein weiterer Vorteil ist der neutrale pH-Wert den das entstehende Material besitzt. Magnesiumphosphatschäume besitzen eine hochporöse offenporige Struktur um zum einen den Knochen nachzuahmen und zum anderen die Steuerung und Bildung von Knochengewebe zu ermöglichen. In der vorliegenden Arbeit wurden die mechanischen Eigenschaften als auch die Zytokompatibilität der hergestellten Schäume untersucht.
Es wurden unterschiedliche Herstellungsverfahren genutzt um Magnesiumphosphatschäume zu erhalten. Zum einen das Replika- Verfahren, die dabei entstandenen Farringtonit Schäume (Mg3(PO4)2, Farringtonit) wurden zu Struvit ((NH4)Mg(PO4)•6H2O) umgewandelt bzw. mit PLGA infiltriert und auf ihre mechanische Eigenschaften hin untersucht. Zum anderen wurde ein proteinbasierter Schaumbildner verwendet. Die Zytokompatibilitätsprüfung wurde mit der Osteosarkomzelllinie MG-63 durchgeführt. Es erfolgte die Untersuchung der Zellproliferation und der Zellaktivität (WST). Zudem wurden Proben mittels Licht- und Elektronenmikroskopie analysiert. Die Feststellung der Proteinexpression erfolgte nach gelelektrophoretischer Auftrennung mittels Western Blot und PCR Analyse.
Thermoplastic polymers have a history of decades of safe and effective use in the clinic as implantable medical devices. In recent years additive manufacturing (AM) saw increased clinical interest for the fabrication of customizable and implantable medical devices and training models using the patients’ own radiological data. However, approval from the various regulatory bodies remains a significant hurdle. A possible solution is to fabricate the AM scaffolds using materials and techniques with a clinical safety record, e.g. melt processing of polymers. Melt Electrowriting (MEW) is a novel, high resolution AM technique which uses thermoplastic polymers. MEW produces scaffolds with microscale fibers and precise fiber placement, allowing the control of the scaffold microarchitecture. Additionally, MEW can process medical-grade thermoplastic polymers, without the use of solvents paving the way for the production of medical devices for clinical applications. This pathway is investigated in this thesis, where the layout is designed to resemble the journey of a medical device produced via MEW from conception to early in vivo experiments. To do so, first, a brief history of the development of medical implants and the regenerative capability of the human body is given in Chapter 1. In Chapter 2, a review of the use of thermoplastic polymers in medicine, with a focus on poly(ε-caprolactone) (PCL), is illustrated, as this is the polymer used in the rest of the thesis. This review is followed by a comparison of the state of the art, regarding in vivo and clinical experiments, of three polymer melt AM technologies: melt-extrusion, selective laser sintering and MEW. The first two techniques already saw successful translation to the bedside, producing patient-specific, regulatory-approved AM implants. To follow in the footsteps of these two technologies, the MEW device parameters need to be optimized. The MEW process parameters and their interplay are further discussed in Chapter 3 focusing on the importance of a steady mass flow rate of the polymer during printing. MEW reaches a balance between polymer flow, the stabilizing electric field and moving collector to produce reproducible, high-resolution scaffolds. An imbalance creates phenomena like fiber pulsing or arcing which result in defective scaffolds and potential printer damage. Chapter 4 shows the use of X-ray microtomography (µCT) as a non-destructive method to characterize the pore-related features: total porosity and the pore size distribution. MEW scaffolds are three-dimensional (3D) constructs but have long been treated in the literature as two-dimensional (2D) ones and characterized mainly by microscopy, including stereo- and scanning electron microscopy, where pore size was simply reported as the distance between the fibers in a single layer. These methods, together with the trend of producing scaffolds with symmetrical pores in the 0/90° and 0/60/120° laydown patterns, disregarded the lateral connections between pores and the potential of MEW to be used for more complex 3D structures, mimicking the extracellular matrix. Here we characterized scaffolds in the aforementioned symmetrical laydown patterns, along with the more complex 0/45/90/135° and 0/30/60/90/120/150° ones. A 2D pore size estimation was done first using stereomicroscopy, followed by and compared to µCT scanning. The scaffolds with symmetrical laydown patterns resulted in the predominance of one pore size, while those with more complex patterns had a broader distribution, which could be better shown by µCT scans. Moreover, in the symmetrical scaffolds, the size of 3D pores was not able to reach the value of the fiber spacing due to a flattening effect of the scaffold, where the thickness of the scaffold was less than the fiber spacing, further restricting the pore size distribution in such scaffolds. This method could be used for quality assurance of fabricated scaffolds prior to use in in vitro or in vivo experiments and would be important for a clinical translation. Chapter 5 illustrates a proof of principle subcutaneous implantation in vivo experiment. MEW scaffolds were already featured in small animal in vivo experiments, but to date, no analysis of the foreign body reaction (FBR) to such implants was performed. FBR is an immune reaction to implanted foreign materials, including medical devices, aimed at protecting the host from potential adverse effects and can interfere with the function of some medical implants. Medical-grade PCL was used to melt electrowrite scaffolds with 50 and 60 µm fiber spacing for the 0/90° and 0/60/120° laydown patterns, respectively. These implants were implanted subcutaneously in immunocompetent, outbred mice, with appropriate controls, and explanted after 2, 4, 7 and 14 days. A thorough characterization of the scaffolds before implantation was done, followed by a full histopathological analysis of the FBR to the implants after excision. The scaffolds, irrespective of their pore geometry, induced an extensive FBR in the form of accumulation of foreign body giant cells around the fiber walls, in a manner that almost occluded available pore spaces with little to no neovascularization. This reaction was not induced by the material itself, as the same reaction failed to develop in the PCL solid film controls. A discussion of the results was given with special regard to the literature available on flat surgical meshes, as well as other hydrogel-based porous scaffolds with similar pore sizes. Finally, a general summary of the thesis in Chapter 6 recapitulates the most important points with a focus on future directions for MEW.
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.
Was vor einigen Jahren undenkbar erschien, könnte zukünftig möglich sein: Krankes Gewebe mit Gesundem ersetzen, das in vitro mit modernsten Biofabrikationstechniken hergestellt wird. Dabei werden bisherige Grenzen überschritten: Während lichtbasierte Biodruckverfahren wie die Zwei-Photonen-Polymerisation Auflösungen bis in den Nanometerbereich erzielen, ermöglicht der Volumetrische Biodruck (VB) den Druck zentimetergroßer Konstrukte in wenigen Sekunden. Diese Geschwindigkeiten erweisen sich unter Biodruckverfahren als konkurrenzlos und werden erreicht, da das Bioharz nicht konsekutiv, sondern zugleich vernetzt wird. Einschränkend gilt bislang nur der Mangel an geeigneten Bioharzen für den VB. Daher beschäftigt sich vorliegende Arbeit mit der Charakterisierung und Modifikation eines dafür geeigneten Bioharzes: Gelatine-Methacrylat (GelMA). Dank seiner Zusammensetzung ähnelt das etablierte Hydrogelsystem der Extratrazellularmatrix: Der Gelatine-Anteil ermöglicht Biokompatibilität und Bioaktivität durch zelladhäsive sowie degradierbare Aminosäure-Sequenzen. Zugleich können durch photovernetzbare Methacryloyl-Substituenten Konstrukte mit einer Formstabilität bei 37 °C erzeugt werden.
Zunächst wurde das Bioharz zellbiologisch charakterisiert, indem mit der embryonalen Mausfibroblasten-Zelllinie NIH-3T3 beladene GelMA-Zylinder gegossen, photopolymerisiert und kultiviert wurden. Im Verlauf einer Woche wurde die Zytokompatibilität der Gele anhand der Proliferationsfähigkeit (PicoGreen-Assay), des Metabolismus (CCK-8-Assay) und der Vitalität (Live/Dead-Assay) der Zellen beurteilt. Dabei wurden Polymerkonzentrationen von 6 – 8 % sowie GelMA-Harze zweier verschiedener Molekulargewichte verglichen. Alle hergestellten Gele erwiesen sich als zytokompatibel, 6 % ige Gele ließen im Inneren jedoch zusätzlich eine beginnende Zellspreizung zu und ein niedriges GelMA-Molekulargewicht verstärkte die gemessene Proliferation. Die sich anschließende mechanische und physikalische Charakterisierung belegte, dass höher konzentrierte Gele einen größeren E-Modul aufwiesen und damit steifer waren. Eine Modifikation der Gele mit Fibronektin beeinflusste die Zellverträglichkeit weder positiv noch negativ und die Zugabe von Kollagen war wegen Entmischungseffekten nicht bewertbar. Es liegt die Vermutung nah, dass eine weitere Reduktion der Polymerkonzentration und damit Verringerung der Gelsteifigkeit der Schlüssel für mehr Zellspreizung und -wachstum ist. Da jedoch die Druckbarkeit des Bioharzes die weitere Senkung des GelMA-Gehalts limitiert, sollten zunächst Methoden entwickelt werden, welche die Netzwerkdichte des GelMAs anderweitig herabsetzen.
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.
Bei der Implantatversorgung von Patienten mit Osteoporose besteht weiterhin eine hohe Komplikationsrate vor allem durch aseptische Prothesenlockerungen. Eine vielversprechende Möglichkeit diese zu minimieren stellt eine Funktionalisierung der Implantate mit Strontium dar.
Ziel der vorliegenden Arbeit war es dabei die Wirkung lokal verfügbaren Strontiums auf osteoklastäre und osteoblastäre Zellen zu untersuchen.
Mittels elektrochemischer Abscheidung erfolgte die Beschichtung von Titanproben mit strontiumdotiertem Struvit, wobei sieben verschiedene Dotierkonzentrationen zwischen 6 µg und 487 µg Strontium pro Probe hergestellt wurden. Die Untersuchungen an osteoklastären RAW 264.7 Zellen erfolgten mittels Bestimmung von Zellzahl und -aktivität, verschiedener mikroskopischer Methoden sowie auf genetischer Ebene. Osteoblastäre MG63-Zellen wurden orientierend anhand von Zellzahl und Zellaktivität untersucht.
Zellbiologisch konnte ein hemmender Einfluss von Strontium auf Differenzierung sowie Proliferation und Aktivität osteoklastärer Zellen gezeigt werden. Die Dotierkonzentration mit den günstigsten Eigenschaften war unter vorliegenden Versuchsbedingungen 487 µg Strontium pro Probe, da sich hierbei zudem eine erhaltene ostoblastäre Proliferation und Aktivität zeigte.
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.
Background: Glioblastoma multiforme (GBM) and metastatic triple-negative breast cancer (TNBC) with PTEN mutations often lead to brain dissemination with poor patient outcome, thus new therapeutic targets are needed. To understand signaling, controlling the dynamics and mechanics of brain tumor cell migration, we implemented GBM and TNBC cell lines and designed 3D aligned microfibers and scaffolds mimicking brain structures. Methods: 3D microfibers and scaffolds were printed using melt electrowriting. GBM and TNBC cell lines with opposing PTEN genotypes were analyzed with RHO-ROCK-PTEN inhibitors and PTEN rescue using live-cell imaging. RNA-sequencing and qPCR of tumor cells in 3D with microfibers were performed, while scanning electron microscopy and confocal microscopy addressed cell morphology. Results: In contrast to the PTEN wildtype, GBM and TNBC cells with PTEN loss of function yielded enhanced durotaxis, topotaxis, adhesion, amoeboid migration on 3D microfibers and significant high RHOB expression. Functional studies concerning RHOB-ROCK-PTEN signaling confirmed the essential role for the above cellular processes. Conclusions: This study demonstrates a significant role of the PTEN genotype and RHOB expression for durotaxis, adhesion and migration dependent on 3D. GBM and TNBC cells with PTEN loss of function have an affinity for stiff brain structures promoting metastasis. 3D microfibers represent an important tool to model brain metastasizing tumor cells, where RHO-inhibitors could play an essential role for improved therapy.
Es erfolgte eine elektrochemische Abscheidung von Bruschitschichten auf Titan in Gegenwart von Kristallisationsinhibitoren. Dabei wurden die Kristallisationsinhibitoren Zitronensäure, treta-Natriumdiphosphat-Decahydrat und Phytinsäure verwendet und die entstandenen Schichten mit denen ohne Inhibitorzugabe verglichen. Um das Ausmaß der Inhibierung zu verifizieren, wurde die Masse aller Schichten gemessen, welche für die Inhibition mit Zitronensäure und Phytinsäure abnahm und für Natriumdiphosphat zunahm. Die kristallographische Zusammensetzung der mit und ohne Inhibierung abgeschiedenen Schichten wurde mit Hilfe der Röntgendiffraktometrie bestimmt und zeigte, dass sich reine Bruschitschichten mit unterschiedlichem amorphem Anteil abschieden. Die daraus entstandenen Werte lieferten zugleich die Informationen über die einzelnen Kristallitgrößen innerhalb der Schichten. Über den Einfluss der Inhibitoren auf die Schichtmorphologie gaben rasterelektronenmikroskopische Aufnahmen weiteren Aufschluss. Die Inhibition verursachte teils mit Rissen durchzogene Schichten, deren Kristallformationen sich von Standardelektrolyt unterschieden. Ausgewählte Proben wurden unter verschiedenen Bedingungen desinfiziert bzw. sterilisiert und nachfolgend erneut gewogen und mittels Röntgendiffraktogrammetrie und Rasterelektronenmikroskopie analysiert. Nach der Desinfektion entstanden reine Bruschitschichten, die an Masse verloren aber trotzdem die typischen Kristallformationen zeigten. Die Sterilisation führte zur Umwandlung von Bruschit in Monetit und Hydroxylapatit. Des Weiteren wurde die biologische Reaktion der Schichten auf humane fötale Osteoblasten-Zelllinien zur Überprüfung der Zellverträglichkeit ermittelt. Die entstandenen Ergebnisse waren nicht verwertbar und enthielt sehr hohe Standardabweichungen.
Objective
As native cartilage consists of different phenotypical zones, this study aims to fabricate different types of neocartilage constructs from collagen hydrogels and human mesenchymal stromal cells (MSCs) genetically modified to express different chondrogenic factors.
Design
Human MSCs derived from bone-marrow of osteoarthritis (OA) hips were genetically modified using adenoviral vectors encoding sex-determining region Y-type high-mobility-group-box (SOX)9,transforming growth factor beta (TGFB) 1or bone morphogenetic protein (BMP) 2cDNA, placed in type I collagen hydrogels and maintained in serum-free chondrogenic media for three weeks. Control constructs contained unmodified MSCs or MSCs expressing GFP. The respective constructs were analyzed histologically, immunohistochemically, biochemically, and by qRT-PCR for chondrogenesis and hypertrophy.
Results
Chondrogenesis in MSCs was consistently and strongly induced in collagen I hydrogels by the transgenesSOX9,TGFB1andBMP2as evidenced by positive staining for proteoglycans, chondroitin-4-sulfate (CS4) and collagen (COL) type II, increased levels of glycosaminoglycan (GAG) synthesis, and expression of mRNAs associated with chondrogenesis. The control groups were entirely non-chondrogenic. The levels of hypertrophy, as judged by expression of alkaline phosphatase (ALP) and COL X on both the protein and mRNA levels revealed different stages of hypertrophy within the chondrogenic groups (BMP2>TGFB1>SOX9).
Conclusions
Different types of neocartilage with varying levels of hypertrophy could be generated from human MSCs in collagen hydrogels by transfer of genes encoding the chondrogenic factorsSOX9,TGFB1andBMP2. This technology may be harnessed for regeneration of specific zones of native cartilage upon damage.
Background
Mesenchymal stem cell (MSC) based-treatments of cartilage injury are promising but impaired by high levels of hypertrophy after chondrogenic induction with several bone morphogenetic protein superfamily members (BMPs). As an alternative, this study investigates the chondrogenic induction of MSCs via adenoviral gene-delivery of the transcription factor SOX9 alone or in combination with other inducers, and comparatively explores the levels of hypertrophy and end stage differentiation in a pellet culture system in vitro.
Methods
First generation adenoviral vectors encoding SOX9, TGFB1 or IGF1 were used alone or in combination to transduce human bone marrow-derived MSCs at 5 x 10\(^2\) infectious particles/cell. Thereafter cells were placed in aggregates and maintained for three weeks in chondrogenic medium. Transgene expression was determined at the protein level (ELISA/Western blot), and aggregates were analysed histologically, immunohistochemically, biochemically and by RT-PCR for chondrogenesis and hypertrophy.
Results
SOX9 cDNA was superior to that encoding TGFB1, the typical gold standard, as an inducer of chondrogenesis in primary MSCs as evidenced by improved lacuna formation, proteoglycan and collagen type II staining, increased levels of GAG synthesis, and expression of mRNAs associated with chondrogenesis. Moreover, SOX9 modified aggregates showed a markedly lower tendency to progress towards hypertrophy, as judged by expression of the hypertrophy markers alkaline phosphatase, and collagen type X at the mRNA and protein levels.
Conclusion
Adenoviral SOX9 gene transfer induces chondrogenic differentiation of human primary MSCs in pellet culture more effectively than TGFB1 gene transfer with lower levels of chondrocyte hypertrophy after 3 weeks of in vitro culture. Such technology might enable the formation of more stable hyaline cartilage repair tissues in vivo.
In this study, we evaluate hydrogels based on oxidized hyaluronic acid, cross-linked with adipic acid dihydrazide, for their suitability as bioinks for 3D bioprinting. Aldehyde containing hyaluronic acid (AHA) is synthesized and cross-linked via Schiff Base chemistry with bifunctional adipic acid dihydrazide (ADH) to form a mechanically stable hydrogel with good printability. Mechanical and rheological properties of the printed and casted hydrogels are tunable depending on the concentrations of AHA and ADH cross-linkers.
In recent decades, hybrid characterization systems have become pillars in the study of cellular biomechanics. Especially, Atomic Force Microscopy (AFM) is combined with a variety of optical microscopy techniques to discover new aspects of cell adhesion. AFM, however, is limited to the early-stage of cell adhesion, so that the forces of mature cell contacts cannot be addressed. Even though the invention of Fluidic Force Microscopy (FluidFM) overcomes these limitations by combining the precise force-control of AFM with microfluidics, the correlative investigation of detachment forces arising from spread mammalian cells has been barely achieved. Here, a novel multifunctional device integrating Fluorescence Microscopy (FL) into FluidFM technology (FL-FluidFM) is introduced, enabling real-time optical tracking of entire cell detachment processes in parallel to the undisturbed acquisition of force-distance curves. This setup, thus, allows for entailing two pieces of information at once. As proof-of-principle experiment, this method is applied to fluorescently labeled rat embryonic fibroblast (REF52) cells, demonstrating a precise matching between identified force-jumps and visualized cellular unbinding steps. This study, thus, presents a novel characterization tool for the correlated evaluation of mature cell adhesion, which has great relevance, for instance, in the development of biomaterials or the fight against diseases such as cancer.
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.
Background
There is a need to establish more cell lines from breast tumors in contrast to immortalized cell lines from metastatic effusions in order to represent the primary tumor and not principally metastatic biology of breast cancer. This investigation describes the simultaneous isolation, characterization, growth and function of primary mammary epithelial cells (MEC), mesenchymal cells (MES) and adipose derived stem cells (ADSC) from four normal breasts, one inflammatory and one triple-negative ductal breast tumors.
Methods
A total of 17 cell lines were established and gene expression was analyzed for MEC and MES (n = 42) and ADSC (n = 48) and MUC1, pan-KRT, CD90 and GATA-3 by immunofluorescence. DNA fingerprinting to track cell line identity was performed between original primary tissues and isolates. Functional studies included ADSC differentiation, tumor MES and MEC invasion co-cultured with ADSC-conditioned media (CM) and MES adhesion and growth on 3D-printed scaffolds.
Results
Comparative analysis showed higher gene expression of EPCAM, CD49f, CDH1 and KRTs for normal MEC lines; MES lines e.g. Vimentin, CD10, ACTA2 and MMP9; and ADSC lines e.g. CD105, CD90, CDH2 and CDH11. Compared to the mean of all four normal breast cell lines, both breast tumor cell lines demonstrated significantly lower ADSC marker gene expression, but higher expression of mesenchymal and invasion gene markers like SNAI1 and MMP2. When compared with four normal ADSC differentiated lineages, both tumor ADSC showed impaired osteogenic and chondrogenic but enhanced adipogenic differentiation and endothelial-like structures, possibly due to high PDGFRB and CD34. Addressing a functional role for overproduction of adipocytes, we initiated 3D-invasion studies including different cell types from the same patient. CM from ADSC differentiating into adipocytes induced tumor MEC 3D-invasion via EMT and amoeboid phenotypes. Normal MES breast cells adhered and proliferated on 3D-printed scaffolds containing 20 fibers, but not on 2.5D-printed scaffolds with single fiber layers, important for tissue engineering.
Conclusion
Expression analyses confirmed successful simultaneous cell isolations of three different phenotypes from normal and tumor primary breast tissues. Our cell culture studies support that breast-tumor environment differentially regulates tumor ADSC plasticity as well as cell invasion and demonstrates applications for regenerative medicine.
The human body has very good self-healing capabilities for numerous different injuries to a variety of different tissues. This includes the main human mechanical framework, the skeleton. The skeleton is limited in its healing without additional aid by medicine mostly by the defect size. When the defect reaches a size above 2.5 cm the regeneration of the defect ends up faulty. Here is where implants, defect fillers and other support approaches developed in medicine can help the body to heal the big defect still successfully.
Usually sturdy implants (auto-/allo-/xenogenic) are implanted in the defect to bridge the distance, but for auto- and allogenic implants a suitable donor site must be found and for all sources the implant needs to be shaped into the defect specific site to ensure a perfect fit, the best support and good healing. This shaping is very time consuming and prone to error, already in the planning phase. The use of a material that is moldable and sets in the desired shape shortly after applying negates these disadvantages. Cementitious materials offer exactly this property by being in a pasty stage after the powder and liquid components have been mixed and the subsequently hardening to a solid implant. These properties also enable the extrusion, and therefore may also enable the injection, of the cement via a syringe in a minimal invasive approach.
To enable a good injection of the cement modifications are necessary. This work aimed to modify commonly used calcium phosphate-based cement systems based on α-TCP (apatitic) and β-TCP (brushitic). These have been modified with sodium phytate and phytic acid, respectively. Additionally, the α-TCP system has been modified with sodium pyrophosphate, in a second study, to create a storable aqueous paste that can be activated once needed with a highly concentrated sodium orthophosphate solution.
The powder phase of the α-TCP cement system consisted of nine parts α-TCP and one part CDHA. These were prepared to have different particle sizes and therefore enable a better powder flowability through the bimodal size distribution. α-TCP had a main particle size of 20 μm and CDHA of 2.6 μm. The modification with sodium phytate led to an adsorption of phytate ions on the surface of the α-TCP particles, where they started to form complexes with the Ca2+ ions in the solution. This adsorption had two effects. The first was to make the calcium ions unavailable, preventing supersaturation and ultimately the precipitation of CDHA what would lead to the cement hardening. The second was the increase of the absolute value of the surface charge, zeta potential, of the powder in the cement paste. Here a decrease from +3 mV to -40 mV could be measured. A strong value for the zeta potential leads to a higher repulsion of similarly charged particles and therefore prevents powder agglomeration and clogging on the nozzle during injection. These two modifications (bimodal particles size distribution and phytic acid) lead to a significant increase in the paste injectability. The unmodified paste was injectable for 30 % only, where all modified pastes were practically fully injectable ~90 % (the residual paste remained in the nozzle, while the syringe plunger already reached the end of the syringe).
A very similar observation could be made for the β-TCP system. This system was modified with phytic acid. The zeta potential was decreased even stronger from -10 ± 1.5 mV to -71.5 ± 12 mV. The adsorption of the phytate ions and subsequent formation of chelate complexes with the newly dissolved Ca2+ ions also showed a retarding effect in the cements setting reaction. Where the unmodified cement was not measurable in the rheometer, as the reaction was faster than the measurement setup (~1.5 min), the modified cements showed a transition through the gel point between 3-6 min. This means the pastes stayed between 2 and 4 times longer viscous than without the modification. Like with the first cement system also here the effects of the phytate addition showed its beneficial influence in the injectability measurement. The unmodified cement was not injectable at all, due to the same issue already encountered at the rheology measurements, but all modified pastes were fully injectable for at least 5 min (lowest phytate concentration) and at least 10 min (all other concentrations) after the mixing of powder and liquid.
The main goal of the last modification with sodium pyrophosphate was to create a paste that was stable in aqueous environment without setting until the activation takes place, but it should still show good injectability as this was the desired way of application after activation. Like before also the zeta potential changed after the addition of pyrophosphate. It could be lowered from -22 ± 2mV down to -61 to -68 ± 4mV (depending on the pyrophosphate concentration). The pastes were stored in airtight containers at room temperature and checked for their phase composition over 14 days. The unmodified paste showed a beginning phase conversion to hydroxyapatite between 7 and 14 days. All other pastes were still stable and unreacted. The pastes were activated with a high concentrated (30 wt%) sodium orthophosphate solution. After the activation the pastes were checked for their injectability and showed an increase from -57 ± 11% for the unmodified paste to -89 ± 3% (practically fully injectable as described earlier) for the best modified paste (PP005).
It can be concluded that the goal of enabling full injection of conventional calcium phosphate bone cement systems was reached. Additional work produced a storage stable paste that still ensures full injectability. Subsequent work already used the storable paste and modified it with hyaluronic acid to create an ink for 3D extrusion printing. The first two cement systems have also already been investigated in cell culture for their influence on osteoblasts and osteoclasts. The next steps would have to go more into the direction of translation. Figuring out what properties still need to be checked and where the modification needs adjustment to enable a clinical use of the presented systems.
A method is reported for making hollow channels within hydrogels decorated with cell‐adhesion peptides exclusively at the channel surface. Sacrificial fibers of different diameters are used to introduce channels within poly(ethylene glycol) hydrogels crosslinked with maleimide‐thiol chemistry, which are backfilled with a cysteine‐containing peptide solution which is conjugated to the lumen with good spatial efficiency. This allows for peptide patterning in only the areas of the hydrogel where they are needed when used as cell‐guides, reducing the amount of required peptide 20‐fold when compared to bulk functionalization. The power of this approach is highlighted by successfully using these patterned hydrogels without active perfusion to guide fibroblasts and olfactory ensheathing cells—the latter having unique potential in neural repair therapies.
Das Ziel der experimentellen Studie war die Erprobung der (bereits in vitro erfolgreich getesteten) Ca(OH)2-Beschichtung In vivo unter dem Aspekt, ob und inwieweit die antibakteriellen und somit auch antiinflammatorischen bzw. entzündungsmoderierenden Eigenschaften der Ca(OH)2-Beschichtung eine sinnvolle und effektive Ergänzung zu den bisher erfolgreich eingesetzten Calciumphosphat(CaP)-Beschichtungen mit bewiesenen, guten proosseointegrativen Eigenschaften bei lasttragenden Implantaten sein können.
Zusammenfassend kann festgestellt werden, dass die Ergebnisse der In vitro Untersuchung durch die In vivo Versuche in den Bereichen 0-100 KBE grundsätzlich als gestützt gelten können. Die Zuverlässigkeit der Wirkung durch Ca(OH)2 nimmt jedoch mit steigender KBE-Zahl ab, sodass weitere Testreihen sinnvoll sind.
Macrophages are key players of the innate immune system that can roughly be divided into the pro-inflammatory M1 type and the anti-inflammatory, pro-healing M2 type. While a transient initial pro-inflammatory state is helpful, a prolonged inflammation deteriorates a proper healing and subsequent regeneration. One promising strategy to drive macrophage polarization by biomaterials is precise control over biomaterial geometry. For regenerative approaches, it is of particular interest to identify geometrical parameters that direct human macrophage polarization. For this purpose, we advanced melt electrowriting (MEW) towards the fabrication of fibrous scaffolds with box-shaped pores and precise inter-fiber spacing from 100 μm down to only 40 μm. These scaffolds facilitate primary human macrophage elongation accompanied by differentiation towards the M2 type, which was most pronounced for the smallest pore size of 40 μm. These new findings can be important in helping to design new biomaterials with an enhanced positive impact on tissue regeneration.