<p>The research presented here represents a quest to understand and address limitations in the field of skeletal muscle tissue engineering, with hopes to better understand the factors involved in producing viable engineered skeletal muscle tissue. The driving force behind this research was to address two of the many factors important in muscle cell proliferation and differentiation, toward developing mature and functional bioartificial skeletal muscles (BAMs). Our work focused on understanding the individual effects of mechanical stimulation and microRNAs (miRNAs), as well as the synergistic relationship between the two factors. We hypothesized that (1) myoblast proliferation and differentiation are modulated by mechanical stimulation via temporally regulated miRNAs and that (2) modulating these miRNAs can enhance skeletal muscle function in a 3D tissue-engineered system.</p><p>We first established a BAM system using C2C12 mouse myoblasts in a collagen gel, showing that these cells were able to produce mature sarcomeres when cultured under steady, passive tension for up to 36 days. Staining muscle-specific proteins and electron microscopy showed distinct striations and myofiber organization as early as 6 days, post-differentiation. At 33 days, cultures contained collagen fibers and showed localization of paxillin at the fiber termini, suggesting that myotendinous junctions were forming.</p><p>We then focused on the effects of mechanical stimulation on C2C12 myoblasts in a more simple, 2D system. In particular, we assessed miRNA and muscle-specific gene expression over time and in response to two cyclic stretch regimens using miRNA microarray technology and quantitative real time RT-PCR. Both miRNAs and certain genes, such as SRF and Mef2c, had differential responses to the two regimens. Over-expression and inhibition studies of one muscle-specific miRNA, miR-1, abrogated the stretch response and suggest that a balancing mechanism is in place to avoid large fluctuations in miRNA levels. </p><p>Finally, since miRNA modulation quenched the stretch-mediated response in myoblasts, we chose to examine 3D BAM function when miRNA levels were altered to promote differentiation. Using the same collagen gel model established previously, a muscle-specific miRNA, miR-133, known to promote proliferation, was transiently inhibited (anti-miR-133) to encourage differentiation. Forces in the anti-miR-133 BAMs were, on average, 20% higher over the negative control. Further, myofiber diameters were significantly greater and striations were more organized in the anti-miR-133 BAMs, suggesting that transient, exogenous delivery of miRNAs may be a viable approach to create a more fully differentiated muscle.</p> / Dissertation

Bone grafts are used to treat more than 300,000 fracture patients yearly, as well as patients with congenital defects, bone tumors, and those undergoing spinal fusion. Given the established limitations of autograft and allograft bone, there is a substantial need for bone graft substitutes. Tissue engineering strategies employing the addition of osteogenic cells and/or osteoinductive factors to porous scaffolds represent a promising alternative to traditional bone grafts. While many bone defects are in load-bearing sites, very little is known about the response of bone grafts and their substitutes to mechanical loading, despite vast documentation on the ability of normal bone to adapt to its mechanical environment. The goal of this research was to quantify the effects of controlled in vivo mechanical stimulation on bone graft repair and bone graft substitutes and identify the local stress/strain environment associated with load-induced changes in bone formation. The global hypothesis that cyclic in vivo mechanical loading improves mineralized matrix formation within bone grafts and bone graft substitutes was addressed in this work using orthotopic and ectopic models specifically designed to facilitate modeling of local stresses and strains. In the first study, a bone defect repair model utilizing an orthotopic implant capable of supplying a controlled mechanical stimulus to a trabecular allograft showed a significant reduction in new bone formation with controlled in vivo mechanical loading. Although the reason remains unclear, loading conditions may not have been ideal for increased bone formation or potential micromotion may have influenced the results. A second study demonstrated for the first time that controlled in vivo mechanical stimulation enhances mineralized matrix production on a mesenchymal stem cell-seeded polymeric construct using a novel subcutaneous implant system. In addition, the local stresses and strains associated with this adaptive response were predicted. The novel subcutaneous implant represents technology which may be adapted for the preparation of tissue-engineered bone constructs, capitalizing on the benefits of mechanical loading and a vascularized in vivo environment. Such an approach may produce larger, stronger, and more homogeneous constructs than could be developed in a static culture system subject to diffusional limitations.

Bone tissue engineering strategies are currently being developed as alternative mechanisms to address the clinical demand for bioactive and biomechanical graft material. To date, these efforts have been largely restricted by inadequate supply of committed osteoprogenitor cells and loss of osteoblastic phenotype expression following in vitro culture and expansion. The objective of this thesis research was to address the cell sourcing limitations of tissue-engineered bone grafts through constitutive and sustained overexpression of the osteoblast-specific transcriptional activator Runx2/Cbfa1 in osteogenic marrow-derived stromal cells using retroviral gene delivery. Runx2 overexpression enhanced expression of multiple osteoblastic genes proteins and, more importantly, significantly up-regulated matrix mineralization in both monolayer culture and following cell seeding in 3-D polymeric scaffolds. To evaluate in vivo performance, Runx2-expressing cells were seeded into 3-D constructs and implanted both subcutaneously and in a critical size craniotomy bone defect model. Notably, in vitro pre-culture of Runx2-transduced cell-seeded constructs prior to implantation significantly enhanced their capacity to form mineralized tissue in the subcutaneous space and induce new bone formation in the critical size defect model compared to control cells. The described series of analyses provided a novel combination of tissue and genetic engineering techniques toward the development of a Runx2-modified stromal cell/polymeric scaffold composite tissue-engineered bone graft substitute.

The growth plate is a cartilaginous tissue responsible for the longitudinal growth of long bones. It is a complex tissue composed of chondrocytes whose maturation and proliferation is tightly regulated by a biochemical feedback loop. Injury to this tissue can result in a limb length discrepancy or angular deformity that may lead to life long disability. Given the recent rise in the number of growth plate injuries and the variability in success of current therapies, there is a significant need for a greater understanding of growth plate injury pathology and the development of improved treatment strategies. Cartilage tissue engineering strategies offer an attractive alternative to regenerating growth plate tissue and restoring growth function. Bone marrow-derived stem cells (BMSCs) have been shown to be able to undergo chondrogenic differentiation and in vitro and in vivo and therefore offers an appealing and abundant cell resource for developing tissue engineering strategies for the treatment of growth plate defects. However, the dependence of chondrogenic differentiation and matrix accumulation on monolayer expansion protocols and three-dimensional (3D) culture environment has received little attention. Prior to developing treatment strategies for growth plate injury repair, it is essential to first understand the interconnection between alterations in growth plate morphology and subsequent limb deformities. To that end, we have established a surgical defect model of growth plate injury in Sprague Dawley rats and developed a novel technique to quantitatively monitor growth plate morphology in health and disease using microcomputed tomography (micro-CT) imaging. In an effort to develop a tissue engineering treatment strategy for growth plate injury, the role of monolayer expansion, 3D scaffold, and growth factor regimen in the chondrogenic differentiation of rat BMSCs was also examined. This research study has demonstrated the utility of micro-CT as a non-invasive imaging modality for assessing growth plate injury and repair. This work has also provided an improved understanding of the interrelationship of monolayer expansion, 3D culture environment, and growth factor regimen in BMSC chondrogenic differentiation. Finally, this work suggests that an injectable in situ gelling hydrogel is a feasible method for decreasing limb length discrepancies, however, neither implantation of agarose alone into the defect nor the inclusion of BMSCs fully corrects growth disruption.

Cartilage tissue engineering represents an exciting potential therapy for providing permanent and functional regeneration of healthy cartilage tissues, but these treatment options have yet to be successfully implemented in a clinical setting. One of the primary obstacles for cartilage engineering is obtaining a sufficient supply of cells capable of regenerating a functional cartilage matrix. Mesenchymal progenitors can easily be isolated from multiple tissues, expanded in vitro, and possess a chondrogenic potential, but it remains unclear what types or combinations of signals are required for lineage-specific differentiation and tissue maturation. The overall goal of this dissertation was to investigate how the coordination of biochemical stimuli with cues from mechanical forces and the extracellular matrix regulate the chondrogenesis of bone marrow stromal cells (BMSCs). These studies explored the potential for cyclic tensile loading and chondrogenic factors, TGF-1 and dexamethsone, to promote fibrochondrocyte-specific differentiation of BMSCs. The application of cyclic tensile displacements to cell-seeded fibrin constructs promoted fibrochondrocyte patterns of gene expression and the development of a fibrocartilage-like matrix. These responses were influenced by the specific loading conditions examined and the differentiation state of the BMSCs. Additionally, the roles of integrin adhesion and cytoskeletal organization in BMSC differentiation were examined within engineered hydrogels presenting controlled densities of biomimetic ligands. Adhesion to the arginine-glycine-aspartic acid (RGD) motif inhibited chondrogenesis in a density-dependent manner and was influenced by interactions with the f-actin cytoskeleton. Together, this research provided fundamental insights into the regulatory mechanisms involved in the chondrogenesis of mesenchymal progenitor cells.

The research presented here deals with synthetic materials for application in tissue engineering, primarily poly(ethylene glycol) (PEG) and poly(dimethyl siloxane)star (PDMS)star. Tissue engineering seeks to repair or replace damaged tissue through implantation of cell encapsulated in an artificial scaffold. Cell differentiation and extracellular matrix (ECM) deposition can be influenced through a wide variety of in vitro culture techniques including biochemical stimuli, cell-cell interactions, mechanical conditioning and scaffold physical properties. In order to systematically optimize in vitro conditions for tissue engineering experiments, the individual effects of these different components must be studied. PEG hydrogels are a suitable scaffold for this because of their biocompatibility and biological "blank slate" nature. This dissertation presents data investigating: the effects of glycosaminoglycans (GAGs) as biochemical stimuli on pig vocal fold fibroblasts (PVFfs); the effects of mechanical conditioning and cell-cell interactions on smooth muscle cells (SMCs); and the effects of scaffold physical properties on SMCs. Results show that GAGs influence PVFf behavior and are an important component in scaffold design. Hyaluronic acid (HA) formulations showed similar production in collagen I and III as well as reduced levels of smooth muscle a-actin (SMa-actin), while chondroitin sulfate (CSC) and heparin sulfate showed enriched collagen III environments with enhanced expression of SMa-actin. A physiological flow system was developed to give comprehensive control over in vitro mechanical conditioning on TEVGs. Experiments performed on SMCs involved creating multi-layered TEVGs to mimic natural vascular tissue. Constructs subjected to mechanical conditioning with an endothelial cell (EC) layer showed enhanced expression of SMC differentiation markers calponin h1 and myocardin and enhanced deposition of elastin. Consistent with other studies, EC presence diminished overall collagen production and collagen I, specifically. Novel PDMSstar-PEG hydrogels were studied to investigate the effects of inorganic content on mesenchymal stem cell differentiation for use in TEVGs. Results agree with previous observations showing that a ratio of 5:95 PDMSstar: PEG by weight enhances SMC differentiation markers; however, statistically significant conclusions could not be made. By studying and optimizing in vitro culture conditions including scaffold properties, mechanical conditioning and multi-layered cell-cell interactions, TEVGs can be designed to maximize SMC differentiation and ECM production.

Vocal fold scarring, caused by injury and inflammation, presents significant treatment challenges. Tissue engineering might be a promising treatment for vocal fold restoration or regeneration. It is important to investigate how scaffold properties alter cell behavior instead of screening thousand of materials, which is fundamental knowledge for rational scaffold design. This work studies how tuning only one parameter, mechanical strength of the hydrogel scaffold, influences the extracellular matrix production of encapsulated porcine vocal fold fibroblast (PVFF). PVFF cells were encapsulated by photopolymerization in 10 wt%, 20 wt%, and 30 wt% poly(ethylene glycol) diacrylate (PEGDA) hydrogels (MW 10,000), with the similar biochemical environment and network structure but different mechanical properties. Cell adhesive peptide, RGDS, was grafted into each hydrogel network to mimic a cell adhesive environment. The glycosaminoglycans (GAGs) production per cell increased from 10 wt% to 20 wt%, 30 wt% gels, with an increase in hydrogel stiffness. The collagen production per cell increased from 10 wt% to 20 wt% gels but no further increase occurred with the increasing modulus from 20 wt% to 30 wt% gels. Interestingly, in hydrogels of intermediate modulus (20% PEGDA hydrogels), the highest elastin per cell was observed compared with gels with higher and lower storage modulus after day 30. Histological analysis showed GAGs, collagen and elastin were distributed pericellularly. However, the organization of collagen type I appeared to be influenced by gel mechanical properties, which was confirmed by immunohistological analysis. Furthermore, the immunohistological analysis showed that the phenotype of PVFF is regulated by the stiffness of the PEG hydrogel. This study demonstrates that different levels of VFF ECM formation may be achieved by varying the mechanical properties of PEG hydrogels and validates a systematic and controlled platform for further research of cell-biomaterials interaction.

In the US alone, more than 500,000 coronary artery bypass procedures are performed annually. Tissue engineering shows the potential to construct functional grafts to overcome the limited availability of autologous saphenous veins, relatively poor elasticity and low compliance of synthetic materials (mainly Dacron and polytetrafluoroethylene). In order to meet the low modulus associate with myocyte differentiation, the high suture retention and an ultimate tensile strength (UTS) sufficient to withstand implantation and peak physiological stresses, we designed and characterized a multi-component scaffold comprised of polyurethane electrospun mesh layers bonded together by a fibrin hydrogel matrix. We have demonstrated this composite construct retains the high tensile strength and suture retention strength but displays a "J-shaped" mechanical response similar to that of native coronary artery. To improve our design, poly(ethylene glycol) diacrylate based hydrogel system was utilized as a blank slate to study the phenotypic regulation by cell material interactions. Fibrinogen, fibronectin, laminin and collagen type IV were incorporated into the hydrogel to mimic the stimuli from extracellular matrix (ECM) proteins. Surprisingly, no significant effect was detected on induction of smooth muscle cell (SMC) differentiation marker expression, activation of mitogen-activated protein (MAP) kinases pathway, or alteration of surface integrin expression profile. However, fibronectin showed repression of undesired phenotypes in SMC differentiation. In contrast to ECM proteins, glycosaminoglycans (GAGs) showed more influence on regulating SMC phenotype. By using a scaffold environment intended to be mimetic of early atherosclerosis, the impact of GAG identity on SMC foam cell formation was explored. We focused on chondroitin sulfate C (CSC), dermatan sulfate (DS), and an intermediate molecular weight hyaluronan (HA_IMW, ~400 kDa), the levels and/or distribution of which are significantly altered in atherosclerosis. CSC and DS hydrogels were associated with greater SMC phagocytosis of apolipoprotein B than HA_IMW gels. However, only SMCs in DS constructs maintained increased expression of adipocyte marker A-FABP relative to HA_IMW gels over 35 days of culture. Combined, our results suggested interesting roles for fibronectin and HA_IMW in repression of undesired phenotypes in SMC differentiation, which could give insights into rational design of novel biomaterials for vascular tissue engineering applications.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 157-172).

Due to the limited intrinsic healing ability of mature cartilage tissue, stem cell therapies offer the potential to restore cartilage lost due to trauma or arthritis. Mesenchymal stem cells (MSCs) are a promising cell source due to their ability to differentiate into various adult tissues under specific biochemical and physical cues. Current MSC chondrogenic differentiation strategies employ large pellets, however, we have previously developed a high-throughput technique to form small MSC aggregates (500-1,000 cells) that may reduce diffusion barriers while maintaining a multicellular structure that is analogous to cartilaginous condensations. The objective of this study was to examine the effects on chondrogenesis of incorporating chondroitin sulfate methacrylate (CSMA) microparticles (MPs) within these small MSC spheroids when cultured in the presence of transforming growth factor-β1 (TGF-β1) over 21 days. Spheroids +MP induced earlier increases in collagen II and aggrecan gene expression (chondrogenic markers) than spheroids -MP, although no large differences in immunostaining for these matrix molecules were observed by day 21. Collagen I and X was also detected in the ECM of all spheroids by immunostaining. Interestingly, histology revealed that CSMA MPs clustered together near the center of the MSC spheroids and induced circumferential alignment of cells and ECM around the material core. Because chondrogenesis was not hindered by the presence of CSMA MPs, this study demonstrates the utility of this culture system to further examine the effects of matrix molecules on MSC phenotype, as well as potentially direct differentiation in a more spatially controlled manner that better mimics the architecture of specific target tissues.