Cartilage tissue engineering is an emerging treatment option for osteoarthritis and trauma related joint injuries. A continuing challenge for cartilage tissue engineering is increasing construct extracellular matrix production and material properties. Shear stress and oxygen tension play an important role in tissue engineering of cartilage. In this select stimulatory conditions using combinations of shear stress and oxygen tension have been used to enhance the construct extracellular matrix deposition and material properties. Additionally, a perfusion concentric cylinder bioreactor has been developed to incorporate multiple fluid flow regimes through the construct. This thesis attempts to elucidate the effect of shear stress and biochemical conditions on cartilage development in vitro to provide functional tissue engineered constructs.

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An increase in coronary disease prevalence and mortality highlights the growing need for therapies to treat atherosclerotic vessels. While current bypass procedures utilize autologous vessels for small caliber grafts, there is a big push towards the use of engineered tissues to bypass diseased portions of arteries. Cardiovascular tissue engineering is the emerging discipline that aims to create a functional substitute. Ideally, a tissue engineered blood vessel would possess the relevant cells and matrix proteins that interact in a physiologic manner and will respond to the environmental cues of the host. A particular obstacle to achieving appropriate vessel structure is the inclusion of elastin in a tissue engineered media equivalent. Rat arterial smooth muscle cells that were retrovirally mediated to overexpress versican V3 have been shown to have an enhanced expression of tropoelastin in vitro as well as in injury models. The unique tropoelastin expression by these adult cells was studied in the context of tissue engineered media equivalents. Changes to the extracellular matrix architecture and composition, stimulation with medium additives, and cyclic distension, were shown to increase tropoelastin synthesis in V3 versican overexpressing cells. This study not only expanded the characterization of V3 versican overexpressing smooth muscle cells, it also explored the novel use of these cells as a tropoelastin source in a tissue engineered media equivalent.

Acetylcholine is a neurotransmitter that regulates neurite branching, induces neurite outgrowth, and synapse formation. Because of its various roles in neuronal activities, acetylcholine-based materials may also be useful in nerve repair. We present a series of biodegradable polymers with varying concentrations of acetylcholine-like motifs. We hypothesize that neurite sprouting and extension can be enhanced by using materials to present biochemical and physical cues. Acetylcholine-like motifs were incorporated by the polycondensation of diglycidyl sebacate, aminoethyl acetate, and leucine ethyl ester, which permitted control over acetylcholine motif concentration. Interactions between the polymers and neurons were characterized using rat dorsal root ganglia explants (DRG). We screened the potential application of these materials in nerve tissue engineering using the following criteria: 1) neurite sprouting, 2) neurite length, and 3) distribution of the neurite lengths. The ability of DRG to sprout neurites was influenced by the concentration of acetylcholine motifs of the polymer. Addition of acetylcholine receptor antagonists to DRG cultured on the polymers significantly decreased neurite sprouting, suggesting acetylcholine receptors mediate sprouting on the polymers. Future studies may examine how neurons on acetylcholine-based polymers exhibit changes in downstream signaling events and cell excitability that are associated with receptor activation. In preparation for testing the acetylcholine-based polymers in vivo, porous scaffolds with longitudinally oriented channels were fabricated using fiber templating and salt leaching. Micro computed tomography, scanning electron microscopy, and cryo-sectioning revealed the presence of longitudinally oriented channels. Channel volume and average pore size of the scaffolds were controlled by the number of fibers and salt fusion time. Future studies may involve testing the effect of acetylcholine-motifs by coating polymers onto such scaffolds or assessing the effect of the scaffold's dimensional properties on nerve regeneration.

Often caused by trauma or tumor removal, large bone defects frequently result in delayed or non-union. The current gold standard for treatment is autograft. However, due to limitations, such as the size and location of the defect, these cannot always be utilized. A common alternative to autograft is the use of BMP-2 with a collagen scaffold, however, this treatment is limited by numerous side effects. In recent years, genetic materials such as microRNAs (miRNAs) have offered possible alternative therapies. MiRNAs are small non-coding RNA molecules that generally range from 20-24 nucleotides, serve as repressors of gene expression, and are involved in a wide range of biological activities. Their functions can be inhibited or upregulated by delivering antagomiRs or miRNA mimics, respectively. Two miRNAs involved in bone regeneration are of particular interest in this study, miR-26a and miR-133a. Previous studies demonstrated miR-26a is involved in osteoblastic differentiation and miR-133 is a negative regulator of Runx2, the key transcription factor of osteogenesis. Therefore, we hypothesized the delivery of miR-26a and antagomiR-133a will increase bone formation in critical-sized bone defects. The research outlined in this thesis investigates the healing efficacy of these genetic cargos delivered by novel peptide nanoparticles, RALA, soak loaded into a collagen-hydroxyapatite scaffold. vi To test this hypothesis, scaffolds soak-loaded with RALA/microRNA were implanted into calvarial defects in Wistar Rats. The defects were then left to heal for 8 weeks and were longitudinally monitored using micro-computed tomography (μCT). At 8 weeks, rats were euthanized and calvaria tissue was harvested for histological analysis. The μCT data demonstrates that the scaffolds with microRNAs show promise as a novel therapy for bone defects. The histological analysis showed the treatments promote healing by normal bone formation activity. While there was no statistical difference (p ≥ 0.11276) between groups for the healing variables, this is believed to be due to the small sample size and low power (60%). All of the miRNA treatment groups had samples with considerably higher healing responses than the gene-free group. In conclusion, the findings of this study support the use of this cell-free implant system as a potential novel clinical therapy, as an alternative to bone grafting, for treating large bone defects.

Tissue engineering is an emerging field that offers novel and unmatched potential medical therapies and treatments. While the vast aim of tissue engineering endeavors is to provide clinically implantable constructs, secondary applications have been developed to utilize tissue-engineered constructs for in-vitro evaluation of devices and therapies. Specifically, in-vitro blood vessel mimics (BVM) have been developed to create a bench-top blood vessel model using human cells that can be used to test and evaluate vascular disease treatments and intravascular devices. Previous BVM work has used fat derived human microvascular endothelial cells (EC) sodded on an ePTFE scaffold. To create a more physiologically accurate model, a dual layer of large vessel endothelial and smooth muscle cells (SMC) on an ePTFE tube is investigated throughout this thesis. Human umbilical vein endothelial cells (HUVEC) and human umbilical vein smooth muscle cells (HUVSMC) were chosen as the large vessel cell types and cultivated according to standard procedures. Before dual sodding, sodding density experiments with HUVSMC were performed to determine the number of cells required to create a confluent cell layer. HUVSMC sodded by trans-luminal pressure at densities ranging from 3.5x10^5 cells/cm^2 to 1.0x10^6 cells/cm^2 were run for one day to observe luminal coverage. After determining the desirable range for HUVSMC sodding, HUVSMC experiments with 5.0x10^5 cells/cm^2 and 7.5x10^5 cells/cm^2 were run over seven days to evaluate progression of the graft over time. Histology and SEM methods were used for analysis. A HUVEC study was next conducted over 7 days to confirm that the large vessel endothelial cell could be sodded and sustained on ePTFE in-vitro. Next, dual sodding was performed by pressure sodding HUVSMC at 7.5x10^5 cells/cm^2 followed by trans-luminal flow for 30 minutes. HUVECs were subsequently trans-luminally pressure sodded at 5.0x10^5 cells/cm^2 followed by an additional 30 minutes of trans-luminal flow; perfusion flow began following the final 30 minutes of trans-luminal flow. Experiments for the dual layered grafts were run for both one and seven days to evaluate and develop the dual sodding protocol as well as observe the co-culture over time. Analysis of the dual layered grafts was performed by SEM, histology, and fluorescence microscopy. HUVECs were incubated with Cell Tracker™ prior to dual sodding and both cell types with bisbenzimide after graft harvest to attempt to distinguish between cell types. Results from the thesis illustrate that large vessel smooth muscle and endothelial cells can be sodded onto ePTFE scaffolds and sustained within the in-vitro BVM system for up to 7 days. Furthermore, cost analysis demonstrates that the addition of a smooth muscle cell layer adds minimal costs to the BVM system. In conclusion, the studies contained within this thesis culminate in a protocol for the dual sodding of smooth muscle and endothelial cells with the aim of creating a physiologically representative co-culture blood vessel mimic.

Osteoarthritis is a joint disease characterized by the breakdown of articular cartilage. The field of tissue engineering is interested in developing methods to produce biological alternatives to current orthopedic procedures. Lubricin is a molecule which is important in the proper lubrication of articular cartilage. It is a challenge in the field of tissue engineering to produce cartilage with sufficient lubricin expression. Developing a reporter cell for lubricin allowed for a more efficient investigation of the conditions wh­­­ich may influence its expression. By comparing "optimized" and traditional media solutions, it was determined that the use of a previously reported type II collagen optimized media would negatively affect the expression of lubricin. This information indicates the need to further evaluate the conditions which are conducive to producing cartilage with both sufficient types of type II collagen and lubricin.

Tissue Engineering A Blood Vessel Mimic While Monitoring Contamination Through Sterility Assurance Testing Navid Djassemi Tissue engineering blood vessel mimics has been proposed as a method to analyze the endothelial cell response to intravascular devices that are used in today’s clinical settings for the treatment of cardiovascular disease. Thus, the development of in vitro blood vessel mimics (BVMs) in Cal Poly’s Tissue Engineering Lab has introduced the possibility of assessing the characteristics of cellular response to past, present, and future intravascular devices that aim at treating coronary artery disease. This thesis aimed at improving the methods and procedures utilized in the BVM model. Initial aspects of this project focused on using an expanded polytetrafluoroethylene (ePTFE) scaffold in conjunction with human endothelial cells to replicate the innermost intimal layer of a blood vessel. Human umbilical vein endothelial cells (HUVECs) were pressure sodded onto ePTFE scaffolds through cell sodding techniques in an attempt to effectively and consistently replicate and assess the intimal layer. Through each study ePTFE grafts were subjected to different culture times and steady flow rates to observe and compare the differences in the endothelial cell deposition. Results were inconsistent, although moderate cell adhesion was noted throughout each of the BVM setups. Each study exhibited a range of cell sodding density rates. In the second phase of the thesis, contamination assessment protocols were implemented in the BVM lab. The intent of this part of the project was to assess the relative sterility in the cell culture lab, a critical component involved with the success or hindrance of cell and tissue cultures. Using microbiological validated methods, microbiological tests were conducted to examine the levels of microbial growth in and around the tissue engineering lab. Results were tracked over a two month period in the lab with several observations of aerobic microorganism growth on various counter and lab surfaces. Higher growth trends were found on surfaces outside the cell culture lab, in the general TE lab area. These findings were used to provide overall suggestions on tracking microbes for long-term durations in ongoing BVM setups to directly improve the overall sterility assurance of the studies. As the project reached its conclusion a look back at all the BVM setups and contamination assessments lead to a few suggestions for improving aseptic techniques within the TE lab, such as monitoring microbial growth in the culture processes, creating limit specifications, and creating a standardized way to regulate quality control within the lab environment. Furthermore, as the development BVM evolves, the findings from this report can be used with related research for improving the culture conditions of various BVM studies.

In the past decade, advances in tissue engineering have allowed researchers to fabricate simple tissues. However, the process of creating these native tissues is a time consuming and inefficient process. A scaffold must first be fabricated then exposed to a sea of cells in the hopes of seeding. Furthermore, even though cells may have attached, more time must be spent in order to allow the cells to migrate to their ideal locations. To deal with this problem, researchers have investigated whether rapid prototyping principals could be adapted to facilitate the cell seeding process by placing cells in their respective locations during scaffold fabrication. The goal for this thesis was to establish the foundation for a cell-compatible printer that, in the future, could fabricate pre-seeded scaffolds. This task included implementing changes to a commercial solenoid-based inkjet system that would allow cells to be loaded into the printer in a sterile fashion. In addition, protocols had to be designed with system limitations in mind. An initial test with the designed system showed a majority of cell viability percentages above 90%. If additional tests confirm this possibility, the system should be further modified to provide cells with a proper culturing environment. Furthermore, additional research would need to be performed in order to determine whether scaffolding materials can be dispensed through the system to fabricate scaffolds.

PREPARATION AND CHARACTERIZATION OF ELECTROSPUN POLY(D,L-LACTIDE-CO-GLYCOLIDE) SCAFFFOLDS FOR VASCULAR TISSUE ENGINEERING AND THE ADVANCEMENT OF AN IN VITRO BLOOD VESSEL MIMIC Tiffany Richelle Peña Currently, an estimated 1 in every 3 adult Americans are affected by one or more cardiovascular complications. The most common complication is coronary artery disease, specifically atherosclerosis. Outcomes of balloon angioplasty treatments have been significantly improved with the addition of drug eluting stents to the process. Although both bare metal and drug eluting stents have greatly increased the effectiveness of angioplasty and decreased the occurrence of restenosis, several complications still exist. For this reason, the stent industry is continually advancing toward better stent and drug-eluting designs, deployment methods, and adjuvant drug therapies, necessitating fast, reliable pre-clinical test methods. Recently, advancements in tissue engineering have led to the development of an in vitro blood vessel mimic (BVM) and the feasibility of evaluating cellular response to intravascular device implantation has been demonstrated. There are several physiological and scalability limitations of the current BVM model that must be addressed before effective use of the model can be initiated. The limiting aspect addressed in this thesis is the use of expanded poly(tetrafluorethylene) [ePTFE] scaffolding for the development of the BVM. There are several disadvantages and limitations to ePTFE including high cost and non-native mechanical properties. The ability to produce and tailor scaffolds in-house would greatly impact the scalability, cost effectiveness, and control over scaffold properties for BVM optimization. Also, in-house fabrication will open up further avenues of research into optimum scaffold design for better cellular responses when cultured in vitro. Electrospinning is a relatively simple and economical method of creating tissue engineering constructs with micro-architecture similar to the native extracellular matrix. Based on the clinical problem and the potential for the BVM, the aim of this thesis is to employ electrospinning for the development of poly(D,L-lactide-co-glycolide) [PLGA] vascular scaffolds as a replacement to ePTFE for the BVM. After primary literature review, PLGA was determined an advantageous polymer for tissue engineering vascular scaffolds and electrospinning based on evidence of adequate endothelial cell attachment, mechanical properties similar to the native vessels, controlled degradation, and good biocompatibility. The first phase of this thesis was to develop an acceptable protocol for the fabrication of electrospun PLGA scaffolds by varying solution concentration, flow rate and applied voltage. Electrospun solutions of 15 wt% PLGA in CHCl3 resulted in continuous un-beaded fibers of 5-6 microns and tensile properties (3-5 MPa) similar to the native vessel. The optimum protocol for electrospinning 15 wt% PLGA incorporated a flow rate of 5.5 ml/hr and an applied voltage of 12,000 V. In the second phase of this thesis, final protocol PLGA scaffolds were cultured in vitro with human umbilical vein endothelial cells (HUVECs) up to 6 days. Fluorescent microscopy and SEM analysis suggest the porous nature of the scaffolds was conducive to sub-luminal cellular penetration. Although results were not optimal for developing an endothelium for the ideal BVM design, the potential of using electrospinning for in-house production of scaffolds for tissue engineering was established. Further optimization of the electrospinning protocol to develop nano-sized structural features could enhance the ability to form an intimal lining of endothelial cells for the next generation BVM design.