For clinical translation of a pancreatic substitute, long-term storage is essential, and cryopreservation is a promising means to achieve this goal. The two main cryopreservation methods are conventional freezing and vitrification, or ice-free cryopreservation. However, as both methods have their potential drawbacks for cryopreservation of a pancreatic substitute, they must be systematically evaluated in order to determine the appropriate method of cryopreservation. Furthermore, previous studies have indicated benefits to encapsulation in 3-D adhesive environments for pancreatic substitutes and that adhesion affects cell response to cryopreservation. Thus, the overall goal of this thesis was to investigate cryopreservation effects on model pancreatic substitutes consisting of cells encapsulated in non-adhesive and adhesive 3-D alginate hydrogels. Murine insulinoma betaTC-tet cells encapsulated in unmodified alginate hydrogels were chosen as the model pancreatic substitute in a non-adhesive 3-D environment. Murine myoblast C2C12 cells, stably transfected to secrete insulin, encapsulated in partially oxidized, RGD-modified alginate hydrogels were chosen as the model pancreatic substitute in a 3-D adhesive environment. With respect to cryopreservation effects on intermediary metabolism of betaTC-tet cells encapsulated in unmodified alginate, results indicate that relative carbon flow through the tricarboxylic acid cycle pathways examined is unaffected by cryopreservation. Additionally, insulin secretory function is maintained in Frozen constructs. However, vitrification by a cryopreservation cocktail referred to as DPS causes impairment in insulin secretion from encapsulated betaTC-tet cells, possibly due to a defect in late-stage insulin secretion. Results from Stable C2C12 cells encapsulated in RGD vs. RGE-alginate indicate that up to one day post-warming, cell-matrix interactions do not affect cellular response to cryopreservation after vitrification or freezing. Although there are differences in metabolic activity and insulin secretion immediately post-warming for DPS-vitrified RGD-encapsulated Stable C2C12 cells relative to Fresh controls, metabolic activity and insulin secretion are maintained at all time points assayed for Frozen constructs. Overall, due to results comparable to Fresh controls and simplicity of procedure, conventional freezing is appropriate for cryopreservation of betaTC-tet cells encapsulated in unmodified alginate or Stable C2C12 cells encapsulated in partially oxidized, RGD-modified alginate.

Currently growth plate fractures account for nearly 18.5% of fractures in children and can lead to stunted bone growth or angular deformation. If the body is unable to heal itself a bony bar forms, preventing normal bone growth. Clinical treatment involves removing the bony bar and replacing it with a filler substance, which causes poor results 60% of the time. Using primarily poly(lactic-co-glycolic acid) (PLGA) as the scaffold material, the goal was to develop an implant that would support to the implant site, allow for cell ingrowth, and degrade away over time. Porous scaffolds were fabricated from PLGA microspheres using the salt leaching method. The first part of this work investigated the effect of sintering the microspheres by studying the mechanical properties, degradation and morphology and their potential applications for hard and soft tissue implants. Growth factor or drugs can be encapsulated into PLGA microspheres, which was the second part of this work. Encapsulated insulin-like growth factor I (IGF-I) was able to withstand the scaffold fabrication process without compromising it’s bioactivity and promoted cell proliferation. The next part of this work experimented with the addition of a hydrogel porogen. Porogen particles were made using a quick degrading poly(beta-amino ester) (PBAE) hydrogel and loaded with ketoprofen. The addition of the porogen creates a dual drug-releasing scaffold with a localized delivery system. The final step of this work involved animal studies to determine the effectiveness of the scaffolds in growth plate regeneration and how they compare to the current clinical treatment option. Gross observation, microCT analysis, angular measurement of bone growth and histological methods were employed to evaluate the scaffolds. The goal was to develop a versatile scaffold that could be used for a wide range of tissue engineering applications. The mechanical properties, degradation profiles and drug delivery capabilities can be all tailored to meet the specific needs of an implant site. One specific application was regenerating the native growth plate that can also encourage the endogenous mesenchymal stem cells to follow the desire linage. By regenerating the native growth plate, angular deformation and stunted limb growth were greatly reduced.

Tissue engineering is a multidisciplinary field that is rapidly emerging as a promising approach for tissue repair and regeneration. In this approach, scaffolds which allow cells to invade the construct and guide the cells grow into specific tissue play a pivotal role. Electrospinning has gained popularity recently as a simple and versatile method to produce fibrous structures with nano- to microscale dimensions. These electrospun fibers have been extensively applied to create nanofiber scaffolds for tissue engineering applications. Specifically for bone and cartilage tissue engineering, polymeric materials have some attractive properties such as the biodegradability. Ceramic scaffolds and implant coatings, such as hydroxyapatite and silica-based bioglass have also been considered as bone graft substitutes for bone repair because of their bioactivity and, in some cases, tunable resorbability. Besides tissue engineering scaffolds, for clinical application, especially for load-bearing artificial implants, metallic materials such as titanium are the most commonly used material. Osseointegration between bone and implants is very essential for implant success. To achieve better osseointegration between bone and the implant surface, three dimensional porous structures can provide enhanced fixation with bone by allowing tissue to grow into the pores. In this study, pre-3D electrospun polymer and ceramic scaffolds with peptide conjugation and 3D titanium scaffolds with different surface morphology were fabricated to testify the osteoblast and mensechymal stem cell attachment and differentiation. The overall goal of this thesis is to determine if the peptide functionalization of polymeric scaffolds and physical parameters of ceramic and metallic scaffold can promote osteoblast maturation and mesenchymal stem cell differentiation in vitro to achieve an optimal scaffold design for greater osseointegration. The results of the studies showed with functionalization of MSC- specific peptide, polymer scaffolds behaved with higher biocompatibility and MSC affinity. For the ceramic and metallic scaffolds, microstructures and nanostructures can synergistically promote osteoblast maturation and 3D micro-environment with micro-roughness is a promising design for osteoblast maturation and MSC differentiation in vitro compared to 2D surfaces.

The current study characterizes the mechanical and biodegradation properties of a polycarbonate polyurethane (PU) electrospun nanofiber scaffold intended for use in the growth of a tissue engineered annulus fibrosus (AF) intervertebral disc component. Both the tensile strength and initial modulus of aligned scaffolds were higher than those of random scaffolds and remained unaffected during a 4 week biodegradation study, suggesting a surface-mediated degradation mechanism. The resulting degradation products were non-toxic. Confined compressive mechanical force of 1kPa, was applied at 1Hz to in vitro bovine AF tissue grown on the scaffolds to investigate the influence of mechanical force on AF tissue production, which was found to decrease significantly at 72 hours relative to 24 hours, independent of any effects from mechanical forces. Overall, the consistent rate of PU degradation, along with mechanical properties comparable to those of native AF tissue, and the absence of cytotoxic effects, make this polymer suitable for further investigation for use in tissue-engineering the AF.

The current study characterizes the mechanical and biodegradation properties of a polycarbonate polyurethane (PU) electrospun nanofiber scaffold intended for use in the growth of a tissue engineered annulus fibrosus (AF) intervertebral disc component. Both the tensile strength and initial modulus of aligned scaffolds were higher than those of random scaffolds and remained unaffected during a 4 week biodegradation study, suggesting a surface-mediated degradation mechanism. The resulting degradation products were non-toxic. Confined compressive mechanical force of 1kPa, was applied at 1Hz to in vitro bovine AF tissue grown on the scaffolds to investigate the influence of mechanical force on AF tissue production, which was found to decrease significantly at 72 hours relative to 24 hours, independent of any effects from mechanical forces. Overall, the consistent rate of PU degradation, along with mechanical properties comparable to those of native AF tissue, and the absence of cytotoxic effects, make this polymer suitable for further investigation for use in tissue-engineering the AF.

High-throughput cell culture is an emerging technology that shows promise as a tool for research in tissue engineering, drug discovery, and medical diagnostics. An important, but overlooked, challenge is the integration of experimental methods with information processing suitable for handling large databases of cell-cell and cell-substrate interactions. In this work the traditional global descriptions of cell behaviors and surface characteristics was shown insufficient for investigating short-distance cell-to-cell and cell-to-surface interactions. This problem was addressed by introducing individual-cell based local metrics that emphasize cell local environment. An individual-cell based local data analysis method was established. Contact inhibition of cell proliferation was used as a benchmark for the effectiveness of the local metrics and the method. Where global, summary metrics were unsuccessful, the local metrics successfully and quantitatively distinguished the contact inhibition effects of MC3T3-E1 cells on PLGA, PCL, and TCPS surfaces. In order to test the new metrics and analysis method, a model of cell contact inhibition was proposed. Monte Carlo simulation was performed for validating the individual-cell based local data analysis method as well as the cell model itself. The simulation results well matched with the experimental observations. The parameters used in the cell model provided new descriptions of both cell behaviors and surface characteristics. Based on the viewpoint of individual cells, the local metrics and local data analysis method were extended to the investigation of cell-surface interactions, and a new high-throughput screening and knowledge discovery method on combinatorial libraries, local cell-feature analysis, was developed. PLGA/PCL combinatorial libraries were used as a prototype and a shaper and holder phenomenon involving MC3T3-E1 cells interacting with PCL islands was discovered. In summary, the viewpoint of individual cells casts new light on the study of cell-cell and cell-surface interactions and represents a novel methodology for developing new data analysis and knowledge discovery methods. The results of contact inhibition study and the shaper and holder model provide new knowledge, while the local data analysis method as well as the cell model of contact inhibition suggested novel approaches to study cell-cell and cell-surface interactions.

Articular cartilage on weight-bearing joints experiences three main forces: fluid-induced shear across the surface, perfusion through the cartilage from the surrounding fluid, and compression during motion of the joint. A new bioreactor that employs two of these forces was developed in this lab to study their effect on tissue-engineered cartilage development. The focus of this research and overall hypothesis is that bioreactors that employ both perfusion and shear will improve chondrogenesis and preservation to produce functionally relevant cartilage by modulating shear stress and introducing exogenous preservation factors. Applying both a low shear stress across the surface of cell-seeded scaffolds and perfusion through them in a perfusion concentric cylinder (PCC) bioreactor may stimulate chondrocytes to undergo chondrogenesis. Experimental data showed that the PCC bioreactor stimulated cartilage growth over the course of four weeks, supported by the appearance of glycosaminoglycan (GAG) and collagen type II, which are markers for articular cartilage. Computational fluid dynamics modeling showed that shear stress across the face of the construct was heterogeneous, and that only the center experienced a relatively uniform shear stress of 0.4 dynes/cm^2 when the outer cup of the bioreactor rotated at 38 rpm. When compared to a concentric cylinder (CC) bioreactor that employed only shear stress, the PCC bioreactor caused a significant increase in cellular proliferation, which resulted in a 12-fold increase in cell number per construct compared to 7-fold increase within the CC bioreactor. However, the PCC bioreactor had a less pronounced effect on glycosaminoglycan and collagen content with 1.3 mg of GAG and 1.8 mg of collagen per construct within the CC bioreactor and 0.7 mg of GAG and 0.8 mg of collagen per construct within the PCC bioreactor after 28 days in culture (p < 0.05). Our results led to an important observation that the PCC bioreactor affected cellular proliferation significantly but not extracellular matrix synthesis. The next objective of this study focused on the PCC bioreactor to evaluate the direct role of perfusion and shear on chondrogenesis in vitro and in vivo.

The development of tissue engineered blood vessel mimics for the testing of intravascular devices in vitro has been established in the Cal Poly tissue engineering lab. Due to the prevalence of cardiovascular disease in diabetic patients and minimal accessible studies regarding the interactions between diabetes and intravascular devices used to treat vascular disease, there is a need for the development of diabetic models that more accurately represents diabetic processes occurring in the blood vessels, primarily endothelial dysfunction. This thesis aimed to create a diabetic blood vessel mimic by implementing a high glucose environment for culturing human endothelial cells from healthy umbilical veins (HUVECs) and from diabetic coronary arteries (DHCAECs). The characterization of these BVMs was achieved using immunofluorescence, scanning electron microscopy (SEM), and qPCR gene expression analysis. From this study, it was determined that HUVECs and DHCAECs are robust enough to be cultured in a high glucose environment – analogous to hyperglycemia – and these cells exhibited different characteristics when evaluated under microscopy and qPCR gene expression. The immunofluorescence and SEM imaging showed presence of cells within each blood vessel mimic. The qPCR gene expression analysis demonstrated that mRNA expression of endothelial nitric oxide synthase (eNOS), platelet endothelial cell adhesion molecule (PECAM), and receptor for advanced glycation end products (RAGE) differs between HUVECs and DHCAECs, as well as between cells cultured in v normal and elevated glucose concentrations. These differences in gene regulation indicate the potential of the diabetic BVM to more accurately represent the endothelial response to diabetes and to the implementation of intravascular devices in the future. It was determined that culturing DHCAECs in a high glucose cell media for use in blood vessel mimics results in a model that differs considerably from HUVECs grown in normal glucose media. It was also determined that there was a difference between DHCAECs cultured in high glucose media and normal glucose media, as well as HUVECs cultured in high glucose media and normal glucose media. This study aided the development of a diabetic BVM; however, there are still improvements to be made, namely the inclusion of vascular smooth muscle cells in the model and improving the confluency of the BVM.

Trotz intensiver Forschung im Rahmen der Bänder- und Sehnenerkrankungen gelten bestimmte Fragestellungen hinsichtlich Erkrankungs- sowie Heilungsmechanismen als unbeantwortet. Verschiedenste Konzepte des Tissue Engineerings sollen helfen entsprechende Fragen zu beantworten und moderne Therapiekonzepte zu etablieren. Für grundlegende Untersuchungen zur Biologie der Tenogenese sowie zum Wirkmechanismus applizierter mesenchymaler Stromazellen (MSC), gewinnt die Anwendung von dezellularisiertem Sehnengewebe immer mehr an Bedeutung. Zudem erscheint der Einsatz dezellularisierter Sehnen- und Bandkonstrukte zur Wiederherstellung der betreffenden erkrankten Organe sehr vielversprechend. In der vorliegenden Arbeit sollte der Grundstein zur Entwicklung eines in vitro-Modells auf Grundlage equiner Beugesehnen gelegt werden. Primäres Ziel war es, ein optimales Dezellularisierungsprotokoll für intakte equine Beugesehnen (oberflächliche und tiefe Beugesehne) zu etablieren. Um die Zytokompatibilität der dezellularisierten Sehnen zu überprüfen, erfolgte nach Präparation von Sehnenstreifen die Besiedlung mit equinen MSC mit Kontrolle des Besiedlungserfolges. Materialien und Methoden: Oberflächliche und tiefe Beugesehnen (OBS und TBS) des Pferdes (n = 6) wurden nach vier verschiedenen Protokollen dezellularisiert. In zwei Protokollen (Protokolle A und B) erfolgte zunächst die Anwendung von Gefrier-Auftau- Zyklen mit anschließender Lagerung in hypertoner Lösung. Protokoll A sah danach eine Inkubation in 1 % Triton X 100 und Protokoll B eine Inkubation in 1 % Sodium-Dodecyl-Sulfat (SDS) enthaltender Lösung vor. Die beiden anderen Protokolle (Protokolle C und D) sahen ein Verbringen in hypertone Lösung ohne vorherige Gefrierzyklen vor. Anschließend erfolgte bei Protokoll C die Inkubation in Triton X 100 und bei Protokoll D die Inkubation in SDS enthaltender Lösung. Die Effektivität der angewandten Dezellularisierungsprotokolle wurde durch histologischer Färbung, Zellzählung nach Kollagenaseverdau, DNA-Quantifizierung und transmissionselektronenmikroskopischer Untersuchung ermittelt. Nach Evaluierung der Effektivität der Protokolle wurden oberflächliche Beugesehnen nach den Protokollen A und B dezellularisiert (n=3). Nach Präparation von Sehnenstreifen in definierter Größe erfolgte die Besiedelung mit Eisenoxid-markierten equinen MSC. Der Besiedlungserfolg wurde mit verschiedenen histologischen und Fluoreszenzfärbungen (Fluoreszenzmikroskopie) und MRT-Untersuchung kontrolliert. Die Prüfung auf statistische Unterschiede zwischen den Protokollen erfolgte mit dem Friedman-Test und im Falle eines statistisch signifikanten Unterschieds mit dem Wilcoxon-Rang-Test. Das Signifikanzniveau wurde mit p < 0,05 festgelegt. Die Auswertung des Besiedlungserfolges erfolgte deskriptiv. Ergebnisse: Für alle angewandten Protokolle konnte ein signifikanter Dezellularisie-rungseffekt in beiden Sehnenstrukturen (OBS und TBS) gezeigt werden. Die Anzahl der vitalen Zellen nach Kollagenaseverdau sowie die histologisch ermittelte Zellzahl der dezellularisierten Sehnen belief sich in Abhängigkeit des jeweiligen Dezellularisie-rungsprotokolls und der Sehne (OBS und TBS) auf 1 bis 21 % (Median) des nativen Gewebes. Der ermittelte DNA-Gehalt nach Anwendung der mit Gefrier-Auftau-Zyklen kombinierten Protokollen A und B entsprach < 24 % (Median) des nativen Gewebes. Die Anwendung der Protokolle C und D führte zu einem DNA-Gehalt von < 47 % (Median). Die Auswertung der transmissionselektronenmikroskopischen Untersuchung zeigte ebenfalls eine effektive Dezellularisierung des Sehnengewebes bei Erhalt der Struktur der extra-zellulären Matrix. Nach Anwendung der Protokolle A und B konnte wiederum tendenziell eine bessere Effektivität der Dezellularisierung festgestellt werden. Eine gelungene Besiedlung der Sehnenstreifen mit equinen MSC konnte anhand der mikroskopischen Untersuchung und MRT-Untersuchung gezeigt werden. Das beobachtete Zellwachstum bei beibehaltender Vitalität der Zellen sprechen für eine gute Zytokompatibilität. Die nach Protokoll A dezellularisierten und besiedelten Sehnenstreifen ließen ein besseres Zellwachstum über eine Kulturdauer von 14 Tagen erkennen. In der vorliegenden Arbeit konnte eine effektive Dezellularisierung von intakten equinen Beugesehnen gezeigt werden. Anhand der Ergebnisse der Besiedlung erwies sich die Dezellularisierung nach Protokoll A (Gefrier-Auftau-Zyklen und Triton X 100) als vielversprechende Grundlage zur Entwicklung eins in vitro Modells auf Grundlage dezellularisierter equiner Beugesehnen.

Osteoarthritis (OA), a degenerative disease of articular joints, is the leading cause of chronic disability in the US and affects more than a third of adults over 65 years old. Due to the obesity epidemic and an aging population, the prevalence of OA is expected to rise in both young and old adults. There are no disease modifying OA drugs. Therefore, providing any treatment options that delay the onset or progression of OA is highly desirable. The scope of this dissertation examines two different strategies to promote translational therapies for OA. The first approach investigated whether Smad ubiquitin regulatory factor 2 (Smurf2), an E3 ubiquitin ligase, could be a potential therapeutic target for OA. The second approach examined the incorporation of small chemical residues to enhance the physical and bioactivity of a bioinert scaffold for cartilage tissue repair. Overexpression of Smurf2 in chondrocytes was shown to accelerate spontaneous OA development in mice. We hypothesized that reduced Smurf2 expression could slow the progression of OA and enhance the performance of cells for cartilage repair. By performing surgical destabilization of the medial meniscus (DMM) on Smurf2-deficient mice, loss of Smurf2 was shown to mitigate OA changes in young mice but this protection diminished in older mice. Assessment of Smurf2-deficient chondrocytes in vitro revealed an upregulation of chondrogenic genes compared to wild-type; however, these differences were not seen at the protein level, deterring its potential use for cell-based therapies. During the course of this study, new insights about how age and sex affects different joint compartments in response to DMM surgery were also uncovered. These results broadened existing understanding of DMM-induced OA in mice but also questioned the validity of such a model to identify disease modifying targets that are translatable to OA in humans with advanced age. Due to a lack of innate repair mechanisms in cartilage, damage to cartilage increases the risk of developing OA early. Tissue engineering provides a unique strategy for repairing damaged cartilage by delivering cells in a well-controlled environment that can promote the formation of neotissue. We hypothesized that synthetic chemical residues could enhance the mechanical properties of a bioinert scaffold and promote matrix production of encapsulated chondrocytes. Covalent incorporation of small anionic or zwitterionic chemical residues in a polyethylene glycol-based hydrogel improved its stiffness and resistance to fluid flow, however, the resulting physical environment can also exert a dominant negative effect on matrix production of encapsulated chondrocytes. These results suggest that modulating the biosynthesis of chondrocytes with biochemical signals requires a concurrent reduction in any conflicting mechanotransduction signaling, emphasizing the importance of a degradable system to promote new cartilage formation. In summary, this dissertation establishes Smurf2 as a modulator of OA progression but implies that other factors such as age or protein(s) with redundant Smurf2 functions may play a role in limiting its effect as a therapeutic target. This work also reveals fundamental biology about how chondrocytes behave in response to physical and chemical cues in their microenvironment, which will aid in the design of better scaffolds for cartilage tissue engineering.