Angeborene oder erworbene Knochendefekte können infolge ihrer Häufigkeit, ihrer oft mangelhaften spontanen Regenerationsfähigkeit sowie ihrer in der Regel langen Heilungsdauer ein erhebliches medizinisches, soziales und ökonomisches Problem darstellen. Zur Lösung dieses Problems stehen standardisierte und seit langen praktizierte Möglichkeiten wie die Osteosynthese oder die Defektauffüllung mit biologischen Knochenersatzmaterialien zur Verfügung. Auch synthetische Knochenersatzmaterialien, zum Teil in Kombinationen mit regenerativmedizinischen Prinzipien, kommen immer häufiger zum Einsatz wenn aufgrund eines großen Substanzverlustes des Knochens Defekte aufgefüllt werden müssen. Ziel dieser Studie am Tiermodell Schwein war es, die Knochenheilung calvärer Knochendefekte kritischer Größe unter Einsatz zweier verschiedener synthetischer Knochenersatzmaterialien, auf ß-Tricalciumphosphat- (β-TCP; Syntricer®, MedArtis Medizinprodukte und Forschung AG, München, Deutschland) bzw. Hydroxyapatit- Basis (HA; Ostim®, Heraeus-Kulzer, Hanau, Deutschland), angewandt sowohl in reiner Form als auch in Kombination mit autologen Stammzellen bzw. autologem Knochenmark, zu optimieren. Zur Untersuchung standen 16 klinisch gesunde weibliche Schweine der Deutschen Landrasse zur Verfügung. Alle Tiere waren zu Versuchsbeginn etwa 6 Monate alt und das durchschnittliche Lebendgewicht betrug zwischen 50 und 60 kg. Sowohl die β-TCP- als auch die HA-Gruppe umfasste 8 Schweine. Diese wurden wiederum in eine Kurz- und eine Langzeitgruppe zu je 4 Schweinen unterteilt, deren Beobachtungszeitraum 6 bzw. 16 Wochen betrug. Je Schwein wurden vier standardisierte Bohrlochdefekte kritischer Größe am Os frontale gesetzt. Ein Defekt wurde leer gelassen und diente als Kontrolldefekt für die physiologische Knochenheilung. Die drei anderen Defekte wurden einmal mit reinem und die anderen beiden mit biotechnologisch modifiziertem Knochenersatzmaterial aufgefüllt. Die biotechnologische Modifikation der Basissubstanzen erfolgte für einen Defekt durch die Anreicherung mit aus dem Knochenmark entnommenen und kultivierten autologen Stammzellen und für den vierten Defekt mit intraoperativ frisch punktiertem autologem Knochenmark. Die Ergebnisse der Knochenheilung wurden mit Hilfe einer computertomographischen Verlaufskontrolle intra vitam, sowie durch eine mikrotomographische und histologische Untersuchung nach der Euthanasie, untersucht. Sowohl Tier- als auch Versuchsmodell erwiesen sich als geeignet zur Untersuchung der Knochenregeneration mit Knochenersatzmaterialien. Die Knochenregeneration mit Ersatzmaterial führte in beiden Gruppen nach 6 Wochen im Vergleich mit der physiologischen Heilung zu besseren Ergebnissen, wobei sich das HA, wenn auch nicht signifikant, dem β-TCP überlegen zeigte. Die mikrotomographische Untersuchung zeigte aufgrund der höheren Detailerkennbarkeit im Vergleich zum CT einen größeren Unterschied zwischen den beiden Gruppen. So liegen die Mittelwerte im CT für die mit HA und mit dessen biotechnologischen Modifikationen gefüllten Defekte im Durchschnitt bei 11,2 ( ) und in der β-TCP-Gruppe bei 10,3 ( ) während sie für die mikrotomographische Untersuchung im Durchschnitt mit 9,4 ( ) für die mit HA und 5,7 ( ) für die mit β-TCP gefüllten Defekte bewertet wurden. Die histologische Bewertung zeigt den Unterschied zwischen beiden Gruppen bezüglich der Knochenregeneration am deutlichsten. So zeigte sich in der β-TCP-Gruppe ein sehr variabler Anteil an neugebildetem Knochengewebe, während in der HA-Gruppe immer mindestens 75% neugebildetes Knochengewebe nachgewiesen werden konnte. Der Zusatz von frischem Knochenmarkpunktat oder Stammzellen zu den Knochenersatzmaterialien hatte keinen erkennbaren Einfluss auf die Regeneration des Knochens. Synthetische resorbierbare Knochenersatzmaterialien, sowohl auf β-TCP- als auch auf HA-Basis, können die Knochenheilung positiv beeinflussen, und führen damit zu einer schnelleren Knochenregeneration als bei der physiologischen Knochenheilung. Im vorliegenden Modell führt die Kombination mit biotechnologischen Modifikationen wie autologen Stammzellen oder frisch punktiertem Knochenmark nicht zu einer zusätzlichen signifikanten Verbesserung der Knochenregeneration. Das pastöse nanopartikuläre Hydroxyapatit erscheint aufgrund besserer Handhabung, schnellerer Resorption sowie besserer Knochenheilung als das überlegene Material. / Inherited or acquired bone defects can, due to their frequency, their low spontaneous regenerative potential, and their generally long healing trajectories, present a significant medical, social and economical problem. Currently available solutions include standardized and well-established methods such as osteosynthesis or bone grafting using biological bone substitutes. In addition, synthetic bone substitutes, sometimes in combination with regenerative medicine, are increasingly used in cases when large bone defects require significant tissue replacement. The goal of this study was to optimize the healing of calvarial bone defects in pigs through the use of two distinct synthetic bone substitutes, -Tricalciumphosphate (β-TCP; Syntricer®, MedArtis Medizinprodukte und Forschung AG, Munich, Germany) and Hydroxyapatite (HA; Ostim®, Heraeus-Kulzer, Hanau, Germany), applied either in their pure form or in combination with either autologous stem cells or autologous bone marrow. Subjects for this study were 16 clinically healthy female domestic pigs (Deutsche Landrasse). All animals were approximately 6 months of age at the beginning of the study, with live weights between 50 and 60 kg. The animals were split into two groups of 8 for the separate study of HA and β-TCP. Each of these groups was further divided into two groups of 4 pigs, for studies of short and long duration (6 and 16 weeks, respectively). Four standardized drill holes of critical size were made in the Os frontale of each pig. One hole was left untreated as a control of physiological bone healing. Among the remaining three holes, one was filled with pure bone substitute (HA or β-TCP), and the other two were filled with biotechnologically modified bone substitute. This modification of the pure substances consisted for one drill hole of enrichment with autologous stem cells, cultured from bone marrow and, for the final hole, with intraoperative freshly extracted bone marrow. The bone healing results were measured intra vitam by computed tomography (CT), and after euthanasia by microtomography and histology. Both the model animal and experimental design proved useful for this study of bone healing using bone substitutes. Bone regeneration with bone substitutes in both experimental groups was enhanced after 6 weeks when compared with the untreated physiologically healed defects, where HA was superior to β-TCP (though not significantly). Owing to a higher resolution, microtomographic analysis revealed a greater difference between the two study groups than did CT. Thus, the mean values as measured by CT for defects filled with HA and biotechnologically modified HA are 11.2, and for 10.3 for the β-TCP group, while they are 9.4 and 5.7, respectively, as measured by microtomography. The histological assessment revealed the greatest difference in bone healing between the two experimental groups. Here, the β-TCP group displayed highly variable amounts of newly formed bone tissue, whereas each subject in the HA group displayed a minimum of 75% newly formed bone tissue. The addition of freshly extracted bone marrow or stem cells to the bone substitutes had no detectable effect on bone regeneration. Synthetic resorptable bone substitutes, on a basis of both β-TCP and HA, can positively influence bone healing, and thereby lead to a faster regeneration of bone tissue than the physiological process. In the present study, biotechnological modification of bone substitutes with autologous stem cells or bone marrow did not further enhance bone regeneration. Given its easy handling, faster resorption, and better bone healing, the nanoparticulate Hydroxyapatite paste appears to be the superior material.

Gelenkknorpel ist ein gefäßloses, hoch spezialisiertes Gewebe mit nur sehr begrenzter Regenerationsfähigkeit. Entstandene Läsionen werden bei natürlicher Heilung durch min-derwertigen Faserknorpel gefüllt. Ein vielversprechender Therapieansatz kommt aus dem Gebiet des Tissue Engineering. Dabei werden isolierte Chondrozyten in vitro vermehrt und anschließend in den Defekt eingebracht. In den letzten Jahren ist hier die 3 D-Kultivierung in patientenspezifischen Biomaterialien zunehmend in den Fokus der Forschung geraten. Ziel der hier vorgestellten Studie war es, die Tauglichkeit von Vollblut und Blutplasma als Aus-gangsmaterial für MACTs aufgrund makroskopischer Eigenschaften, Zellzahlentwicklung im Konstrukt, Zellvitalität und Syntheseleistung charakteristischer EZM-Marker zu untersuchen. Es wurde für diese Studie Knorpel aus den Fesselgelenken vier geschlachteter Pferde (2-16 Jahre) entnommen, mechanisch zerkleinert und anschließend mit Kollagenase A verdaut. Von einem 6 jährigen klinikeigenen Wallach wurde Vollblut in Citratröhrchen gewonnen, ein Teil zu Plasma weiterverarbeitet und beides bei -80 °C bis zur weiteren Verwendung Schock gefroren. Zur Herstellung der walzenförmigen Konstrukte mit den Maßen 9,6 cm2 x 4,7 mm, wurden 4,5 ml Vollblut bzw. Plasma mit je 3x106 Chondrozyten suspensiert und durch Zuga-be von CaCl2 zur Koagulation gebracht. Der Kultivierungszeitraum betrug 28 Tage in DMEM, angereichert mit 10% allogenem Serum und 1% Antibiotika. Die Konstrukte wurden an den Tagen 1, 14 und 28 auf Zellzahl, -vitalität und mithilfe qRT-PCR auf hyaline Knorpelmarker wie Kollagen Typ II und Aggrekan untersucht. Zudem wurden histologische und immunhisto-chemische Präparate der Konstrukte angefertigt. Die Zellvitalität betrug sowohl in den VB-, als auch in den BP-Konstrukten ≥95% bei steigen-der Zellzahl (bis zu 5x106 im Vollblutkonstrukt). Die MACTs beider Ausgangsmaterialien schrumpften auf eine Größe von 2 cm² x 2 mm. Histologisch konnten in beiden Konstruktar-ten mit der Alzianblaufärbung sGAG belegt werden. Darüber hinaus wurde der sGAG Gehalt mit dem DMMB Assay quantitativ ermittelt. Aggrekan, C-4-S, C-6-S und COMP wurden als wichtige Bestandteile der EZM immunhistochemisch angefärbt und waren ebenfalls in beiden Konstruktarten nachweisbar. Mit der qRT PCR konnte die Genexpression von Aggrekan, Kol II und Kol I über den zeitlichen Verlauf ermittelt werden. Es stellte sich heraus, dass sich sowohl die Genexpression von Aggrekan als auch die von Kol II, den beiden Indikatorprotei-nen EZMs des hyalinen Gelenkknorpels über den Kultivierungszeitraum absenkten. Dies deutet auf eine Dedifferenzierung der Chondrozyten hin. Die Expression von Kol I dagegen stieg um ein Vielfaches an. Auch das kennzeichnet eine Dedifferenzierung. Zieht man ande-re Studien heran, so ist die festgestellte Umstellung der Genexpression aber vergleichsweise niedrig, eine Dedifferenzierung weg vom chondrogenen Phänotyp in der hier vorliegenden Arbeit also weniger stark ausgebildet. Biokompatibilität mit und Abbaubarkeit im Empfängerorganismus konnten in dieser in vitro Untersuchung nicht evaluiert werden. Zusammenfassend kann man festhalten, dass VB und BP als Ausgangsbiomaterialien zur Herstellung von MACT geeignet sind. Inwieweit es gelingen wird, den chondrogenen Phäno-typ beispielweise durch mechanische Stimulation der eingesäten Zellen stärker zu erhalten, muss in folgenden Studien geklärt werden. Ebenfalls weiterer Forschungsbedarf ist bei den Eigenschaften Elastizität und Steifheit gegeben. Grundsätzlich gilt, dass MACTs auf VB- und BP-Basis einen einfachen, kostengünstigen und patientenspezifisch herstellbaren Therapie-ansatz für die Behandlung von Knorpeldefekten darstellen. / Articular cartilage is a vessel-free, highly specialized tissue with only very limited regenera-tive capacity. Resulting lesions are filled with inferior fibrocartilage by natural healing. A promising therapeutic approach comes from the field of tissue engineering. Therefore iso-lated chondrocytes are expanded in vitro and then placed into the defect. In the last few years the 3-D culturing in patient-specific biomaterials has come increasingly into the focus of research. Purpose of the present study was to evaluate the suitability of whole blood and blood plasma as a basic material for MACT based on macroscopic properties, development of cell number in the construct, cell viability and synthetic performance of characteristic markers. For this study cartilage from the four fetlock joints of slaughtered horses (2-16 years) were removed, crushed mechanically and then digested with Collagenase A. Whole blood was obtained in citrate tubes of a 6 year old gelding owned by the clinic, finished part to both plasma and shock frozen at -80°C until further use. To prepare the cylindrical constructs, measuring 9.6 cm2 x 4.7 mm, 4.5 ml of whole blood or plasma, each with 3x106 chondro-cytes were suspended and coagulated by the addition of CaCl2. The cultivation period was 28 days in DMEM supplemented with 10% allogenic serum and 1% antibiotics. The con-structs were evaluated on days 1, 14 and 28 on cell number, viability, and using qRT-PCR examination for hyaline cartilage markers such as collagen type II and aggrecan. In addition, histological and immunohistochemical preparations of the constructs were made. The cell vitality was in the WB, as well as in the BP constructs ≥ 95% with increasing cell number (up to 5x106 in whole blood construct). The MACTs of both basic materials shrink to a size of 2 cm x 2 mm². Histologically sGAG could be verified in both construct species by Alzianblau staining. In addition, the GAG content was determined with the DMMB assay quantitatively. Aggrecan, chondroitin-4-sulphate, chondroitin-6-sulphate and COMP as major components of the ECM were stained immunohistochemically and were also detectable in both types of constructs. Aggrecan, collagen II and collagen I were determined on the time course by qRT PCR gene expression. It turned out that the gene expressions of both, aggre-can and of collagen II, the two ECM protein indicators of hyaline cartilage lowered over the cultivation period. This indicates a dedifferentiation of chondrocytes. The expression of colla-gen I on the other hand increased to a multiple, also featuring a dedifferentiation. If one approached other studies, the observed change in gene expression is comparatively low. In the present work the dedifferentiation from the chondrogenic phenotype is less distinctive. Biocompatibility and biodegradability in the recipient organism could not be evaluated in this in vitro investigation. In summary, one should notice that VB and BP are suitable basic materials for the production of MACT. It has to be clarified by following studies, to what extent the chondrogenic pheno-type can be strengthened, for example by mechanical stimulation of cells sown. Also further research is needed on the given properties, e.g. elasticity and stiffness. Generally MACT based on WB and BP- is a simple, inexpensive and patient-specific produced therapeutic approach for the treatment of cartilage defects.

Development of combination products such as tissue engineered constructs which combine biomaterials with biologics has prompted the need to clarify the role of biomaterial in potentiating the immune response towards the biological component due to adjuvant effect. In tissue engineering applications, immune responses are to be minimized while vaccine strategies seek to enhance the protective immune response. Thesis project presented herein showed that adjuvant effect of poly(lactic-co-glycolic acid) (PLGA) is mediated in part by maturation of dendritic cells (DCs), immune cells that orchestrate adaptive immune response. Maturation of human peripheral blood monocyte-derived DCs in response to PLGA contact was demonstrated in vitro and in vivo by increased co-stimulatory and MHC molecule expression, mixed lymphocyte reaction, cytokine release, and delayed type hypersensitivity reaction. In contrast to PLGA, agarose did not induce DC maturation, in accordance with its low inflammatory effect. Roles of various receptors involved in DC maturation and recognition of biomaterials were assessed by in vitro receptor blocking studies. In particular, role of Toll-like receptors were further investigated using DCs derived from bone marrows of murine model of Toll-like receptor 4 deficiency (C3H/HeJ). While PLGA induced maturation of DCs from C57BL6 mice, maturation was not observed in DCs from C3H/HeJ strain or control strain, C3H/HeOuJ, perhaps due to particular haplotypes of these animals. Collectively, these results establish the differential adjuvant effects of agarose and PLGA on the level of DC maturation, and begin to elucidate the mechanisms of biomaterial adjuvant effect. In addition, assays developed herein provide methods to screen for biomaterials to be used in combination products, such that biomaterials with desired levels of adjuvanticity as measured by DC maturation effects may be selected for given application.

Atherosclerosis is an inflammatory disease which develops focally in regions of the vasculature where there is dysfunction of endothelial cells modulated in part by shear stress from flowing blood. To address the clinical crisis of atherosclerosis, tissue engineering has focused on development of a living blood vessel substitute for use as a vascular graft in bypass surgery. Despite substantial progress in understanding the biological basis and developing clinical treatments for cardiovascular disease, critical challenges remain. As a novel strategy to improve understanding of basic human vascular biology and develop superior tissue engineered grafts, this dissertation combines the scientific and clinical approaches by using a tissue engineered blood vessel as a more physiologic in vitro model to study endothelial cell biology. Through the use of transcriptional profiling, results demonstrate significant changes in endothelial cell gene expression using the tissue engineered blood vessel model. Furthermore, the presence of a more physiologic substrate alters the cellular response to shear stress which is a critical mediator of vascular pathology. A case study of endothelial cell function in this system focuses on cell-cell communication through gap junctions. Endothelial cell connexins which form gap junctions are shown to be differentially regulated by substrate and shear stress. Moreover, gap junction communication between endothelial cells is modulated by the mechanical environment. Studies using RNA interference to knockdown expression of individual connexin isotypes demonstrate integrated regulation of connexins yet unique roles in endothelial cell function. Collectively, results exemplify the sensitivity of endothelial cell phenotype to substrate and shear stress and underline the importance of using more physiologic models in the study of basic cell biology.

Tissue engineering has emerged as a promising alternative to conventional orthopaedic grafting therapies. The general paradigm for this approach, in which phenotype-specific cells and/or bioactive growth factors are integrated into polymeric matrices, has been successfully applied in recent years toward the development of bone, ligament, and cartilage tissues in vitro and in vivo. Despite these advances, an optimal cell source for skeletal tissue repair and regeneration has not been identified. Furthermore, the lack of robust, functional orthopaedic tissue interfaces, such as the bone-ligament enthesis, severely limits the integration and biological performance of engineered tissue substitutes. This works aims to address these limitations by spatially controlling the genetic modification and differentiation of fibroblasts into a mineralizing osteoblastic phenotype within three-dimensional polymeric matrices. The overall objective of this project was to investigate transcription factor-based gene therapy strategies for the differentiation of fibroblasts into a mineralizing cell source for orthopaedic tissue engineering applications. Our central hypothesis was that fibroblasts genetically engineered to express Runx2 via conventional and biomaterial-mediated ex vivo gene transfer approaches will differentiate into a mineralizing osteoblastic phenotype. We have demonstrated that a combination of retroviral Runx2 overexpression and glucocorticoid hormone treatment synergistically induces osteoblastic differentiation and biological mineral deposition in primary dermal fibroblasts cultured in monolayer. We report for the first time that glucocorticoids induce osteoblastic differentiation in this model system by modulating the phosphorylation state of a negative regulatory serine residue (Ser125) on Runx2 through an MKP-1-dependent mechanism. Furthermore, we utilized these Runx2-genetically engineered fibroblasts to create mineralized templates for bone repair in vitro and in vivo. Finally, we engineered a heterogeneous bone-soft tissue interface with a novel biomaterial-mediated gene transfer approach. Overall, these results are significant toward the ultimate goal of regenerating complex, higher-order orthopaedic grafting templates which mimic the cellular and microstructural characteristics of native tissue. Cellular therapies based on primary dermal fibroblasts would be particularly beneficial for patients with a compromised ability to recruit progenitors to the sight of injury as result of traumatic injury, radiation treatment, or osteodegenerative disease.

Stem cell synthesized extracellular matrix (ECM) may serve as a replacement for current bone grafting techniques. The overall goal of this thesis is to quantify the osteoinductivity of the ECM produced by human amniotic fluid stem cells (AFS cells), compare it to that of human mesenchymal stem cells (MSC), and assess its potential for use in bone tissue engineering therapies. Each stem cell type was cultured in osteogenic media to produce the ECM, which was then decellularized via freeze/thaw cycling and DNase treatment. The success of the decellularization was confirmed with live/dead staining and DNA quantification. A series of in vitro studies were performed to evaluate the characteristics of the ECM relevant to a bone tissue engineering therapy. Reseeded MSCs were able to attach to and proliferate on both ECM types in both 2D and 3D culture. In 2D, cells cultured on both ECM types showed increased levels of calcium deposition. Additionally, cells cultured on the MSC ECM showed increased alkaline phosphatase activity. A synergistic effect on osteogenic differentiation was observed when the osteoinductive factor dexamethasone was added to the culture. In 3D, both ECM types increased the mineralized matrix production of reseeded MSCs. The AFS ECM had a greater effect than the MSC ECM. When ECM was used to treat a rat femoral segmental defect in vivo, it was found that each ECM type increased the rate of bridging of the defect when compared to collagen coated scaffolds. However the ECM did not have a significant effect on the volume of mineralized matrix within the defect site in this study.

The host response toward biomaterial component of tissue-engineered devices has been extensively investigated. The objective of this research was to understand the response of dendritic cells (DCs) to different biomaterials upon contact and identify biomaterials suitable for use in tissue engineering constructs for rheumatoid arthritis (RA) applications. Differential levels of functional DC maturation were observed depending on the type of biomaterial in 2-dimensional films or 3-dimensional scaffolds used to treat immature DCs; Poly(lactic-co-glycolic acid) (PLGA) or chitosan supported higher levels of DC maturation, as compared to immature DCs. Alginate supported moderate levels of DC maturation. Agarose did not support DC maturation whereas hyaluronic acid inhibited DC maturation. Further, these DCs treated with different biomaterials induced differential phenotype and polarization of autologous T cells upon co-culture of DCs and T cells; DCs treated with PLGA induced T helper type I with immunogenic response while DCs treated with agarose did T helper type II with tolerogenic response. Effect of different biomaterials (PLGA and agarose) was assessed in vivo upon implantation of them into the knee joint of RA-induced rabbit. Total leukocyte concentrations in the peripheral blood or in the joint lavage of the left knees (untreated control) were observed in differential levels depending on the biomaterial implant, possibly due to the systemic circulation of the peripheral blood. Furthermore, cartilage and bone healing progression was differentially observed in the osteochondral defect of the knee joint of RA-induced rabbit, depending on type of biomaterial scaffold implanted into the defect. Collectively, these results demonstrate the multifunctional impacts of inherently different biomaterials on in vitro immunomodulation of phenotype and polarization of DCs and autologous T cells. Furthermore, taken together with these immunomodulatory impacts of biomaterials, in vivo effects of different biomaterial scaffolds on RA environment shown in this study can suggest the criteria of selection and design of biomaterials for orthopedic tissue engineering, which may ultimately be best integrated into the diseased cartilage and bone.

Biomimetic materials that recapitulate the complex mechanical and biochemical cues in load-bearing tissues are of significant interest in regenerative medicine and tissue engineering applications. Several investigators have endeavored to not only emulate the mechanical properties of the vasculature, but to also mimic the biologic responsiveness of the blood vessel in creating vascular substitutes. Previous studies in our lab generated the elastin-like protein polymer LysB10, which was designed with the capability of physical and chemical crosslinks, and was shown to display a range of elastomeric properties that more closely matched those of the native artery. While extensive validation of the mechanical properties of elastin-mimetic polymers has demonstrated their functionality in a number of tissue engineering applications, limited cell growth on the surfaces of the polymers has motivated further optimization for biological interaction. Recent biologically-inspired surface strategies have focused on functionalizing material surfaces with extracellular matrix molecules and bioactive motifs in order to encourage integrin-mediated cellular responses that trigger precise intracellular signaling processes, while limiting nonspecific biomaterial interactions. Consequently, this dissertation addresses three approaches to modulating cellular behavior on elastin-mimetic analogs with the goal of promoting vascular wall healing and tissue regeneration: genetic engineering of elastin-like protein polymers (ELPs) with cell-binding domains, biofunctionalization of elastin-like protein polymers via chemoselective ligation of bioactive ligands, and incorporation of matrix protein fibronectin for engineering of cell-seeded multilamellar collagen-reinforced elastin-like constructs. The synthesis of recombinant elastin-like protein polymers that integrate biologic functions of the extracellular matrix provides a novel design strategy for generating clinically durable vascular substitutes. Ultimately, the synthesis of model protein networks provides new insights into the relationship between molecular architecture, biomimetic ligand presentation, and associated cellular responses at the cell-material interface. Understanding how each of these design parameters affects cell response will contribute significantly to the rational engineering of bioactive materials. Potential applications for polymer blends with enhanced mechanical and biological properties include surface coatings on vascular grafts and stents, as well as composite materials for tissue engineered scaffolds and vascular substitutes.

Flocktechnologie ist eine im Bereich der Textiltechnik angewandte Methode, bei der kurze Fasern nahezu senkrecht auf ein vorher mit Klebstoff beschichtetes Substrat aufgebracht werden. In der vorliegenden Arbeit wurde die elektrostatische Beflockung als Methode zur Herstellung von porösen, dreidimensionalen Scaffolds für das Tissue Engineering von Knorpel und Knochen etabliert. Dieser neuartige Scaffoldtyp wurde eingehend charakterisiert und in Zellversuchen im Hinblick auf seine Biokompatibilität untersucht. Dabei zeigte sich, dass verschiedene Zellen im Scaffold proliferieren und differenzieren können. Die in der Arbeit beschriebenen Flockscaffolds stellen somit eine vielversprechende Matrix für die Therapie von Gelenkknorpeldefekten dar.

Articular cartilage which covers the surfaces of synovial joints is designed to allow smooth contact between long bones and to absorb shock induced during joint movement. Tissue engineering, a means of combining cells, biomaterials, bioreactors and bioactive agents to produce functional tissue replacements suitable for implantation, represents a potential long-term strategy for cartilage repair. The interplay between environmental factors, however, gives rise to complex culture conditions that influence the development of tissue-engineered constructs. A fibrous capsule that is composed of abundant type I collagen molecules and resembles fibrocartilage usually forms at the outer edge of neocartilage, yet the understanding of its modulation by environmental cues is still limited. Therefore, this dissertation was aimed to characterize the capsule formation, development and function through manipulation of biochemical parameters present in a hydrodynamic environment while a chemically reliable media preparation protocol for hydrodynamic cultivation of tissue-engineered cartilage was established. To this end, a novel wavy-wall bioreactor (WWB) that imparts turbulent flow-induced shear stress was employed as the model system and polyglycolic acid scaffolds seeded with bovine primary chondrocytes were cultivated under varied biochemical conditions. The results demonstrated that tissue morphology, biochemical composition and mechanical strength of hydrodynamically engineered cartilage were maintained as the serum content decreased by 80% (from 10% to 2%). Transient exposure of the low-serum constructs to exogenous insulin-like growth factor-1 (IGF-1) or transforming growth factor-β1 (TGF-β1) further accelerated their development in comparison with continuous treatment with the same bioactive molecules. The process of the capsule formation was found to be activated and modulated by the concentration of serum which contains soluble factors that are able to induce fibrotic processes and the capsule development was further promoted by fluid shear stress. Moreover, the capsule formation in hydrodynamic cultures was identified as a potential biphasic process in response to concentrations of fibrosis-promoting molecules such as TGF-β. Comparison between the capsule-containing and the capsule-free constructs, both of which had comparable tissue properties and were produced by utilizing the WWB system in combination with IGF-1 and TGF-β1, respectively, showed that the presence of the fibrous capsule at the construct periphery effectively improved the ability of engineered cartilage to integrate with native cartilage tissues, but evidently compromised its tissue homogeneity. Characterization of the fibrous capsule and elucidation of the conditions under which it is formed provide important insights for the development of tissue engineering strategies to fabricate clinically relevant cartilage tissue replacements that possess optimized tissue homogeneity and properties while retaining a minimal capsule thickness required to enhance tissue integration.