No description available.

Bänder und Sehnen sind bradytrophe Gewebe die eine limitierte intrinsische Heilungskapazität aufweisen. Trotz einer primären Nahtrekonstruktion kann es zur Ausbildung eines mechanisch insuffizienten Narbengewebes kommen. Die Verwendung autologer oder allogener Sehnen-/Bandersatzplastiken bei Vorliegen substantieller Defekte bergen die Gefahr der donor site morbidity und antigener/allergischer Reaktionen. Besonders das Tissue Engineering kann hier zur Entwicklung innovativer Therapieansätze beitragen. Die Verwendung autologer mesenchymaler Vorläuferzellen und biomimetischer Zellträger zu Generierung eines Sehnen- /Bandersatzes ex vivo ist eine vielversprechende Alternative. Ziel der vorliegenden Arbeit war die Generierung von biomimetischen Zellträgern auf der Basis von Kollagen Typ I mittels Elektrospinning. Dabei orientierte sich das Scaffolddesign am Aufbau der EZM von nativem Band- und Sehnengewebe. In einem zweiten Schritt wurde die Auswirkung unterschiedlicher Scaffoldarchitektur auf die tenogene Differenzierung von humanen MSZ untersucht. Hierzu wurden MSZ aus dem Knochenmark isoliert, amplifiziert, die Zellträger mit diesen Zellen besiedelt und für einen definierten Zeiträum (21 Tage) kultiviert. Die Kollagen I-Ausgangskonzentration hatte entscheidenden Einfluss auf den Faserdurchmesser. Wobei unter Verwendung einer 5-8%-igen Kollagenlösung der Faserdurchmesser im Bereich von nativen Kollagenfasern in natürlichem Sehnengewebe erzielt werden konnte. Unter Verwendung eines rotierenden Metallzylinders als Kollektor konnte mit steigender Rotationgeschwindigkeit eine zunehmende parallele Faserausrichtung in den NFS erreicht werden. Ein Einfluss auf die Morphologie und die Proliferation der MSZ auf NFS mit unterschiedlicher Faserdicke zeigte sich nicht. Ausgerichtete Fasern führten zu einer signifikant parallelen Ausrichtung der MSZ mit langgezogenem schlanken Zellkörper, im Unterschied zu einer polygonalen MSZ-Morphologie auf nicht ausgerichteten NF. Die tenogene Differenzierung der Zellen in den NFS wurde mittels RT-PCR- Analyse untersucht. Hierbei wurde die Expression der tendogenen Markergene Tenascin C, Elastin, Kollagen I und Skleraxis bestimmt. Zusätzlich wurden immunfluoreszens- und histochemische Färbungen durchgeführt, um die Infiltration der Zellen in die Zellträger und den Einfluss unterschiedlicher Faserparameter auf die Morphologie der MSZ nachzuweisen. Unter Verwendung von ausgerichteten Kollagen I-NFS zeigte sich eine signifikant höhere tenogene Markergenexpression für Skleraxis und Tenascin C in der Frühphase und im weiteren Verlauf ebenfalls für Col I und Elastin im Vergleich zu nicht ausgerichteten NFS. Elektrospinning von Kollagen I unter Verwendung eines rotierenden Kollektors ermöglicht die Herstellung biomimetischer NFS mit paralleler Faserausrichtung analog zu nativem Sehnengewebe. Die so hergestellten NFS zeichnen sich im Vergleich zu nicht ausgerichteten NFS durch eine signifikant höher mechanische Zugfestigkeit und die Induktion einer tenogenen Markergenexpression in MSZ aus. Prinzipiell haben Kollagen I-NFS das Potential bestehende Therapiestrategien zu Rekonstruktion substantieller Sehnenrupturen im Rahmen Stammzell-basierter Ansätze zu unterstützen. Die generelle Eignung in vivo muss aber zunächst in adäquaten Großtiermodellen (z. B. Rotatorenmanschettendefekt im Schaf) überprüft werden. Die vorliegende Arbeit zeigt die Bedeutung eines Zielgewebe-gerichteten Designs von Zellträgern für die Entwicklung innovativer Strategien im Tissue Engineering. Bei der Regeneration muskuloskelettaler Gewebe, wie dem Sehnenegewebe, spielen nicht nur strukturelle Aspekte sondern auch die biochemische Zusammensetzung des zu erneuernden Gewebes eine entscheidende Rolle, die bei der Scaffold-Herstellung zu berücksichtigen sind. / In vitro studies of tenogenous differentiation of human mesenchymal stem cells in collagen -I-nanofaserscaffolds for the replacement of tendons

The limited treatment options available for heart disease patients has lead to increased interest in the development of embryonic stem cell (ESC) therapies to replace heart muscle. The challenges of developing usable ESC therapeutic strategies are associated with the limited ability to obtain a pure, defined population of differentiated cardiomyocytes, and the design of in vivo cell delivery platforms to minimize cardiomyocyte loss. These challenges were addressed in Chapter 2 by designing a cardiomyocyte selectable progenitor cell line that permitted evaluation of a collagen-based scaffold for its ability to sustain stem cell-derived cardiomyocyte function (“A P19 Cardiac Cell Line as a Model for Evaluating Cardiac Tissue Engineering Biomaterials”). P19 cells enriched for cardiomyocytes were viable on a transglutaminase cross-linked collagen scaffold, and maintained their cardiomyocyte contractile phenotype in vitro while growing on the scaffold. The potential for a novel cell-surface marker to purify cardiomyocytes within ESC cultures was evaluated in Chapter 3, “Dihydropyridine Receptor (DHP-R) Surface Marker Enrichment of ES-derived Cardiomyocytes”. DHP-R is demonstrated to be upregulated at the protein and RNA transcript level during cardiomyogenesis. DHP-R positive mouse ES cells were fluorescent activated cell sorted, and the DHP-R positive cultured cells were enriched for cardiomyocytes compared to the DHP-R negative population. Finally, in Chapter 4, mouse ESCs were characterized while growing on a clinically approved collagen I/III-based scaffold modified with the RGD integrin-binding motif, (“Collagen (+RGD and –RGD) scaffolds support cardiomyogenesis after aggregation of mouse embryonic stem cells”). The collagen I/III RGD+ and RGD- scaffolds sustained ESC-derived cardiomyocyte growth and function. Notably, no significant differences in cell survival, cardiac phenotype, and cardiomyocyte function were detected with the addition of the RGD domain to the collagen scaffold. Thus, in summary, these three studies have resulted in the identification of a potential cell surface marker for ESC-derived cardiomyocyte purification, and prove that collagen-based scaffolds can sustain ES-cardiomyocyte growth and function. This has set the framework for further studies that will move the field closer to obtaining a safe and effective delivery strategy for transplanting ESCs onto human hearts.

The limited treatment options available for heart disease patients has lead to increased interest in the development of embryonic stem cell (ESC) therapies to replace heart muscle. The challenges of developing usable ESC therapeutic strategies are associated with the limited ability to obtain a pure, defined population of differentiated cardiomyocytes, and the design of in vivo cell delivery platforms to minimize cardiomyocyte loss. These challenges were addressed in Chapter 2 by designing a cardiomyocyte selectable progenitor cell line that permitted evaluation of a collagen-based scaffold for its ability to sustain stem cell-derived cardiomyocyte function (“A P19 Cardiac Cell Line as a Model for Evaluating Cardiac Tissue Engineering Biomaterials”). P19 cells enriched for cardiomyocytes were viable on a transglutaminase cross-linked collagen scaffold, and maintained their cardiomyocyte contractile phenotype in vitro while growing on the scaffold. The potential for a novel cell-surface marker to purify cardiomyocytes within ESC cultures was evaluated in Chapter 3, “Dihydropyridine Receptor (DHP-R) Surface Marker Enrichment of ES-derived Cardiomyocytes”. DHP-R is demonstrated to be upregulated at the protein and RNA transcript level during cardiomyogenesis. DHP-R positive mouse ES cells were fluorescent activated cell sorted, and the DHP-R positive cultured cells were enriched for cardiomyocytes compared to the DHP-R negative population. Finally, in Chapter 4, mouse ESCs were characterized while growing on a clinically approved collagen I/III-based scaffold modified with the RGD integrin-binding motif, (“Collagen (+RGD and –RGD) scaffolds support cardiomyogenesis after aggregation of mouse embryonic stem cells”). The collagen I/III RGD+ and RGD- scaffolds sustained ESC-derived cardiomyocyte growth and function. Notably, no significant differences in cell survival, cardiac phenotype, and cardiomyocyte function were detected with the addition of the RGD domain to the collagen scaffold. Thus, in summary, these three studies have resulted in the identification of a potential cell surface marker for ESC-derived cardiomyocyte purification, and prove that collagen-based scaffolds can sustain ES-cardiomyocyte growth and function. This has set the framework for further studies that will move the field closer to obtaining a safe and effective delivery strategy for transplanting ESCs onto human hearts.

Polymer scaffold use has become commonplace in tissue engineering strategies. Scaffolds provide sturdy interfaces that securely anchor tissue engineered constructs to their designated locations. Researchers have used scaffolds to provide support to developing tissues as well as a growth template to aid the development of the desired phenotypic structure. In addition to using scaffolds for their mechanical support, scaffolds can be used as a diagnostic tool by attaching sensors. Strain gauge sensors have been attached to scaffolds to monitor compression and elongation. These polybutylterphalate (PBT) scaffolds were used in a cartilage tissue-engineering project for femoral cartilage repair. The aim of this project was to measure native cartilage pressure in normal canine stifle joints using strain gauge scaffolds. By using pressure sensitive films to confirm joint surface pressures determined with strain gauge measurements, "sensate" scaffolds were created to be able to provide in vivo joint loading measurements. An understanding of the in vivo pressures in the menisco-femoral joint space will facilitate the development of tissue engineered cartilage by determining chondrocyte mechanical triggers as well as helping define reasonable expectations for engineered articular cartilage tissue that is required for successful cartilage repair.

Subglottic stenosis, malignancy and traumatic injury to the trachea require surgical resection. When defects are less than 50% of the tracheal length in adults and 1/3 in children, a circumferential resection and primary anastomosis affords excellent results. For longer lesions, on the other hand, there are no currently acceptable solutions leading to patients requiring permanent tracheostomies or palliative treatment. Tracheal replacement approaches with synthetic prosthesis and scaffolds have all led to inflammation, obstruction, mucous build-up and eventual restenosis. Tissue-engineering approaches using recipients’ own stem cells and biologic scaffolds derived from decellularized donor trachea have shown great promise. They have the potential to abrogate the need for immunosuppressive therapy. Our research focuses on three major limitations in this field including the structural integrity, the immunogenicity and the recellularization of donor tracheae. We compared three decellularization protocols, quantified and qualified the extracellular matrix (ECM) components and performed compliance measurements on large circumferential tracheal scaffolds following cyclical decellularization techniques and illustrated significant differences in ECM composition and resultant structural integrity of decellularized tracheal scaffolds depending on the protocol. In addition, we investigated the immunogenicity of decellularized and recellularized tracheal allografts at a protein level and in vitro and in vivo T cell proliferation. Decellularization is associated with a delay in leukocyte infiltration and recellularization promoted cartilage preservation and the recruitment of regulatory T cells. We described a dramatic increase of TGF-β1 in recellularized scaffolds. Moreover, we designed a dual-chamber bioreactor for recellularization of tracheal allografts. Our method allowed for dynamic perfusion seeding, confirmed adherence of two different cell types and achieved higher cell numbers and homogeneous structures compared to traditional static seeding methods. In summary, we have identified and addressed three major limitations for tissue-engineering of long-segment decellularized tracheal scaffolds relating to structural integrity, immunogenicity and recellularization techniques.

Subglottic stenosis, malignancy and traumatic injury to the trachea require surgical resection. When defects are less than 50% of the tracheal length in adults and 1/3 in children, a circumferential resection and primary anastomosis affords excellent results. For longer lesions, on the other hand, there are no currently acceptable solutions leading to patients requiring permanent tracheostomies or palliative treatment. Tracheal replacement approaches with synthetic prosthesis and scaffolds have all led to inflammation, obstruction, mucous build-up and eventual restenosis. Tissue-engineering approaches using recipients’ own stem cells and biologic scaffolds derived from decellularized donor trachea have shown great promise. They have the potential to abrogate the need for immunosuppressive therapy. Our research focuses on three major limitations in this field including the structural integrity, the immunogenicity and the recellularization of donor tracheae. We compared three decellularization protocols, quantified and qualified the extracellular matrix (ECM) components and performed compliance measurements on large circumferential tracheal scaffolds following cyclical decellularization techniques and illustrated significant differences in ECM composition and resultant structural integrity of decellularized tracheal scaffolds depending on the protocol. In addition, we investigated the immunogenicity of decellularized and recellularized tracheal allografts at a protein level and in vitro and in vivo T cell proliferation. Decellularization is associated with a delay in leukocyte infiltration and recellularization promoted cartilage preservation and the recruitment of regulatory T cells. We described a dramatic increase of TGF-β1 in recellularized scaffolds. Moreover, we designed a dual-chamber bioreactor for recellularization of tracheal allografts. Our method allowed for dynamic perfusion seeding, confirmed adherence of two different cell types and achieved higher cell numbers and homogeneous structures compared to traditional static seeding methods. In summary, we have identified and addressed three major limitations for tissue-engineering of long-segment decellularized tracheal scaffolds relating to structural integrity, immunogenicity and recellularization techniques.

A variety of synthetic bone graft materials such as the polymer poly(lactic-co-glycolic acid) (PLGA) have been investigated as alternatives to current tissue based bone graft materials. In this study, efforts have been made to improve the tissue-PLGA interface by immobilizing bone morphogenetic protein-2 (BMP-2) in an oriented manner on scaffolds using covalently immobilized heparin. The results demonstrated a four-fold increase in covalently immobilized heparin compared to non-specific heparin attachment. Furthermore, the scaffolds with covalently attached heparin retained approximately three-fold more BMP-2 than did either scaffolds with no heparin attached or scaffolds with non-specific heparin attachment. The activity of scaffolds with BMP-2 immobilized in various manners was examined using an alkaline phosphatase assay on C3H10T1/2-seeded scaffolds. These results indicated approximately twice the amount of activity with scaffolds that had BMP-2 immobilized with covalently attached heparin than on scaffolds with adsorption of BMP-2 and a three-fold increase in activity when compared to scaffolds that had non-specific heparin attachment as the mechanism for BMP-2 immobilization. These results demonstrated that PLGA with covalently linked heparin has potential to immobilize BMP-2 in a specific orientation that is favorable for cell-receptor binding, leading to the more efficient use of the bone-growth factor.

The limited treatment options available for heart disease patients has lead to increased interest in the development of embryonic stem cell (ESC) therapies to replace heart muscle. The challenges of developing usable ESC therapeutic strategies are associated with the limited ability to obtain a pure, defined population of differentiated cardiomyocytes, and the design of in vivo cell delivery platforms to minimize cardiomyocyte loss. These challenges were addressed in Chapter 2 by designing a cardiomyocyte selectable progenitor cell line that permitted evaluation of a collagen-based scaffold for its ability to sustain stem cell-derived cardiomyocyte function (“A P19 Cardiac Cell Line as a Model for Evaluating Cardiac Tissue Engineering Biomaterials”). P19 cells enriched for cardiomyocytes were viable on a transglutaminase cross-linked collagen scaffold, and maintained their cardiomyocyte contractile phenotype in vitro while growing on the scaffold. The potential for a novel cell-surface marker to purify cardiomyocytes within ESC cultures was evaluated in Chapter 3, “Dihydropyridine Receptor (DHP-R) Surface Marker Enrichment of ES-derived Cardiomyocytes”. DHP-R is demonstrated to be upregulated at the protein and RNA transcript level during cardiomyogenesis. DHP-R positive mouse ES cells were fluorescent activated cell sorted, and the DHP-R positive cultured cells were enriched for cardiomyocytes compared to the DHP-R negative population. Finally, in Chapter 4, mouse ESCs were characterized while growing on a clinically approved collagen I/III-based scaffold modified with the RGD integrin-binding motif, (“Collagen (+RGD and –RGD) scaffolds support cardiomyogenesis after aggregation of mouse embryonic stem cells”). The collagen I/III RGD+ and RGD- scaffolds sustained ESC-derived cardiomyocyte growth and function. Notably, no significant differences in cell survival, cardiac phenotype, and cardiomyocyte function were detected with the addition of the RGD domain to the collagen scaffold. Thus, in summary, these three studies have resulted in the identification of a potential cell surface marker for ESC-derived cardiomyocyte purification, and prove that collagen-based scaffolds can sustain ES-cardiomyocyte growth and function. This has set the framework for further studies that will move the field closer to obtaining a safe and effective delivery strategy for transplanting ESCs onto human hearts.

The ultimate goal of this research was to develop a “self-fitting” shape memory polymer (SMP) scaffold for the repair of craniomaxillofacial (CMF) bone defects. CMF defects may be caused by trauma, tumor removal or congenital abnormalities and represent a major class of bone defects. Their repair with autografts is limited by availability, donor site morbidity and complex surgical procedures. In addition, shaping and positioning of these rigid grafts into irregular defects is difficult. Herein, we have developed SMP scaffolds which soften at T > ~56 °C, allowing them to conformally fit into a bone defect. Upon cooling to body temperature, the scaffold becomes rigid and mechanically locks in place. This research was comprised of four major studies. In the first study, photocrosslinkable acrylated (AcO) SMP macromers containing a poly(ε-caprolactone) (PCL) segment and polydimethylsiloxane (PDMS) segments were synthesized with the general formula: AcO-PCL40-block-PDMSm-block-PCL40-OAc. By varying the PDMS segment length (m), solid SMPs with highly tunable mechanical properties and excellent shape memory abilities were prepared. In the second study, porous SMP scaffolds were fabricated based on AcO-PCL40-block-PDMS37-block-PCL40-OAc via a revised solvent casting particulate leaching (SCPL) method. By tailoring scaffold parameters including salt fusion, macromer concentration and salt size, scaffold properties (e.g. pore features, compressive modulus and shape memory behavior) were tuned. In the third study, porous SMP scaffolds were produced from macromers with variable PDMS segment lengths (m = 0 – 130) via an optimized SCPL method. The impact on pore features, thermal, mechanical, and shape memory properties as well as degradation rates were investigated. In the final study, a bioactive polydopamine coating was applied onto pore surfaces of the SMP scaffold prepared from PCL diacrylate. The thin coating did not affect intrinsic bulk properties of the scaffold. However, the coating significantly increased its bioactivity, giving rise to the formation of “bone-bonding” hydroxyapatite (HAp) when exposed to simulated body fluid (SBF). It was also shown that the coating largely enhanced the scaffold’s capacities to support osteoblasts adhesion, proliferation and osteogenesis. Thus, the polydopamine coating should enhance the performance of the “self-fitting” SMP scaffolds for the repair of bone defects.