Thesis (M.S.)--University of Michigan, 2001. / Includes bibliographical references.

Due to the inability of intra-articular injuries to adequately self-heal, current therapies are largely focused on palliative care and restoration of joint function rather than true regeneration. Subsequently tissue engineering of chondral and osteochondral tissue constructs has emerged as a promising strategy for the repair of partial and full-thickness intra-articular defects. Unfortunately, the fabrication of large tissue constructs is plagued by poor nutrient transport to the interior of the tissue resulting in poor tissue growth and necrosis. Further, for the specific case of osteochondral grafts, the presence of two distinct tissue types offers additional challenges related to cell sourcing, scaffolding strategies, and bioprocessing. To overcome these constraints, this dissertation was focused on the development and validation of a microfluidic hydrogel platform which reduces nutrient transport limitations within an engineered tissue construct through a serpentine microfluidic network embedded within the developing tissue. To this end, a microfluidic hydrogel was designed to meet the nutrition requirements of a developing tissue and validated through the cultivation of chondral tissue constructs of clinically relevant thicknesses. Additionally, optimal bioprocessing conditions with respect to morphogen delivery and hydrodynamic loading were pursued for the production of bony and cartilaginous tissue from bone marrow derived mesenchymal stem cells. Finally, the optimal bioprocessing conditions were implemented within MSC laden microfluidic hydrogels to spatially engineer the matrix composition of a biphasic osteochondral graft through directed differentiation.

published_or_final_version / Mechanical Engineering / Master / Master of Philosophy

No description available.

In vitro grown cartilage is functionally inferior to native tissue, and improvements in its quality should be attempted so it can be used therapeutically. In these studies we investigated the effects of cell shape on tissue quality through alteration of substrate geometry and application of mechanical stimuli. Articular chondrocytes were isolated and cultured on the surface Ti-6Al-4V substrates with various geometries. When cultured on fully porous titanium alloy substrates, chondrocyte spreading was enhanced over those grown on substrates with solid bases. Chondrocytes which remained round did not synthesize significant amounts of matrix and were thus unable to form cartilaginous tissue. In contrast, chondrocytes which were directed to spread to a limited amount, resulting in a polygonal morphology, accumulated significantly more matrix molecules and in time formed cartilage-like tissue. Computational fluid dynamics analyses demonstrated that cells on fully porous substrates experience time-dependent shear stresses that differ from those experienced by cells on substrates with solid bases where media flow-through is restricted. Integrin-blocking experiments revealed that integrins are important regulators of cell shape, and appeared to influence the accumulation of collagen and proteoglycans by chondrocytes. Furthermore, compressive mechanical stimulation induced a rapid, transient increase in chondrocyte spreading by 10 minutes, followed by a retraction to pre-stimulated size within 6 hours. This has been shown to be associated with increased accumulation of newly synthesized proteoglycans. Blocking the α5β1 integrin, or its β1 subunit, inhibited cell spreading and resulted in a partial inhibition of compression-induced increases in matrix accumulation, thereby substantiating the role of β1 integrins in this process. These results suggest that both fluid induced shear forces and compressive forces regulate chondrocyte matrix accumulation by altering cell morphology, which is mediated by integrins. Identifying the molecular mechanisms that influence chondrocyte shape and thus tissue formation may ultimately lead to the development of a tissue that more closely resembles native articular cartilage.

Coronary heart disease is a leading cause of death among Americans for which coronary artery bypass graft (CABG) surgery is a standard surgical treatment. The success of CABG surgery is impaired by the compliance mismatch between vascular grafts and native vessels. Tissue engineered vascular grafts (TEVGs) have the potential to be compliance matched and thereby reduce the risk of graft failure. Glutaraldehyde (GLUT) vapor-crosslinked gelatin/fibrinogen constructs were fabricated and mechanically tested in a previous study by our research group at 2, 8, and 24 hours of GLUT vapor exposure. Constructs electrospun with tropoelastin in addition to gelatin and fibrinogen fibers were also fabricated and tested for the same amounts of GLUT vapor exposure. The current study details a computational method that was developed to predict the material properties of our constructs for crosslinking times between 2 and 24 hours by interpolation and regression of the 2, 8, and 24 hour crosslinking time data. Matlab and Abaqus were used to determine the optimal combination of fabrication parameters to produce compliance matched constructs. The validity of the method was first tested on a 16 hour crosslinked gelatin/fibrinogen construct of 130μm thickness. The predicted compliance was 0.00059 mmHg-1 while the experimentally determined compliance was 0.00065 mmHg-1, a relative difference of 9.2%. Prior data in our laboratory has shown the compliance of the left anterior descending porcine coronary (LADC) artery to be 0.00071 ± 0.0003 mmHg-1. The optimization algorithm predicts that a 258μm thick construct that is GLUT vapor crosslinked for 8.1 hours would match LADC compliance. The algorithm was expanded to predict the compliance of constructs consisting of alternating layers of tropoelastin/gelatin/fibrinogen and gelatin/fibrinogen. A four layered graft was designed and fabricated using this optimization routine. The layered construct was found to have a compliance of 0.00051 mmHg-1 while the predicted compliance was 0.00061 mmHg-1, a difference of 16%. This is a promising method for matching the compliance of our TEVGs with the native tissue of various specimens.

The development of bone regeneration materials to support new bone formation is an active field of research. This report describes the development and characterization of a novel composite scaffold made of a chitosan-alginate co-polymer hydrogel matrix and eggshell (ES) particles. Scaffolds with ES particles or with nanotextured ES (NTES) particles following treatment with phosphoric acid were compared to scaffolds without particles. The scaffolds with particles exhibited a higher porosity and a larger median pore size. Their mechanical strength remained low, but both scaffold types were more resistant to deformation following compression than the scaffolds without particles. The osteogenic potential of the scaffolds was then evaluated with human bone-marrow derived mesenchymal stem cells (MSCs) from four different donors. Results showed that the inclusion of ES or NTES particles significantly increased MSC adherence and viability, as well as alkaline phosphatase activity in the scaffolds. A change of cell morphology and a small, although not statistically significant, increase of osteogenic protein expression (RUNX2 and osteopontin) were also observed at later time points (days 14 and 21). Overall, this research highlights the potential of ES for bone regeneration applications, opening the door for a high-value repurposing of a current industrial waste product.

Der Ersatz von Knochengewebe durch die Methode des Tissue Engineerings stellt eine viel versprechende Alternative zu konventionellen Therapieformen dar. Jedoch müssen die bisherigen Kulturbedingungen verbessert werden, um das Differenzierungsverhalten von Zellen optimal steuern zu können. Dabei spielt nicht nur die Wahl eines geeigneten Scaffolds und der zu verwendenden Zellen, sondern auch die des Kultursystems eine entscheidende Rolle. In einem dynamischen Kultursystem zirkuliert Medium und bietet gegenüber einem statischen Kultursystem veränderte Bedingungen bezüglich Nährstoffversorgung und Stimulation durch Flüssigkeitsscherstress. Um die Einflüsse der veränderten Bedingungen zu analysieren, wird in dieser Arbeit ein dynamisches Kultursystem etabliert. Dazu werden Calciumphosphat(CaP)-Scaffolds mit dem 3D Powder Printing System gedruckt und mit Zellen der Osteosarkomzelllinie MG63 oder der Fibroblastenzelllinie L-929 besiedelt. In 17 Versuchsreihen werden die zellbesiedelten Scaffolds bei unterschiedlichen Fließgeschwindigkeiten und über unterschiedliche Kultivierungszeiträume kontinuierlich perfundiert. Anhand der Wachstumsparameter Zellzahl und Zellviabiltät, sowie der Morphologie und räumlichen Verteilung der Zellen werden die Qualitäten der Kultursysteme untersucht und mit statischen Kultursystemen verglichen. Die mit dem 3D Powder Printing System gedruckten Scaffolds erweisen sich als geeignet: Nach 6-tägiger Kultur können unter dem Rasterelektronenmikroskop auf den CaP-Scaffolds eine reichliche Zellbesiedelung mit morphologisch gesunden Zellen, die in das Porensystem hineinwachsen, beobachtet werden. Bei beiden Zelllinien nehmen in beiden Kultursystemen die Wachstumsparameter über einen 6-tägigen Kultivierungszeitraum stetig zu und eine Langzeitkultur über 30 Tage kann in beiden Kultursystemen am Leben erhalten werden. Die kontinuierliche Perfusion in einem dynamischen Kultursystem wirkt sich auf das Zellwachstum günstig aus. Im Vergleich von dynamischen zu statischem Kultursystem über einen 6-tägigen Kultivierungszeitraum wachsen beide Zelllinien im dynamischen Kultursystem besser. Dabei spielt die Fließgeschwindigkeit im dynamischen Kultursystem auf die verbesserte Nährstoffversorgung und Stimulation durch Flüssigkeitsscherstress eine Rolle. Außerdem ist zu beachten, dass der Einfluss der Fließgeschwindigkeit des Mediums auf die einzelnen Scaffolds innerhalb des Kulturcontainers unterschiedlich ist. Dies hängt vom Strömungsprofil im Container ab und macht sich durch eine erhöhte Standardabweichung der Messwerte gegenüber der statischen Kultur bemerkbar. / The replacement of bone tissue by the method of tissue engineering represents a promising alternative to conventional forms of therapy. However, current culture conditions have to be optimized in order to control the differentiation behavior of cells. In this context, the choice of the appropriate scaffolds and cells as well as of a suitable culture system play a crucial role. In contrast to static culture systems, medium circulates in a dynamic culture system which offers changed conditions regarding nutrition and stimulation by fluid induced shear stress. To analyze the effects of changed conditions, a dynamic culture system is established in this work. For this purpose, calcium phosphate (CaP) scaffolds printed by the 3D Powder Printing System were populated with cells of the MG63 osteosarcoma cell line or of the fibroblast cell line L-929. In 17 experiments, the cultured scaffolds were perfused continuously at different flow rates and for different cultivation periods. Based on the growth parameters cell number and cell viability, as well as on the morphology and the spatial distribution of the cells, the qualities of the dynamic culture systems were compared to static culture systems. The scaffolds printed by the 3D Powder Printing System proved to be suitable: After a 6-day culture period, the CaP scaffolds showed an abundant cell colonization with morphologically healthy cells growing into the pore system which was observed under a scanning electron microscope. Using both cell lines in both culture systems, the growth parameters increased continuosly during a 6-day cultivation period and it was possible to keep a long-term culture over 30 days in both culture systems alive. The continuous perfusion in a dynamic culture system has a favorable effect on cell growth. In comparison of dynamic to static culture systems over a 6-day culture period, both cell lines grew better in the dynamic culture system. Here the flow rate in the dynamic culture system plays a major role controlling the improved nutrition and stimulation by fluid induced shear stress. Furthermore, the influence of the flow rate of the medium on the individual scaffolds within the culture container varies for the different scaffold positions. This depends on the flow profile of the container and is indicated by an increased standard deviation of the measured values when compared to the static culture.

Scaffold materials play an essential role in tissue engineering field due to its function of accommodate and guide cell proliferation. In this study, I investigated different types of crosslinked protein membranes that can be produced in microfluidic channels and a number of various types of PLGA electrospun composite nanofiber scaffold to examine their potentials as scaffold materials in tissue engineering. A simplified fabrication technique has been developed to produce a large surface area of crosslinked protein membranes to fulfill the purpose of cell culture experiments. Bovine serum albumin is used along with two acyl chloride crosslinkers, i.e. TCL and IDCL, respectively to accomplish the cross-linking. On the other hand, PLGA is dissolved in HFIP and enhanced with silk fibroin and carbon nanotubes to make composite electrospun materials. The morphology, physicochemical properties and biocompatibility of the membranes are studied. The biocompatibility of the membranes is investigated using cell proliferation of the PC12, ADSCs and neurons cultured on the membranes. Our results show that compared to crosslinked protein membranes, the electrospun materials are easier to prepare, less toxic and more suitable for mass production. Moreover, the electrospun materials are seen to have better biocompatibility in our cell culture study. Furthermore, the composite electrospun materials with high CNTs concentrations demonstrate positive effects on the proliferation of neurons.

Strong, durable terminal regions that can be easily handled by researchers and surgeons are a key factor in the successful fabrication of tissue engineered blood vessels (TEBV). The goal of this study was to fabricate and characterize electrospun cuffs made of poly-caprolactone (PCL) combined with gelatin that reinforce and strengthen each end of cell-derived vascular tissue tubes. PCL is ideal for vascular tissue engineering applications due to its mechanical properties; however, PCL alone does not support cell attachment. Therefore, we introduced gelatin, a natural matrix-derived protein, into the electrospun material to promote cell adhesion. This work compared the effects of two different methods for introducing gelatin into the PCL materials: gelatin coating and gelatin co-electrospinning. Porosity, pore size, fiber diameter, and mechanical properties of the electrospun materials were measured in order to compare the features of gelatin PCL composites that have the greatest impact on cellular infiltration. Porosity was quantified by liquid intrusion, fiber diameter and pore size were measured using scanning electron microscopy, and tensile mechanical testing was used to evaluate strength, elastic modulus, and extensibility. Attachment and outgrowth of smooth muscle cells onto cuff materials was measured to evaluate differences in cellular interactions between materials by using a metabolic attachment assay and a cellular outgrowth assay. Finally, cuffs were fused with totally cell-derived TEBV and the integration of cuffs with tissue was evaluated by longitudinal pull to failure testing and histological analysis. Overall, these cuffs were shown to be able to add length and increase strength to the ends of TEBV for tube cannulation and manipulation during in vitro culture. In particular, PCL:gelatin cospun cuffs were shown to improve cellular attachment and cuff fusion compared to pure PCL cuffs, while still increasing the strength of the TEBV terminal ends.