THERMORESPONSIVE, REDOX-POLYMERIZED CELLULOSIC HYDROGELS UNDERGO IN SITU GELATION AND RESTORE INTERVERTEBRAL DISC BIOMECHANICS POST DISCECTOMY

Back and neck pain are commonly associated with intervertebral disc (IVD) degeneration. Structural augmentation of diseased nucleus pulposus (NP) tissue with biomaterials could restore degeneration-related IVD height loss and degraded biomechanical behaviors; however, effective NP replacement biomaterials are not commercially available. This study developed a novel, crosslinked, dual-polymer network (DPN) hydrogel comprised of methacrylated carboxymethylcellulose (CMC) and methylcellulose (MC), and used in vitro, in situ and in vivo testing to assess its efficacy as an injectable, in situ gelling, biocompatible material that matches native NP properties and restores IVD biomechanical behaviors. Thermogelling MC was required to enable consistent and timely gelation of CMC in situ within whole IVDs. The CMC-MC hydrogel was tuned to match compressive and swelling NP tissue properties. When injected into whole IVDs after discectomy injury, CMC-MC restored IVD height and compressive biomechanical behaviors, including range of motion and neutral zone stiffness, to intact levels. Subcutaneous implantation of the hydrogels in rats further demonstrated good biocompatibility of CMC-MC with a relatively thin fibrous capsule, similar to comparable biomaterials. In conclusion, CMC-MC is an injectable, tunable and biocompatible hydrogel with strong potential to be used as an NP replacement biomaterial since it can gel in situ, match NP properties, and restore IVD height and biomechanical function. Future investigations will evaluate herniation risk under severe loading conditions and assess long-term in vivo performance

Osteochondral Tissue Engineering for the TMJ Condyle Using a Novel Gradient Scaffold

The articulation of the temporomandibular joint (TMJ), or the jaw joint, is one of the most complex and least studied joints of the musculoskeletal system. Painful disorders of the TMJ, known as temporomandibular disorders (TMDs), have considerable prevalence with over 10 million patients in the United States alone, which may severely interfere with everyday activities like chewing, yawning, talking, and laughing. Within the TMJ, the inferior joint space, which includes the mandibular condyle, typically sustains the greatest damage in TMDs. The objective of this thesis was to characterize the condylar cartilage biomechanics, and to explore novel routes to fabricate integrated gradient-based osteochondral constructs. Pioneering efforts were made toward understanding structure-function correlations for the condylar cartilage. A greater stiffness of the condylar cartilage in the anteroposterior direction than in the mediolateral direction under tension was observed, corresponding to the never before seen anteroposterior organization of collagen fibers. A positive correlation between the thickness and stiffness of the cartilage under compression suggested that their regional variations may be related phenomena caused in response to cartilage loading patterns. Beyond these vital biomechanical characterization efforts, novel microsphere-based gradient scaffolds were developed to address functional osteochondral tissue regeneration. Novel microsphere sintering routes, using ethanol as an anti-solvent or sub-critical CO2 for melting point depression, were established to construct microsphere-based scaffolds. A technique to create opposing macroscopic gradients of encapsulated growth factors using poly(D,L-lactide-co-glycolic acid) microspheres was developed, and in vitro studies with human umbilical cord stem cells provided promising results for osteochondral tissue regeneration. By encapsulating nanoparticles in the microspheres, a proof-of-concept was provided for creating functional scaffolds with a gradient in stiffness. This thesis lays down the foundation for a combined growth factor-stiffness gradient approach that could lead to a translational-level regenerative solution to osteochondral tissue regeneration with extended applications in other areas, including tissue engineering of heterogeneous/interfacial tissues

Optimization and Translation of MSC-Based Hyaluronic Acid Hydrogels for Cartilage Repair

Traumatic injury and disease disrupt the ability of cartilage to carry joint stresses and, without an innate regenerative response, often lead to degenerative changes towards the premature development of osteoarthritis. Surgical interventions have yet to restore long-term mechanical function. Towards this end, tissue engineering has been explored for the de novo formation of engineered cartilage as a biologic approach to cartilage repair. Research utilizing autologous chondrocytes has been promising, but clinical limitations in their yield have motivated research into the potential of mesenchymal stem cells (MSCs) as an alternative cell source. MSCs are multipotent cells that can differentiate towards a chondrocyte phenotype in a number of biomaterials, but no combination has successfully recapitulated the native mechanical function of healthy articular cartilage. The broad objective of this thesis was to establish an MSC-based tissue engineering approach worthy of clinical translation. Hydrogels are a common class of biomaterial used for cartilage tissue engineering and our initial work demonstrated the potential of a photo-polymerizable hyaluronic acid (HA) hydrogel to promote MSC chondrogenesis and improved construct maturation by optimizing macromer and MSC seeding density. The beneficial effects of dynamic compressive loading, high MSC density, and continuous mixing (orbital shaker) resulted in equilibrium modulus values over 1 MPa, well in range of native tissue. While compressive properties are crucial, clinical translation also demands that constructs stably integrate within a defect. We utilized a push-out testing modality to assess the in vitro integration of HA constructs within artificial cartilage defects. We established the necessity for in vitro pre-maturation of constructs before repair to achieve greater integration strength and compressive properties in situ. Combining high MSC density and gentle mixing resulted in integration strength over 500 kPa, nearly 10-fold greater than previous reports of integration with MSC-based constructs. Furthermore, we demonstrated the durability of this repair system by applying dynamic loading and showed its functional contribution to the distribution of compressive loads across the repair space. Overall, the studies contained within this thesis offer the first MSC-based tissue engineering strategy that successfully recapitulates native mechanical function while also demonstrating the potential for complete functional cartilage repair

Analysis of the Effect of Sequential Injection Molding on Weld Lines Properties

This study analyzes a sequential injection molding process to improve weld lines properties and morphology using in-flow phenomena. varioterm technology has been applied and the combination of these techniques led to significant improvements on mechanical properties and weld line appearance. Fiber orientation has been improved and the interfacial bonding at weld line surface has been promoted, resulting in better local properties. weld line skin marks could possibly be eliminated

Development and Characterization of Photocrosslinkable Hyaluronic Acid Hydrogels for Cartilage Regeneration

Damage to cartilage from general wear, disease, or injury can lead to joint pain and tissue degeneration. With its limited ability for self-repair, cartilage has become a target for tissue engineering (TE). As current treatments have yet to provide long-term functional cartilage repair, this dissertation introduces the development and use of photopolymerizable hyaluronic acid (HA) based hydrogels for TE to optimize cellular interactions and neocartilage formation. By altering hydrogel design parameters (e.g., molecular weight and macromer concentration), a wide range of hydrogel properties were obtained. These hydrogels all preserved the rounded morphology of chondrocytes, but cell viability and neocartilage formation were dependent on hydrogel design, where increased crosslinking resulted in cell death and increased macromer molecular weight yielded inhomogeneities in cell and ECM distribution within the hydrogel. These variables also influenced the formed neocartilage properties. The ability of HA hydrogels to promote neocartilage formation was also dependent on cell source and culture. The expansion of chondrocytes in 2D in vitro affected neocartilage formation in HA hydrogels after the second passage, as construct properties further decreased with continued passage. Chondrocytes from different tissue sources also behaved variably in the hydrogels; auricular chondrocytes excelled in static culture and subcutaneous culture over articular chondrocytes, while articular chondrocytes were stimulated in a mechanically loaded environment. As the use of chondrocytes for cartilage TE is limited clinically, we turned to mesenchymal stem cells (MSCs). In vitro culture of MSC-laden HA hydrogels demonstrated that these HA hydrogels not only supported, but enhanced chondrogenesis when compared to relatively inert hydrogels, potentially due to receptor interactions with HA. However, in these hydrogels, ECM was localized to pericellular regions. To accelerate the diffusion and distribution of ECM proteins, hydrolytically degradable HA macromers were synthesized to create a dynamic environment. When degradation complemented ECM deposition, ECM distribution and ultimately the functional maturation of the construct were improved. While this dissertation focused on material development to improve cartilage regeneration, growth factor delivery optimization and successful implementation of these hydrogels in cartilage defect models remain, towards our goal of a successful long-term repair solution to cartilage damage

Shape Memory Assisted Self Healing (Smash) Polymeric and Composite Systems

My research aims to develop a novel approach that uses the shape memory (SM) effect to aid self healing (SH) polymeric systems that are able to simultaneously close and re-bond cracks with a single thermal stimulus. This new concept is termed shape memory assisted self healing (SMASH). Additionally, a new type of shape memory termed reversible plasticity shape memory (RPSM) was also developed where both the elastic and plastic deformation found after deformation completely recover upon a thermal stimulation. I aim to utilize a broad range of polymeric and composite systems that include a single phase semi-crystalline system, a single phase amorphous blend, and a combination of these two polymers in a composite elastomer system to prove the versatility of the SMASH and RPSM effects. Chapter 1 gives a polymer science background along with SM and SH material overview. Chapter 2 discusses the fabrication and analysis of miscible blends that show the SMASH and RPSM effect using a semi-crystalline polymer, poly(e-caprolactone) (PCL) to construct a SM PCL network (n-PCL) and PCL thermoplastic used as the SH agent (l-PCL). The PCL thermoplastic SH agent interpenetrated the n-PCL for form a single phase semi interpenetrating polymer network (SIPN). Films were made for testing to prove the SM and SH effects by varying the amount of SM network and SH agent to optimize both effects. Thermo-mechanical, tensile, and SH experiments were conducted to study the fixing, recovery and healing properties of the polymeric system. Chapter 3 focuses on a unique system for the fabrication of clear thin SMASH SIPN coatings that were developed for optical industrial applications. Here, an amorphous polymer composition, poly(tert-butyl acrylate) (poly(tBA)), was used in a blend of two forms, a network form for shape memory (n-tBA) and a linear form for self-healing (l-tBA), that, together, form a single phase SIPN. Thermal, thermo-mechanical, SM and SH scratch experiments were conducted to investigate both SM and SH mechanisms as influenced by the relative concentrations of n-tBA and l-tBA in the SIPN materials. Chapter 4 introduces for the first time an innovative smart polymeric soft material where aligned nanofibers are used to construct anisotropy embedded in an elastomeric matrix. This system, termed Anisotropic Shape Memory Elastomeric Composite (A-SMEC) was investigated for RPSM and SMASH properties. In addition, the anisotropic mechanical and shape memory properties were investigated and interpreted in light of the underlying structure. Chapter 5 builds upon the results of Chapter 4, presenting the fabrication and testing of laminated A-SMEC biomorphs that were designed to exploit anisotropic in RPSM behavior to yield predictably curled and twisted structures upon deformation. More specifically, the out-of-plane curvature and pitch were analyzed as a function of biomorph orientational lay-up. All polymeric systems described in this dissertation are examples of smart polymers that can be used to tailor mechanical performance while introducing new phenomena, such as self-healing, RPSM, and stretch-induced twisting. Chapter 6 discusses the conclusions followed by future work that are sub-sectioned for each chapter of the dissertation