Thesis (M.S.)--University of Missouri--Columbia, 2004. / "July 2004." Typescript. Includes bibliographical references (leaves 63-70). Also issued on the Internet. Also available on the Internet.

Thesis (M.S.)--University of Missouri--Columbia, 2004. / Typescript. Includes bibliographical references (leaves 63-70). Also issued on the Internet. Also available on the Internet.

Thesis (M.S.)--University of Missouri--Columbia, 2003. / "December 2003." Typescript. Vita. Includes bibliographical references (leaves 84-96). Also issued on the Internet.

A reproducible in vitro model of mechanically injured (scalpel cut) articular cartilage was developed in this work utilising bovine and human osteochondral tissue. Using fluorescence-mode confocal laser scanning microscopy (CLSM), the model allowed (1) spatial and temporal quantification of in situ (within the matrix) chondrocyte viability following a full thickness cartilage injury and (2) serial evaluation of three chondroprotective strategies in injured bovine and human articular cartilage: (a) medium osmolarity (b) medium calcium concentration and, (c) subchondral bone attachment to articular cartilage. Medium osmolarity significantly influenced superficial zone chondrocyte death in injured (scalpel cut) bovine and human articular cartilage. Greatest percentage cell death occurred at 0 mOsm (distilled water). Conversely, a raised medium osmolarity (600 mOsm) was chondroprotective. The majority of in situ cell death occurred within 2.5 hours of the experimental injury, with no further increase over 7 days. Exposure of articular cartilage to calcium-free media significantly decreased superficial zone chondrocyte death in injured (scalpel cut) articular cartilage compared with exposure to calcium-rich media (2-20 mM). In calcium-rich media, the extent of percentage cell death increased with increasing medium calcium concentration but remained localised to the superficial zone of injured articular cartilage over 7 days. However, in calcium-free media, there was an increase in percentage cell death within deeper zones of injured articular cartilage over 7 days. Excision of subchondral bone from injured (scalpel cut) articular cartilage resulted in an increase in chondrocyte death at 7 days that occurred in the superficial zone of injured as well as the adjacent uninjured regions of articular cartilage. However, the presence of subchondral bone in the culture medium prevented this increase in chondrocyte death within the superficial zone. Subchondral bone may have interacted with articular cartilage via soluble mediator(s) that influenced chondrocyte survival. In human articular cartilage, healthy subchondral bone also interacted with articular cartilage in explant culture and promoted in situ chondrocyte survival, while sclerotic subchondral bone was detrimental to chondrocyte viability. These findings are of translational relevance to fluid management systems used during open and arthroscopic articular surgery, clinical and experimental research into cartilage injury, repair and degeneration as well as current techniques of tissue engineering.

Articular cartilage lining joints, such as in the knee, functions to reduce friction and absorb shock. Collagen type II is the largest constituent in the extracellular matrix of articular cartilage and its restoration is of the highest interest to tissue engineers. Cartilage has little ability to naturally regenerate due to the absence of vascularity and the inability of the chondrocytes to proliferate at a high rate. It would be ideal to create a mimicking extracellular matrix/scaffold from type II collagen that could possibly be used to replace damaged articular cartilage that has the same function and morphology. Three different groups of cartilage chips were utilized to extract type II collagen. The yield of the three groups was compared. The extracted type II collagen from the three groups was electrospun at the concentrations of 0.06, 0.08, 0.10 and 0.12 g/mL. Both the pore size and fiber diameter were analyzed. A SDS-Page was performed on the material to assure it was pure type II collagen and that no collagen type I contamination was present.

Articular cartilage is an extraordinary tissue, allowing frictionless movements of articulated joints, and acting as a load-bearing cushion to protect joints from damage. Breakdown of articular cartilage may result in crippling diseases such as osteoarthritis (OA) and, since articular cartilage has a limited repair capacity, a greater understanding of the mechanisms of joint homeostasis and its response to injury are of great clinical need. In this project the hypothesis that endogenous mesenchymal stem cells (MSCs) may contribute to the healing process of a full-thickness articular cartilage defect was investigated by combining a mouse model of joint surface injury and repair with a nucleoside analogue labelling scheme in DBA/1 mice. Following injury, proliferative responses of nucleoside analogue-retaining cells were detected between 4 and 12 days post injury (dpi) in both the bone marrow and the synovial membrane of the knee joint. Phenotypic analysis of these label-retaining cells using immunofluorescence staining revealed an MSC-compatible phenotype (CD44+, CD105+, CD146+, PDGFRα+ and p75NGFR+), with differences observed between the two tissues in expression of CD105 and CD146. The response of the label-retaining cells to the injury was associated with early activation of Notch signalling (4 dpi), followed by BMP signalling at 8 dpi and TGF-β at 12 dpi. Conversely, canonical Wnt signalling, which was active in uninjured knee joints and in injured knee joints up to 8 dpi, was attenuated at 12 dpi. The contribution of nerve growth factor (NGF), known as a pain mediator in OA, to the repair process was then investigated in vitro. NGF was released by both cartilage explants and femoral head cultures following injury. Using a Transwell-based cell migration assay, NGF was revealed to have a chemotactic effect on human bone marrow derived MSCs, but not synovial membrane derived MSCs. High-density micromass cultures also revealed NGF had a potent stimulatory effect on the chondrogenic differentiation of mesenchymal cells. The data presented here demonstrate a contribution of endogenous MSCs to the repair of articular cartilage in vivo and suggest a possible new therapeutic strategy: stimulation of in vivo recruitment of MSCs by modulating signalling pathways activated during the healing process. Furthermore, a novel role for NGF as a factor involved in migration and the chondrogenic differentiation of MSCs is suggested.

Articular cartilage is a mechanically important soft tissue whose organisation at the micro- and nanoscale is critical for healthy joint function and where degeneration is associated with widespread disorders such as osteoarthritis. The tissue possesses a complex, graded and depth-dependent structure and at the nanoscale, cartilage mechanical functionality is dependent on the collagen and hydrated proteoglycans that form the extracellular matrix. The structure and in situ dynamic response of the collagen fibrils at the nanoscale, however, remain unclear. Here we utilise small angle X-ray diffraction to measure the depth-wise structure of the fibrillar architecture whilst performing time-resolved measurements during compression of bovine and human cartilage explants. We demonstrate the existence of a depth-dependent fibrillar pre-strain as determined by the D-periodicity, estimated at approximately 1-2%, due to osmotic swelling pressure from the proteoglycans. Furthermore, we reveal a rapid reduction and recovery of this pre-strain during stress relaxation, approximately 60 seconds after onset of peak load. Selective proteoglycan removal disrupts both collagen fibril pre-strain and transient responses during stress relaxation. Additionally, we show that IL-1β induced tissue inflammation also results in a reduction in fibrillar pre-strain and altered fibrillar mechanics. Cyclic loading induces a dynamic reduction and recovery in the D-period that is present regardless of loading rate or treatment, along with changes in diffraction peak intensities and widths. These findings suggest that the fibrils respond to loading via intra- and inter-fibrillar disordering alongside a transient response that is mediated by changes in hydration. These are the first studies to highlight previously unknown transient and cyclic responses to loading at the fibrillar level, and are likely to transform our understanding of the role of collagen fibril nano-mechanics in cartilage and other hydrated soft tissues. These methods can now be used to better understand cartilage in aging and other muscoskeletal diseases.

The articular cartilage has a limited capacity to regenerate. Cartilage lesions often result in degeneration, leading to osteoarthritis. Current treatments are mostly palliative and reparative, and fail to restore cartilage function in the long term due to the replacement of hyaline cartilage with fibrocartilage. Although a stem-cell based approach towards regenerating the articular cartilage is attractive, cartilage generated from human mesenchymal stem cells (hMSCs) often lack the function, organization and stability of the native cartilage. Thus, there is a need to develop effective methods to engineer physiologic cartilage tissues from hMSCs in vitro and assess their outcomes in vivo. This dissertation focused on three coordinated aims: establish a simple in vivo model for studying the maturation of osteochondral tissues by showing that subcutaneous implantation in a mouse recapitulates native endochondral ossification (Aim 1), (ii) develop a robust method for engineering physiologic cartilage discs from self-assembling hMSCs (Aim 2), and (iii) improve the organization and stability of cartilage discs by implementing spatiotemporal control during induction in vitro (Aim 3). First, the usefulness of subcutaneous implantation in mice for studying the development and maintenance of osteochondral tissues in vivo was determined. By studying juvenile bovine osteochondral tissues, similarities in the profiles of endochondral ossification between the native and ectopic processes were observed. Next, the effects of extracellular matrix (ECM) coating and culture regimen on cartilage formation from self-assembling hMSCs were investigated. Membrane ECM coating and seeding density were important determinants of cartilage disc formation. Cartilage discs were functional and stratified, resembling the native articular cartilage. Comparing cartilage discs and pellets, compositional and organizational differences were identified in vitro and in vivo. Prolonged chondrogenic induction in vitro did not prevent, but expedited endochondral ossification of the discs in vivo. Finally, spatiotemporal regulation during induction of self-assembling hMSCs promoted the formation of functional, organized and stable hyaline cartilage discs. Selective induction regimens in dual compartment culture enabled the maintenance of hyaline cartilage and potentiated deep zone mineralization. Cartilage grown under spatiotemporal regulation retained zonal organization without loss of cartilage markers expression in vivo. Instead, cartilage discs grown under isotropic induction underwent extensive endochondral ossification. Together, the methods established in this dissertation for investigating cartilage maturation in vivo and directing hMSCs towards generating physiologic cartilage in vitro form a basis for guiding the development of new treatment modalities for osteochondral defects.

There are limited treatment options available today to slow down progression of osteoarthritis in its early stages and most interventions, such as highly invasive partial and total joint replacement surgeries, are performed only at the late stages of the disease. Understanding the mechanism of early articular cartilage stress-mediated wear and failure can aid in the design of new treatment options that are introduced at earlier stages of the disease, presenting the potential to slow down osteoarthritis progression and thus significantly improve patient outcomes. This dissertation aims to provide a basic science understanding of wear propagation and repair of articular cartilage in the absence of traumatic events under the normal reciprocal sliding motion of the articular layers at physiologic load magnitudes. In this dissertation there are three main thrusts: (1) characterize cartilage delamination wear under normal sliding (2) define a chemical environment that promotes cartilage explant homeostasis to enable long-term wear-and-repair studies (3) investigate a practical treatment modality capable of stopping or slowing down structural degeneration of articular cartilage in OA. We hypothesize that the mode of cartilage damage is delamination wear that progresses by fatigue failure of the extracellular matrix (ECM) under physiologic sliding, even when cartilage layers are subject to physiologic load magnitudes and contact stresses and even when the friction coefficient μ remains low (H1a). Based on prior literature findings regarding the role of synovial fluid (SF) boundary lubricants on the reduction of friction and wear, we also test the hypothesis (H1b) that SF delays the onset of cartilage delamination when compared to physiological buffered saline (PBS). We then test a third hypothesis (H1c) that loading cartilage against cartilage delays the onset of delamination wear compared to testing glass on cartilage, since contacting porous cartilage layers exhibit a much smaller solid-on-solid contact area fraction than impermeable glass contacting porous cartilage. Next, we hypothesize that the homeostatic dysregulation previously observed in cultured immature cartilage explants results from the presence of non-physiologic levels of important metabolic mediators in the culture medium. To this end, we hypothesize that: (H2a) immature bovine cartilage explants cultured in native synovial fluid will maintain homeostasis as characterized by maintenance of their mechanical properties and ECM contents at initial (post- explantation) levels, and (H2b) explants cultured in a physiologic-based medium, consisting of physiologic levels of key metabolic mediators, will maintain a similar homeostasis over long- term culture. Finally, a laser treatment strategy is explored that has the capability to reform collagen crosslinks, replacing those lost during OA progression. This novel therapy acts without injuring the cells and without any chemical additive or thermal ablation. The laser treatment protocol used in this application can specifically target the subsurface region, located 200 μm of the articular surface. By strengthening this region with enhanced crosslinking, we hypothesize (H3a) that cartilage will demonstrate greater resistance to fatigue failure than untreated controls. We then hypothesized (H3b) that this treatment protocol would also be effective on devitalized fibrillated human articular cartilage from OA joints with overall Outerbridge score OS1-3. We find that for both cartilage-on-cartilage and glass-on-cartilage sliding configurations at physiologic applied loads, long-term sliding with a low friction coefficient causes wear in the form of delamination. We show that the use of synovial fluid as a lubricant delays the onset of wear; and, similarly, that sliding with a cartilage counterface also reduces the incidence of wear. In subsequent studies we fully characterize a homeostatic culture medium to emulate cartilage in vitro behavior in synovial fluid. We show that explants cultured in this medium can maintain their properties for at least one month and have no loss in cell viability. Laser treatment is then tested on both living and devitalized bovine and devitalized human cartilage and the treatment is shown to improve the wear resistance of the tissue without harming embedded cells. Overall this work has led to novel insights that have clinical applicability. One strength of the in vitro investigations described in this body of work is the ability to separate out mechanically-mediated events from biochemically-mediated events, which would be impossible in vivo. Parsing out such specific mechanisms of cartilage wear can help guide better understanding of disease progression and drive therapeutic intervention. Intervening during the early stages of OA offers the promise of preventive care that currently does not exist and could provide significant benefits to a patient’s quality of life. This dissertation asserts that focusing on delaying or preventing wear by improving the resiliency of the extant intact cartilage in early OA is a viable strategy to improve patient outcomes and offers an innovative approach over existing regenerative techniques.

Articular cartilage is the connective tissue lining the ends of long bones, providing a dynamic surface that bears load while providing a smooth surface for articulation. When damaged, however, this tissue exhibits a poor capacity for repair, lacking the lymphatics and vasculature necessary for remodeling. Osteoarthritis (OA), a growing health and economic burden, is the most common disease afflicting the knee joint. Impacting nearly thirty million Americans and responsible for approximately $90 billion in total annual costs, this disease is characterized by a progressive loss of cartilage accompanied by joint pain and dysfunction. Moreover, while generally considered to be a disease of the elderly (65 years and up), evidence suggests the disease may be traced to joint injuries in young, active individuals, of whom nearly 50% will develop signs of OA within 20 years of the injury. For these reasons, significant research efforts are directed at developing tissue-engineered cartilage as a cell-based approach to articular cartilage repair. Clinical success, however, will depend on the ability of tissue-engineered cartilage to survive and thrive in a milieu of harsh mechanical and chemical agents. To this end, previous work in our laboratory has focused on growing tissues appropriate for repair of focal defects and entire articular surfaces, thereby investigating the role of mechanical and chemical stimuli in tissue development. While we have had success at producing replacement tissues with certain qualities appropriate for clinical function, engineered cartilage capable of withstanding the full range of insults in vivo has yet to be developed. For this reason, and in an effort to address this shortcoming, the work described in this dissertation aims to (1) further characterize and (2) optimize the response of tissue-engineered cartilage to physical loading and the concomitant chemical insult found in the injured or diseased diarthrodial joint, as well as (3) provide a clinically relevant strategy for joint resurfacing. Together, this holistic approach maximizes the chances for in vivo success of tissue-engineered cartilage. Regular joint movement and dynamic loads are important for the maintenance of healthy articular cartilage. Extensive work has been done demonstrating the impact of mechanical load on the composition of the extracellular matrix and the biosynthetic activity of resident chondrocytes in explant cultures as well as in tissue-engineered cartilage. In further characterizing the response of tissue-engineered cartilage to mechanical load, the work in this dissertation demonstrated the impact of displacement-controlled and load-controlled stimulation on the mechanical and biochemical properties of engineered cartilage. Additionally, these studies captured tension-compression nonlinearity in tissue-engineered cartilage, highlighting the role of the proteoglycan-collagen network in the ability to withstand dynamic loads in vivo, and optimized a commercial bioreactor for use with engineered cartilage. The deleterious chemical environment of the diseased joint is also well investigated. It is therefore essential to consider the impact of pro-inflammatory cytokines on the mechanical and biochemical development of tissue-engineered cartilage, as chemical injury is known to promote degradation of extracellular matrix constituents and ultimately failure of the tissue. Combining expertise in interleukin-1\alpha, dexamethasone, and drug delivery systems, a dexamethasone drug delivery system was developed and demonstrated to provide chondroprotection for tissue-engineered cartilage in the presence of supraphysiologic doses of pro-inflammatory cytokines. These results highlight the clinical relevance of this approach and indicate potential success as a therapeutic strategy. Clinical success, however, will not only be determined by the mechanical and biochemical properties of tissue-engineered cartilage. For engineered cartilage to bear loads in vivo, it is necessary to match the natural topology of the articular surface, recapitulating normal contact geometries and load distribution across the joint. To ensure success, a method for fabricating a bilayered engineered construct with biofidelic cartilage and subchondral bone curvatures was developed. This approach aims to create a cell-based cartilage replacement that restores joint congruencies, normalizes load distributions across the joint, and serves as a potential platform for the repair of both focal defects and full joint surfaces. The research described in this dissertation more fully characterizes the benefits of mechanical stimulation, prescribes a method for chondroprotection in vivo, and provides a strategy for creating a cartilage replacement that perfectly matches the native architecture of the knee, thus laying the groundwork for clinical success of tissue-engineered cartilage.