Filtern
Volltext vorhanden
- ja (122)
Gehört zur Bibliographie
- ja (122)
Erscheinungsjahr
Dokumenttyp
Sprache
- Englisch (122) (entfernen)
Schlagworte
- melt electrowriting (13)
- 3D printing (12)
- additive manufacturing (10)
- biofabrication (9)
- hyaluronic acid (7)
- melt electrospinning writing (7)
- Hydrogel (6)
- bioprinting (6)
- 3D-Druck (5)
- bioink (5)
- polycaprolactone (5)
- tissue engineering (5)
- Polymere (4)
- Tissue Engineering (4)
- hydrogel (4)
- hydrogels (4)
- magnesium phosphate cement (4)
- phytic acid (4)
- scaffold (4)
- thiol-ene (4)
- Biofabrication (3)
- Biomaterial (3)
- Calciumphosphate (3)
- Knochenzement (3)
- biomaterials (3)
- bone (3)
- breast cancer (3)
- calcium phosphate cement (3)
- chondrogenic differentiation (3)
- electrohydrodynamics (3)
- extracellular matrix (3)
- melt electrowriting (MEW) (3)
- 3D powder printing (2)
- Calciumphosphat (2)
- Cement (2)
- Dihydrooxazole (2)
- Electrospinning (2)
- Fluorescence Microscopy (2)
- Knochenersatz (2)
- MSC (2)
- Melt Electrowriting (2)
- Melt electrowriting (2)
- Nanopartikel (2)
- Oberflächenfunktionalisierung (2)
- Polyethylenglykole (2)
- Polyoxazoline (2)
- Ringöffnungspolymerisation (2)
- Tissue engineering (2)
- Zement (2)
- angiogenesis (2)
- arteriovenous loop (2)
- baghdadite (2)
- biocompatibility (2)
- biomedical materials (2)
- bone adhesive (2)
- bone cement (2)
- calcium phosphate (2)
- cartilage (2)
- cortical neurons (2)
- dual setting (2)
- dual setting system (2)
- dual-stage crosslinking (2)
- electrohydrodynamic (2)
- in vitro (2)
- inflammation (2)
- polymer processing (2)
- polymer-peptide-conjugate (2)
- printability (2)
- rheology (2)
- shape fidelity (2)
- therapy (2)
- (AB)\(_{n}\) segmented copolymers (1)
- 2-Aminoethylphosphonic acid (1)
- 316L stainless-steel (1)
- 3D Bioprinting (1)
- 3D Printing (1)
- 3D cultures (1)
- 3D electrophysiology (1)
- 3D microfiber (1)
- 3D model systems (1)
- 3D neuronal networks (1)
- 3D scaffolds (1)
- 3D tumor model (1)
- ACPC (1)
- ALI culture (1)
- Additive Fertigung (1)
- Adenovirus (1)
- Adhäsion (1)
- Adipose Tissue (1)
- Anisotropic structures (1)
- Arteriosklerose (1)
- Artery Models (1)
- Aspergillose (1)
- Aspergillosis (1)
- Atherosclerosis (1)
- Baghdadite (1)
- Beschichtung (1)
- Biofabrication of hydrogels (1)
- Biofabrikation (1)
- Bioink (1)
- Biologischer Abbau (1)
- Blockcopolymere (1)
- Bone marrow (1)
- Bone-replacement (1)
- Breast cancer (1)
- Ca\(^{2+}\)-Imaging (1)
- Calcium Phosphate (1)
- Calcium phosphate (1)
- Calcium phosphate-based biomaterials (1)
- Calciumhydroxid (1)
- Calciumphosphatzemente (1)
- Cartilage (1)
- Cell Sheet Engineering (1)
- Cell adhesion (1)
- Ceramic polymer composite (1)
- Chelatbildner (1)
- Chemical modification of biopolymers (1)
- Chondrogenesis (1)
- Chondrogenic differentiation (1)
- Chondrozyten (1)
- Colloidal Stability (1)
- Correlative microscopy (1)
- Dectin-1 (1)
- Degradation (1)
- Dual setting system (1)
- Dual-setting (1)
- ECM (1)
- Elektrospinnen (1)
- Elektrostatisches Verspinnen (1)
- Extrazelluläre Matrix (1)
- Fettgewebe (1)
- Fibrin (1)
- Fibroblasten (1)
- FluidFM (1)
- FluidFM technology (1)
- Freezing (1)
- Functional hydrophilic polymers (1)
- Funktionalisierung <Chemie> (1)
- Funktionalisierung von elektrogesponnenen Fasern (1)
- Gefrierstrukturierung (1)
- Gelatine (1)
- Gene therapy (1)
- Gerichtete Erstarrung (1)
- Gewebe (1)
- Geweberegeneration (1)
- Glycidol (1)
- Gold-Nanoparticles (1)
- HA modifiedCNF membranes (1)
- HASH (1)
- HEMA (1)
- Hemodynamics (1)
- Hyaluronic Acid (1)
- Hyaluronsäure (1)
- Hypertrophy (1)
- Hämodynamik (1)
- IP6 (1)
- Implantat (1)
- In vitro model (1)
- Injectability (1)
- Iron Oxide Nanoparticles (1)
- Isocyanate (1)
- Janus fibers (1)
- Knochen (1)
- Knochenkleber (1)
- Knochenwachs (1)
- Knochenzemente (1)
- Knorpel (1)
- Konjugate (1)
- Korrelative Mikroskopie (1)
- MPI (1)
- MPS (1)
- MRI (1)
- Macrophage extracellular traps (1)
- Magnesiumphosphate (1)
- Makrophagen (1)
- Melt electrospinning (1)
- Mesenchymal stem cell (1)
- Modification (1)
- Modifikation von Biokeramiken (1)
- Morphology (1)
- Multifunctionalisability (1)
- NCO-sP(EO-stat-PO) (1)
- NLRP3 (1)
- Nanofaser (1)
- Nanogels (1)
- Nanoröhre (1)
- Nanotubular coatings (1)
- Nanotubuläre Beschichtungen (1)
- Normal breast (1)
- Oxygen diffusion hardening (1)
- PCL (1)
- PEG (1)
- PLGA (1)
- PTEN (1)
- PVD Beschichtung (1)
- PVD coatings (1)
- PVD-Verfahren (1)
- Poly(2-oxazoline) (1)
- Poly(glycidol)s (1)
- Polycaprolacton (1)
- Polylactid-co-Glycolid (1)
- Polymer-peptide-conjugate (1)
- Polymers (1)
- Polymilchsäure (1)
- Polyoxazolines (1)
- Primary cell lines (1)
- QCM (1)
- RAFT (1)
- RHO (1)
- ROCK (1)
- Rapid Prototyping <Fertigung> (1)
- Regeneration (1)
- Rheology (1)
- SOX9 (1)
- Sauerstoffdiffusionshärtung (1)
- Scaffold (1)
- Scaffold <Tissue Engineering> (1)
- Schiff base chemistry (1)
- Schleuderguss (1)
- Setting Control (1)
- Silica precursor (1)
- Sol-gel (1)
- Staphylococcus aureus / drug effects (1)
- Star-shaped poly(ethylene glycol) (1)
- Stem cells plasticity (1)
- Sternpolymere (1)
- Tantal (1)
- Therapeutisches System (1)
- Thermoresponsive Polymere (1)
- Thioether-Poly(glycidol) (1)
- Ti(Ag) Beschichtungen (1)
- Ti(Ag) coatings (1)
- Unidirectional Freezing (1)
- Vancomycin / administration & dosage (1)
- Vancomycin / chemistry (1)
- Vancomycin / pharmacology (1)
- Vascularization (1)
- Vaskularisation (1)
- Verbundwerkstoff (1)
- WST test (1)
- Wasserlösliche Polymere (1)
- Wirkstoff-Träger-System (1)
- Wirkstofffreisetzung (1)
- ZIP (1)
- acrylate-endcapped urethane-based polymer (AUP) (1)
- actin (1)
- acute myeloid leukaemia (1)
- adhesion dynamics (1)
- adipose tissue (1)
- adipose-derived stromal cells (1)
- agarose (1)
- alginate (1)
- alveolar‐capillary barrier (1)
- amoeboid cell migration (1)
- animal model (1)
- anisotropic porous structures (1)
- anti-bacterial agents / administration & dosage (1)
- anti-bacterial agents / chemistry (1)
- anti-bacterial agents / pharmacology (1)
- antibacterial (1)
- antibakteriell (1)
- arthritis (1)
- artificial bones (1)
- astrocytes (1)
- atomic force microscopy (1)
- axial vascularization (1)
- azlactone (1)
- balloon kyphoplasty (1)
- bending strength (1)
- bio-printability (1)
- bioceramic (1)
- bioceramics (1)
- biocompatible Materials (1)
- biodegradable polymer (1)
- biodegradable polymers (1)
- biofabricated vascular graft (1)
- bioinorganic (1)
- bioinspired interface (1)
- bioinspired membrane (1)
- biological models (1)
- biomaterial ink (1)
- biomechanical evaluation (1)
- biomechanics (1)
- biomedical engineering (1)
- biophysics (1)
- biosensor (1)
- biosensors (1)
- blood-plasma (1)
- bohrbar (1)
- bone and cartilage tissue engineering (1)
- bone critical size defect (1)
- bone graft substitutes (1)
- bone replacement material (1)
- bone wax (1)
- bone wires (1)
- brain cancer (1)
- breast cancer model (1)
- brushite cement (1)
- calcaneus (1)
- calcium phosphate cements (1)
- calcium-phosphate (1)
- calciumhydroxide (1)
- carbon fiber reinforcement (1)
- cardiovascular pharmacology (1)
- cartilage repair (1)
- cell coagulation (1)
- cell counting (1)
- cell elongation (1)
- cell guidance (1)
- cell migration (1)
- cell morphology (1)
- cell printing (1)
- cell self-assembled monolayers (1)
- cell transplantation (1)
- cell viability (1)
- cement (1)
- centrifugally casting (1)
- ceramic polymer composite (1)
- ceramics (1)
- chemical crosslinking (1)
- chemokines (1)
- chemoselective (1)
- chemosensitivity (1)
- chondrocyte (1)
- chondrocytes (1)
- chondroprogenitors (1)
- click chemistry (1)
- co-culture (1)
- coating (1)
- coatings (1)
- cochlea (1)
- collagen (1)
- collagen sponge (1)
- combination of physical vapor deposition and electrochemical etching (1)
- composite material (1)
- compressive strength (1)
- connective tissue (1)
- crosslinked coating (1)
- cryostructured scaffolds (1)
- cyclic mechanical stretch (1)
- cyclic stretch (1)
- cyclic testing (1)
- cyto-compatibility (1)
- cytotoxicity (1)
- damage tolerant cement (1)
- deafness (1)
- defects (1)
- defined humanized test system (1)
- definition (1)
- degradable bone substitutes (1)
- degradable implant (1)
- degradation (1)
- detachment force quantification (1)
- dicalcium phosphate cement (1)
- differentation (1)
- digitization (1)
- dihydrate cement (1)
- directional solidification (1)
- disease models (1)
- drillability (1)
- drillable (1)
- drillable bone cement (1)
- drop on demand (1)
- drug delivery (1)
- drug liberation (1)
- drug release (1)
- dual abbindend (1)
- durotaxis (1)
- electroactive (1)
- electroactive polymers (1)
- electron microscopy (1)
- electrospinning (1)
- electrospun fibers (1)
- eletrhydrodynamic (1)
- embedded templating (1)
- endothelial progenitor cells (1)
- expression (1)
- extrusion of hiMPC-containing bioinks alginate + collagen type I (1)
- extrusion-based 3D printing (1)
- factor-I (1)
- farringtonite (1)
- fiber (1)
- fiber reinforcement (1)
- fibers (1)
- fiber–matrix interaction (1)
- fixation (1)
- focal adhesions (1)
- force (1)
- fracture (1)
- free radical polymerization (1)
- functionalization (1)
- funktionale Präpolymere (1)
- gels (1)
- gels and hydrogels (1)
- gentamicin-loaded poly (methyl methacrylate) bone cement (1)
- granules (1)
- growth (1)
- guided bone regeneration (1)
- heart-failure (1)
- hemocompatibility (1)
- heterotypic scaffold design (1)
- human iPSC-derived mesodermal cells (hiMPCs) (1)
- human macrophages (1)
- human neutrophil elastase (HNE) (1)
- hybrid fabrication (1)
- hybrid materials (1)
- hybrid polymers (1)
- hydraulic reactivity (1)
- hydrogel formation (1)
- hydrophilic polymers (1)
- hydroxyapatite (1)
- hypoxia (1)
- imaging agents (1)
- immunomodulation (1)
- implant (1)
- implants (1)
- in vitro cell‐stretch model (1)
- in-vivo Micro-Computed Tomography (1)
- inflammatory response (1)
- inner ear (1)
- inositol hexaphosphate (1)
- intercellular adhesion (1)
- interface control (1)
- iron release (1)
- lectin (1)
- left-ventricular function (1)
- load to failure testing (1)
- lung cell model (1)
- macrophage polarization (1)
- macrophages (1)
- magnetic properties (1)
- magnetoactive materials (1)
- materials testing (1)
- mature cell-cell contacts (1)
- mechanical activation (1)
- mechanical performance (1)
- mechanical properties (1)
- mechanical reinforcement (1)
- medical device (1)
- medical-grade poly(ε-caprolactone) (1)
- medicine (1)
- melanoma (1)
- melt electrowriting (1)
- melt electrofibrillation (1)
- melt electrospinning (1)
- mesenchymal stem cells (1)
- microfibers (1)
- microfibres (1)
- microfluidics (1)
- microstructures (1)
- microvasculature (1)
- migration (1)
- minipig (1)
- modelling (1)
- morphology controls (1)
- mucin (1)
- multilayered vessel wall with intimate, media and adventitia (1)
- nanofibers (1)
- nanostructures (1)
- nanotopographical surfaces (1)
- native chemical ligation (1)
- networks (1)
- neurite growth (1)
- organ-on-a-chip (1)
- organoids (1)
- osseointegration (1)
- osteoblasts (1)
- osteochondral defect (1)
- osteoporosis (1)
- oxidation (1)
- particle study (1)
- peptide conjugation (1)
- peptide immobilization (1)
- phase conversion (1)
- photo-crosslinking (1)
- photopolymerization (1)
- physicochemical characterization (1)
- plasticizers (1)
- platelet-adhesion (1)
- platelets (1)
- poly(2-ethyl-2-oxazoline) (1)
- poly(2-oxazoline)s (1)
- poly(ethylene glycol) (1)
- poly(glycidol) (1)
- poly(lactic-co-glycolic acid) (1)
- poly(lactide-co-glycolide) (1)
- polyacrylic acid (1)
- polyethylene glycol (1)
- polyethylenglykole (1)
- polyglycidol (1)
- polylactid (1)
- polymer (1)
- polymer-analogue functionalization (1)
- polymeric matrix (1)
- polymers (1)
- polyoxazoline (1)
- polyoxazolines (1)
- porosity (1)
- postsurgical adhesion (1)
- pre-crosslinking (1)
- premixed (1)
- primary vascular smooth muscle‐like cells (vSMCs) (1)
- proliferation (1)
- prostheses and implants (1)
- protein adsorption (1)
- präfabriziert (1)
- radiopacity (1)
- rat model (1)
- recruitment (1)
- remodelling (1)
- repair (1)
- rhBMP–2 (1)
- ring opening polymerisation (1)
- ring-opening polymerization (1)
- sPEG (1)
- sacrificial printing (1)
- sanders (1)
- scaffolds (1)
- screw (1)
- self-assembled monolayers (1)
- serial block face EM (1)
- setting reaction (1)
- shear stress (1)
- shear thinning (1)
- signaling (1)
- silicones (1)
- sodium alginate (1)
- solution electrospinning (1)
- spheroids (1)
- spinning (1)
- stanfieldite (1)
- starPEG (1)
- starPEG hydrogel (1)
- stem cells (1)
- stimulation (1)
- stress (1)
- subcutaneous implanation (1)
- sugar glass printing (1)
- surface coating (1)
- surface functionalisation (1)
- surface functionalization (1)
- surfaces (1)
- sutures (1)
- synbones (1)
- synergistic reinforcement (1)
- synthetic hydrogels (1)
- systematic investigations (1)
- tension (1)
- tethering (1)
- thermoresponsive hydrogel (1)
- thrombin (1)
- thrombosis (1)
- tibial head depression fracture (1)
- tissue morphogenesis (1)
- tissue regeneration (1)
- topotaxis (1)
- total joint arthroplasty (1)
- total knee arthroplasty (1)
- traction (1)
- tranexamic acid (1)
- transforming growth factor-beta 1 (1)
- transplantation (1)
- tubular constructs (1)
- tumor heterogeneity (1)
- tunable ultra‐thin biphasic membrane (1)
- unidirectional freezing (1)
- vascular biofabrication (1)
- vascular network and hierarchical organized vessels (1)
- x-ray micro computed tomography (1)
- zinc (1)
- zonal (1)
- zonal construct (1)
- α-tricalcium phosphate (1)
Institut
- Abteilung für Funktionswerkstoffe der Medizin und der Zahnheilkunde (122) (entfernen)
Sonstige beteiligte Institutionen
Within this thesis, three main approaches for the assessment and investigation of altered hemodynamics like wall shear stress, oscillatory shear index and the arterial pulse wave velocity in atherosclerosis development and progression were conducted:
1. The establishment of a fast method for the simultaneous assessment of 3D WSS and PWV in the complete murine aortic arch via high-resolution 4D-flow MRI
2. The utilization of serial in vivo measurements in atherosclerotic mouse models using high-resolution 4D-flow MRI, which were divided into studies describing altered hemodynamics in late and early atherosclerosis
3. The development of tissue-engineered artery models for the controllable application and variation of hemodynamic and biologic parameters, divided in native artery models and biofabricated artery models, aiming for the investigation of the relationship between atherogenesis and hemodynamics
Chapter 2 describes the establishment of a method for the simultaneous measurement of 3D WSS and PWV in the murine aortic arch at, using ultra high-field MRI at 17.6T [16], based on the previously published method for fast, self-navigated wall shear stress measurements in the murine aortic arch using radial 4D-phase contrast MRI at 17.6 T [4]. This work is based on the collective work of Dr. Patrick Winter, who developed the method and the author of this thesis, Kristina Andelovic, who performed the experiments and statistical analyses. As the method described in this chapter is basis for the following in vivo studies and undividable into the sub-parts of the contributors without losing important information, this chapter was not split into the single parts to provide fundamental information about the measurement and analysis methods and therefore better understandability for the following studies. The main challenge in this chapter was to overcome the issue of the need for a high spatial resolution to determine the velocity gradients at the vascular wall for the WSS quantification and a high temporal resolution for the assessment of the PWV without prolonging the acquisition time due to the need for two separate measurements. Moreover, for a full coverage of the hemodynamics in the murine aortic arch, a 3D measurement is needed, which was achieved by utilization of retrospective navigation and radial trajectories, enabling a highly flexible reconstruction framework to either reconstruct images at lower spatial resolution and higher frame rates for the acquisition of the PWV or higher spatial resolution and lower frame rates for the acquisition of the 3D WSS in a reasonable measurement time of only 35 minutes. This enabled the in vivo assessment of all relevant hemodynamic parameters related to atherosclerosis development and progression in one experimental session. This method was validated in healthy wild type and atherosclerotic Apoe-/- mice, indicating no differences in robustness between pathological and healthy mice.
The heterogeneous distribution of plaque development and arterial stiffening in atherosclerosis [10, 12], however, points out the importance of local PWV measurements. Therefore, future studies should focus on the 3D acquisition of the local PWV in the murine aortic arch based on the presented method, in order to enable spatially resolved correlations of local arterial stiffness with other hemodynamic parameters and plaque composition.
In Chapter 3, the previously established methods were used for the investigation of changing aortic hemodynamics during ageing and atherosclerosis in healthy wild type and atherosclerotic Apoe-/- mice using the previously established methods [4, 16] based on high-resolution 4D-flow MRI. In this work, serial measurements of healthy and atherosclerotic mice were conducted to track all changes in hemodynamics in the complete aortic arch over time. Moreover, spatially resolved 2D projection maps of WSS and OSI of the complete aortic arch were generated. This important feature allowed for the pixel-wise statistical analysis of inter- and intragroup hemodynamic changes over time and most importantly – at a glance. The study revealed converse differences of local hemodynamic profiles in healthy WT and atherosclerotic Apoe−/− mice, with decreasing longWSS and increasing OSI, while showing constant PWV in healthy mice and increasing longWSS and decreasing OSI, while showing increased PWV in diseased mice. Moreover, spatially resolved correlations between WSS, PWV, plaque and vessel wall characteristics were enabled, giving detailed insights into coherences between hemodynamics and plaque composition. Here, the circWSS was identified as a potential marker of plaque size and composition in advanced atherosclerosis. Moreover, correlations with PWV values identified the maximum radStrain could serve as a potential marker for vascular elasticity. This study demonstrated the feasibility and utility of high-resolution 4D flow MRI to spatially resolve, visualize and analyze statistical differences in all relevant hemodynamic parameters over time and between healthy and diseased mice, which could significantly improve our understanding of plaque progression towards vulnerability. In future studies the relation of vascular elasticity and radial strain should be further investigated and validated with local PWV measurements and CFD.
Moreover, the 2D histological datasets were not reflecting the 3D properties and regional characteristics of the atherosclerotic plaques. Therefore, future studies will include 3D plaque volume and composition analysis like morphological measurements with MRI or light-sheet microscopy to further improve the analysis of the relationship between hemodynamics and atherosclerosis.
Chapter 4 aimed at the description and investigation of hemodynamics in early stages of atherosclerosis. Moreover, this study included measurements of hemodynamics at baseline levels in healthy WT and atherosclerotic mouse models. Due to the lack of hemodynamic-related studies in Ldlr-/- mice, which are the most used mouse models in atherosclerosis research together with the Apoe-/- mouse model, this model was included in this study to describe changing hemodynamics in the aortic arch at baseline levels and during early atherosclerosis development and progression for the first time. In this study, distinct differences in aortic geometries of these mouse models at baseline levels were described for the first time, which result in significantly different flow- and WSS profiles in the Ldlr-/- mouse model. Further basal characterization of different parameters revealed only characteristic differences in lipid profiles, proving that the geometry is highly influencing the local WSS in these models. Most interestingly, calculation of the atherogenic index of plasma revealed a significantly higher risk in Ldlr-/- mice with ongoing atherosclerosis development, but significantly greater plaque areas in the aortic arch of Apoe-/- mice. Due to the given basal WSS and OSI profile in these two mouse models – two parameters highly influencing plaque development and progression – there is evidence that the regional plaque development differs between these mouse models during very early atherogenesis.
Therefore, future studies should focus on the spatiotemporal evaluation of plaque development and composition in the three defined aortic regions using morphological measurements with MRI or 3D histological analyses like LSFM. Moreover, this study offers an excellent basis for future studies incorporating CFD simulations, analyzing the different measured parameter combinations (e.g., aortic geometry of the Ldlr-/- mouse with the lipid profile of the Apoe-/- mouse), simulating the resulting plaque development and composition. This could help to understand the complex interplay between altered hemodynamics, serum lipids and atherosclerosis and significantly improve our basic understanding of key factors initiating atherosclerosis development.
Chapter 5 describes the establishment of a tissue-engineered artery model, which is based on native, decellularized porcine carotid artery scaffolds, cultured in a MRI-suitable bioreactor-system [23] for the investigation of hemodynamic-related atherosclerosis development in a controllable manner, using the previously established methods for WSS and PWV assessment [4, 16]. This in vitro artery model aimed for the reduction of animal experiments, while simultaneously offering a simplified, but completely controllable physical and biological environment. For this, a very fast and gentle decellularization protocol was established in a first step, which resulted in porcine carotid artery scaffolds showing complete acellularity while maintaining the extracellular matrix composition, overall ultrastructure and mechanical strength of native arteries. Moreover, a good cellular adhesion and proliferation was achieved, which was evaluated with isolated human blood outgrowth endothelial cells. Most importantly, an MRI-suitable artery chamber was designed for the simultaneous cultivation and assessment of high-resolution 4D hemodynamics in the described artery models. Using high-resolution 4D-flow MRI, the bioreactor system was proven to be suitable to quantify the volume flow, the two components of the WSS and the radStrain as well as the PWV in artery models, with obtained values being comparable to values found in literature for in vivo measurements. Moreover, the identification of first atherosclerotic processes like intimal thickening is achievable by three-dimensional assessment of the vessel wall morphology in the in vitro models. However, one limitation is the lack of a medial smooth muscle cell layer due to the dense ECM. Here, the utilization of the laser-cutting technology for the generation of holes and / or pits on a microscale, eventually enabling seeding of the media with SMCs showed promising results in a first try and should be further investigated in future studies. Therefore, the proposed artery model possesses all relevant components for the extension to an atherosclerosis model which may pave the way towards a significant improvement of our understanding of the key mechanisms in atherogenesis.
Chapter 6 describes the development of an easy-to-prepare, low cost and fully customizable artery model based on biomaterials. Here, thermoresponsive sacrificial scaffolds, processed with the technique of MEW were used for the creation of variable, biomimetic shapes to mimic the geometric properties of the aortic arch, consisting of both, bifurcations and curvatures. After embedding the sacrificial scaffold into a gelatin-hydrogel containing SMCs, it was crosslinked with bacterial transglutaminase before dissolution and flushing of the sacrificial scaffold. The hereby generated channel was subsequently seeded with ECs, resulting in an easy-to-prepare, fast and low-cost artery model. In contrast to the native artery model, this model is therefore more variable in size and shape and offers the possibility to include smooth muscle cells from the beginning. Moreover, a custom-built and highly adaptable perfusion chamber was designed specifically for the scaffold structure, which enabled a one-step creation and simultaneously offering the possibility for dynamic cultivation of the artery models, making it an excellent basis for the development of in vitro disease test systems for e.g., flow-related atherosclerosis research. Due to time constraints, the extension to an atherosclerosis model could not be achieved within the scope of this thesis. Therefore, future studies will focus on the development and validation of an in vitro atherosclerosis model based on the proposed bi- and three-layered artery models.
In conclusion, this thesis paved the way for a fast acquisition and detailed analyses of changing hemodynamics during atherosclerosis development and progression, including spatially resolved analyses of all relevant hemodynamic parameters over time and in between different groups. Moreover, to reduce animal experiments, while gaining control over various parameters influencing atherosclerosis development, promising artery models were established, which have the potential to serve as a new platform for basic atherosclerosis research.
In vitro models mimic the tissue-specific anatomy and play essential roles in personalized medicine and disease treatments. As a sophisticated manufacturing technology, 3D printing overcomes the limitations of traditional technologies and provides an excellent potential for developing in vitro models to mimic native tissue. This thesis aims to investigate the potential of a high-resolution 3D printing technology, melt electrowriting (MEW), for fabricating in vitro models. MEW has a distinct capacity for depositing micron size fibers with a defined design. In this thesis, three approaches were used, including 1) extending the MEW polymer library for different biomedical applications, 2) developing in vitro models for evaluation of cell growth and migration toward the different matrices, and 3) studying the effect of scaffold designs and biochemical cues of microenvironments on cells.
First, we introduce the MEW processability of (AB)n and (ABAC)n segmented copolymers, which have thermally reversible network formulation based on physical crosslinks. Bisurea segments are combined with hydrophobic poly(dimethylsiloxane) (PDMS) or hydrophilic poly(propylene oxide)-poly(ethylene oxide)-poly(propylene oxide) (PPO-PEG-PPO) segments to form the (AB)n segmented copolymers. (ABAC)n segmented copolymers contain all three segments: in addition to bisurea, both hydrophobic and hydrophilic segments are available in the same polymer chain, resulting in tunable mechanical and biological behaviors. MEW copolymers either support cells attachment or dissolve without cytotoxic side effects when in contact with the polymers at lower concentrations, indicating that this copolymer class has potential in biological applications. The unique biological and surface properties, transparency, adjustable hydrophilicity of these copolymers could be beneficial in several in vitro models.
The second manuscript addresses the design and development of a melt electrowritten competitive 3D radial migration device. The approach differs from most of the previous literature, as MEW is not used here to produce cell invasive scaffolds but to fabricate an in vitro device. The device is utilized to systematically determine the matrix which promotes cell migration and growth of glioblastoma cells. The glioblastoma cell migration is tested on four different Matrigel concentrations using a melt electrowritten radial device. The glioblastoma U87 cell growth and migration increase at Matrigel concentrations 6 and 8 mg mL-1 In the development of this radial device, the accuracy, and precision of melt electrowritten circular shapes were investigated. The results show that the printing speed and design diameter are essential parameters for the accuracy of printed constructs. It is the first instance where MEW is used for the production of in vitro devices.
The influence of biochemical cues and scaffold designs on astrocytes and glioblastoma is investigated in the last manuscript. A fiber comprising the box and triangle-shaped pores within MEW scaffolds are modified with biochemical cues, including RGD and IKVAV peptides using a reactive NCO-sP(EO-stat-PO) macromer. The results show that astrocytes and glioblastoma cells exhibit different phenotypes on scaffold designs and peptide-coated scaffolds.
This thesis identifies how the printing conditions for a high-resolution additive manufacturing technique, melt electrowriting (MEW), needs to be adjusted to process electroactive polymers (EAPs) into microfibers. Using EAPs based on poly(vinylidene difluoride) (PVDF), their ability to be MEW-processed is studied and expands the list of processable materials for this technology.
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.
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.
As a major component of the articular cartilage extracellular matrix, hyaluronic acid is a widely used biomaterial in regenerative medicine and tissue engineering. According to its well-known interaction with multiple chondrocyte surface receptors which positively affects many cellular pathways, some approaches by combining mesenchymal stem cells and hyaluronic acid-based hydrogels are already driven in the field of cartilage regeneration and fat tissue. Nevertheless, a still remaining major problem is the development of the ideal matrix for this purpose. To generate a hydrogel for the use as a matrix, hyaluronic acid must be chemically modified, either derivatized or crosslinked and the resulting hydrogel is mostly shaped by the mold it is casted in whereas the stem cells are embedded during or after the gelation procedure which does not allow for the generation of zonal hierarchies, cell density or material gradients. This thesis focuses on the synthesis of different hyaluronic acid derivatives and poly(ethylene glycol) crosslinkers and the development of different hydrogel and bioink compositions that allow for adjustment of the printability, integration of growth factors, but also for the material and biological hydrogel, respectively bioink properties.
Polymeric Janus Fibers
(2023)
Janus fibers are a class of composite materials comprising mechanical and chemical to biological functionality. Combining different materials and functionalities in one micro- or even nanoscale fiber enables otherwise unreachable synergistic physicochemical effects with unprecedented opportunities for technical or biomedical applications. Here, recent developments of processing technologies and applications of polymeric Janus fibers will be reviewed. Various examples in the fields of textiles, catalysis, sensors as well as medical applications, like drug delivery systems, tissue engineering and antimicrobial materials, are presented to illuminate the outstanding potential of such high-end functional materials for novel applications in the upcoming future.
Mineral biocements are brittle materials, which usually results in catastrophic failure during mechanical loading. Here, previous works demonstrated the feasibility of reducing brittleness by a dual-setting approach, in which a silica sol was simultaneously gelled during the setting of a brushite forming cement. The current thesis aimed at further improving this concept by both using a novel silicate based cement matrix for an enhanced bonding between cement and silica matrix as well as multifunctional silica precursors to increase the network density of the gel. Due to its well-known biocompatibility and osteogenic regeneration capacity, baghdadite was chosen as mineral component of such composites. This required in a first approach the conversion of baghdadite ceramics into self-setting cement formulations. This was investigated initially by using baghdadite as reactive filler in a brushite forming cement (Chapter 4). Here, the ß-TCP component in a equimolar mixture of ß-TCP and acidic monocalcium phosphate anhydrous was subsequently replaced by baghdadite at various concentrations (0, 5, 10, 20, 30, 50, and 100 wt%) to study the influence on physicochemical cement properties such as mechanical performance, radiopacity, phase composition and microstructure. X-ray diffraction profiles demonstrated the dissolution of baghdadite during the cement reaction without affecting the crystal structure of the precipitated brushite phase. In addition, EDX analysis showed that calcium is homogeneously distributed in the cement matrix, while zirconium and silicon form cluster-like aggregates ranging in size from a few micrometers to more than 50 µm. X-ray images and µ-CT analyses indicate improved X-ray visibility with increased incorporation of baghdadite in brushite cement, with an aluminum equivalent thickness nearly doubling at a baghdadite content of 50 wt%. At the same time, the compressive strength of brushite cement increased from 12.9 ± 3.1 MPa to 21.1 ± 4.1 MPa at a baghdadite content of 10 wt%. Cell culture medium conditioned with powdered brushite cement approached physiological pH values when increasing amounts of baghdadite were added to the cement (pH = 6.47 for pure brushite, pH = 7.02 for brushite with 20 wt% baghdadite substitution). Baghdadite substitution also affected the ion content in the culture medium and thus the proliferation activity of primary human osteoblasts in vitro. The results demonstrated for the first time the suitability of baghdadite as a reactive cement additive for improving the radiopacity, mechanical performance, and cytocompatibility of brushite cements.
A second approach (Chapter 5) aimed to produce single component baghdadite cements by an increase of baghdadite solubility to initiate a self-setting cement reaction. For this, the material was mechanically activated by longer grinding times of up to 24h leading to both a decrease in particle and crystallite size as well as a partial amorphization of baghdadite. Baghdadite cements were formed by adding water at a powder to liquid ratio of 2.0 g/ml. Maximum compressive strengths were determined to be ~2 MPa after 3 days of setting for a 24-hour ground material. Inductively coupled plasma mass spectrometry (ICP-MS) measurements showed an incongruent dissolution profile of the set cements, with preferential dissolution of calcium and only minor release of zirconium ions. Cement formation occurs under alkaline conditions, with the unground raw powder resulting in a pH of 11.9 during setting, while prolonged grinding increases the pH to about 12.3.
Finally, mechanically activated baghdadite cements were combined with inorganic silica networks (Chapter 6) to create dual-setting cements with a further improvement of mechanical performance. While a modification of the cement pastes with a TEOS derived sol was already thought to improve strength, it was hypothesized that using multi-arm silica precursors can further enhance their mechanical performance due to a higher network density. In addition, this should also reduce pore size of both gels and cement and hence will be able to adjust the release kinetics of incorporated drugs. For this, multi-armed silica precursors were synthesized by the reaction of various multivalent alcohols (ethylene glycol, glycerine, pentaerythrit) with an isocyanate modified silica precursor. After hydrolysis under acidic conditions, the sols were mixed with baghdadite cement powders in order to allow a simultaneous gel formation and cement setting. Since the silica monomers have a high degree of linkage sites, this resulted in a branched network that interpenetrated with the growing cement crystals. In addition to minor changes in the crystalline phase composition as determined by X-ray diffraction, the novel composites exhibited improved mechanical properties with up to 20 times higher compressive strength and further benefit from an about 50% lower overall porosity than the reference pure baghdadite cement. In addition, the initial burst release of the model drug vancomycin was completely inhibited by the added silica matrix. This observation was verified by testing for the antimicrobial activity with Staphylococcus aureus by measuring the inhibition zones of selected samples after 24 h and 48 h, whereas the antimicrobial effectiveness of a constant vancomycin release could be demonstrated.
The current thesis clearly demonstrated the high potential of baghdadite as a cement formulation for medical application. The initially poor mechanical properties of such cements can be overcome by special processing techniques or by combination with silica networks. The achieved mechanical performance is > 10 MPa and hence suitable for bone replacement under non-load bearing conditions. The high intrinsic radiopacity as well as the alkaline pH during setting may open the way ahead to further dental applications, e.g. as root canal sealers or filler in dental composites. Here, the high pH is thought to lead to antimicrobial properties of such materials similar to commonly applied calcium hydroxide or calcium silicates, however combined with an intrinsic radiopacity for X-ray imaging. This would simplify such formulations to single component materials which are less susceptible to demixing processes during transport, storage or processing.
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.
Augmenting the vascular supply to generate new tissues, a crucial aspect in regenerative medicine, has been challenging. Recently, our group showed that calcium phosphate can induce the formation of a functional neo-angiosome without the need for microsurgical arterial anastomosis. This was a preclinical proof of concept for biomaterial-induced luminal sprouting of large-diameter vessels. In this study, we investigated if sprouting was a general response to surgical injury or placement of an inorganic construct around the vessel. Cylindrical biocement scaffolds of differing chemistries were placed around the femoral vein. A contrast agent was used to visualize vessel ingrowth into the scaffolds. Cell populations in the scaffold were mapped using immunohistochemistry. Calcium phosphate scaffolds induced 2.7–3 times greater volume of blood vessels than calcium sulphate or magnesium phosphate scaffolds. Macrophage and vSMC populations were identified that changed spatially and temporally within the scaffold during implantation. NLRP3 inflammasome activation peaked at weeks 2 and 4 and then declined; however, IL-1β expression was sustained over the course of the experiment. IL-8, a promoter of angiogenesis, was also detected, and together, these responses suggest a role of sterile inflammation. Unexpectedly, the effect was distinct from an injury response as a result of surgical placement and also was not simply a foreign body reaction as a result of placing a rigid bioceramic next to a vein, since, while the materials tested had similar microstructures, only the calcium phosphates tested elicited an angiogenic response. This finding then reveals a potential path towards a new strategy for creating better pro-regenerative biomaterials.
Present surgical situations require a bone adhesive which has not yet been developed for use in clinical applications. Recently, phosphoserine modified cements (PMC) based on mixtures of o-phosphoserine (OPLS) and calcium phosphates, such as tetracalcium phosphate (TTCP) or α-tricalcium phosphate (α-TCP) as well as chelate setting magnesium phosphate cements have gained increasing popularity for their use as mineral bone adhesives. Here, we investigated new mineral-organic bone cements based on phosphoserine and magnesium phosphates or oxides, which possess excellent adhesive properties. These were analyzed by X-ray diffraction, Fourier infrared spectroscopy and electron microscopy and subjected to mechanical tests to determine the bond strength to bone after ageing at physiological conditions. The novel biomineral adhesives demonstrate excellent bond strength to bone with approximately 6.6–7.3 MPa under shear load. The adhesives are also promising due to their cohesive failure pattern and ductile character. In this context, the new adhesive cements are superior to currently prevailing bone adhesives. Future efforts on bone adhesives made from phosphoserine and Mg2+ appear to be very worthwhile.
Zinc (Zn2+) is considered as important mediator of immune cell function, thrombosis and haemostasis. However, our understanding of the transport mechanisms that regulate Zn2+ homeostasis in platelets is limited. Zn2+ transporters, ZIPs and ZnTs, are widely expressed in eukaryotic cells. Using mice globally lacking ZIP1 and ZIP3 (ZIP1/3 DKO), our aim was to explore the potential role of these Zn2+ transporters in maintaining platelet Zn2+ homeostasis and in the regulation of platelet function. While ICP-MS measurements indicated unaltered overall Zn2+ concentrations in platelets of ZIP1/3 DKO mice, we observed a significantly increased content of FluoZin3-stainable free Zn2+, which, however, appears to be released less efficiently upon thrombin-stimulated platelet activation. On the functional level, ZIP1/3 DKO platelets exhibited a hyperactive response towards threshold concentrations of G protein-coupled receptor (GPCR) agonists, while immunoreceptor tyrosine-based activation motif (ITAM)-coupled receptor agonist signalling was unaffected. This resulted in enhanced platelet aggregation towards thrombin, bigger thrombus volume under flow ex vivo and faster in vivo thrombus formation in ZIP1/3 DKO mice. Molecularly, augmented GPCR responses were accompanied by enhanced Ca2+ and PKC, CamKII and ERK1/2 signalling. The current study thereby identifies ZIP1 and ZIP3 as important regulators for the maintenance of platelet Zn2+ homeostasis and function.
The use of bone-cement-enforced osteosynthesis is a growing topic in trauma surgery. In this context, drillability is a desirable feature for cements that can improve fracture stability, which most of the available cement systems lack. Therefore, in this study, we evaluated a resorbable and drillable magnesium-phosphate (MgP)-based cement paste considering degradation behavior and biocompatibility in vivo. Two different magnesium-phosphate-based cement (MPC) pastes with different amounts of phytic acid (IP 6) as setting retarder (MPC 22.5 and MPC 25) were implanted in an orthotopic defect model of the lateral femoral condyle of New Zealand white rabbits for 6 weeks. After explantation, their resorption behavior and material characteristics were evaluated by means of X-ray diffraction (XRD), porosimetry measurement, histological staining, peripheral quantitative computed tomography (pQCT), cone-beam computed tomography (CBCT) and biomechanical load-to-failure tests. Both cement pastes displayed comparable results in mechanical strength and resorption kinetics. Bone-contact biocompatibility was excellent without any signs of inflammation. Initial resorption and bone remodeling could be observed. MPC pastes with IP 6 as setting retardant have the potential to be a valuable alternative in distinct fracture patterns. Drillability, promising resorption potential and high mechanical strength confirm their suitability for use in clinical routine.
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. ...
3D bioprinting often involves application of highly concentrated polymeric bioinks to enable fabrication of stable cell-hydrogel constructs, although poor cell survival, compromised stem cell differentiation, and an inhomogeneous distribution of newly produced extracellular matrix (ECM) are frequently observed. Therefore, this study presents a bioink platform using a new versatile dual-stage crosslinking approach based on thiolated hyaluronic acid (HA-SH), which not only provides stand-alone 3D printability but also facilitates effective chondrogenic differentiation of mesenchymal stromal cells. A range of HA-SH with different molecular weights is synthesized and crosslinked with acrylated (PEG-diacryl) and allylated (PEG-diallyl) polyethylene glycol in a two-step reaction scheme. The initial Michael addition is used to achieve ink printability, followed by UV-mediated thiol–ene reaction to stabilize the printed bioink for long-term cell culture. Bioinks with high molecular weight HA-SH (>200 kDa) require comparably low polymer content to facilitate bioprinting. This leads to superior quality of cartilaginous constructs which possess a coherent ECM and a strongly increased stiffness of long-term cultured constructs. The dual-stage system may serve as an example to design platforms using two independent crosslinking reactions at one functional group, which allows adjusting printability as well as material and biological properties of bioinks.
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.
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.
3D neuronal cultures attempt to better replicate the in vivo environment to study neurological/neurodegenerative diseases compared to 2D models. A challenge to establish 3D neuron culture models is the low elastic modulus (30–500 Pa) of the native brain. Here, an ultra-soft matrix based on thiolated hyaluronic acid (HA-SH) reinforced with a microfiber frame is formulated and used. Hyaluronic acid represents an essential component of the brain extracellular matrix (ECM). Box-shaped frames with a microfiber spacing of 200 µm composed of 10-layers of poly(ɛ-caprolactone) (PCL) microfibers (9.7 ± 0.2 µm) made via melt electrowriting (MEW) are used to reinforce the HA-SH matrix which has an elastic modulus of 95 Pa. The neuronal viability is low in pure HA-SH matrix, however, when astrocytes are pre-seeded below this reinforced construct, they significantly support neuronal survival, network formation quantified by neurite length, and neuronal firing shown by Ca\(^{2+}\) imaging. The astrocyte-seeded HA-SH matrix is able to match the neuronal viability to the level of Matrigel, a gold standard matrix for neuronal culture for over two decades. Thus, this 3D MEW frame reinforced HA-SH composite with neurons and astrocytes constitutes a reliable and reproducible system to further study brain diseases.
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.
In 3D bioprinting for cartilage regeneration, bioinks that support chondrogenic development are of key importance. Growth factors covalently bound in non-printable hydrogels have been shown to effectively promote chondrogenesis. However, studies that investigate the functionality of tethered growth factors within 3D printable bioinks are still lacking. Therefore, in this study, we established a dual-stage crosslinked hyaluronic acid-based bioink that enabled covalent tethering of transforming growth factor-beta 1 (TGF-β1). Bone marrow-derived mesenchymal stromal cells (MSCs) were cultured over three weeks in vitro, and chondrogenic differentiation of MSCs within bioink constructs with tethered TGF-β1 was markedly enhanced, as compared to constructs with non-covalently incorporated TGF-β1. This was substantiated with regard to early TGF-β1 signaling, chondrogenic gene expression, qualitative and quantitative ECM deposition and distribution, and resulting construct stiffness. Furthermore, it was successfully demonstrated, in a comparative analysis of cast and printed bioinks, that covalently tethered TGF-β1 maintained its functionality after 3D printing. Taken together, the presented ink composition enabled the generation of high-quality cartilaginous tissues without the need for continuous exogenous growth factor supply and, thus, bears great potential for future investigation towards cartilage regeneration. Furthermore, growth factor tethering within bioinks, potentially leading to superior tissue development, may also be explored for other biofabrication applications.