Acetaminophen has been safely used for analgesia for many years. Literature suggests that a plasma acetaminophen level of 5��g/ml is necessary to maintain analgesic relief in humans. Current dosing regiments are inconvenient (every 4-6 hours) and do not maintain this minimum plasma level. Simulations were conducted to examine various doses and input rates for sustained release formulations of acetaminophen. Once parameters were selected from the simulations, sample formulations were prepared and tested using standard dissolution techniques. Investigations into dose/size relationships, hydroxypropylmethylcellulose (HPMC) percentage for erosion matrix tablets, compression force, tablet shape, tablet divisibility, and granulation methods were performed for non-disintegrating hydrophilic matrix tablets. Tablets containing 5% and 7.5% HPMC were selected for pharmacokinetic study in 10 healthy human subjects. Tylenol Extra Strength and Tylenol Extended Relief tablets were administered as control formulations. Pharmacokinetic fitting of the kinetic profiles of all four formulations were performed using Win Nonlin. The formulations were best described by a 1-compartment open model with first order input and first order elimination. The 5% HPMC sustained release acetaminophen formulation was selected for Phase II clinical trials. Patients with osteoarthritis of the knee were recruited for a double blind crossover study of 5% HPMC sustained release acetaminophen formulations and immediate release acetaminophen. Patients received two tablets of study medication, four times a day for 4 weeks. After a seven day wash-out period patients were then crossed over to the other treatment. Patients were evaluated using a twelve question questionnaire and the time to walk 50 feet was measured. Thirty patients were enrolled in the study and seventeen patients completed the study. The sustained release formulations were statistically superior to the baseline treatments in reducing pain level, decreasing disability, and improving the duration of pain relief. Additional, larger scale studies are needed to confirm these findings. / Graduation date: 2000

Joint disease is detrimental to basic quality of life. Articular cartilage is responsible for reducing friction and distributing loads in joints as they undergo large, repetitive load cycles each day, but damaged tissue has very limited intrinsic regenerative ability. Osteoarthritis (OA), the most common joint disease, affects over 500 million people worldwide, contributes more than $27 billion dollars in annual healthcare expenditures, and has increased in prevalence by nearly 50% since 1990 with our aging population. In spite of all this, OA remains a chronic degenerative condition lacking in effective treatment strategies. For cartilage repair in late-stage disease, synthetic joint replacements carry risk of altered loading and metal hypersensitivity, while clinically approved autografts or autologous chondrocyte implantation procedures suffer from lack of donor tissue and donor site morbidities. Prior to surgical intervention, OA management is focused on analgesia rather than preventing or slowing early-stage disease. Disease-modifying OA drugs are yet to successfully complete clinical trials, in part due to the widespread use of animal models for therapeutic discovery rather than high-fidelity human models. Alleviating the burden of cartilage damage will require improvements in both early-stage therapeutic interventions and late-stage repair. Tissue engineering has the potential to offer more biologically faithful cartilage derived with minimal invasiveness, but the resulting cartilage currently lacks the organization or maturity of native tissue. Thus, the central concept of my thesis work was to introduce biologically inspired spatiotemporal cues to guide engineered cartilage formation, establishing novel methods for cartilage tissue engineering that would provide (i) cartilage-bone grafts for regenerative implantation and (ii) advanced in vitro models for studying osteochondral disease. United by the central theme of cartilage, this dissertation spanned three complementary and interacting areas of tissue engineering: regenerative medicine in Aim 1, tools and technological development in Aim 2, and organs on a chip in Aim 3. In Aim 1, we created patient-specific cartilage-bone constructs with native-like features at a clinical scale, using decellularized bone matrix, autologous adipose-derived stem/stromal cells, and dual-chamber perfusion bioreactors to recapitulate the anatomy and zonal organization of the temporomandibular ramus-condyle unit with its fibrocartilage. We validated key tissue engineering strategies for achieving in vivo cartilage regeneration, with the cartilage-bone grafts serving as templates for remodeling and regeneration, rather than providing direct replacements for the native tissue. To enable precise in vitro manipulation of TGF-β signaling, a key pathway in cartilage development, in Aim 2 we developed an optogenetic system in human induced pluripotent stem cells and used light-activated TGF-β signaling to direct differentiation into smooth muscle, tenogenic, and chondrogenic lineages. This optogenetic platform served as a versatile tool for selectively activating TGF-β signaling with precise spatiotemporal control. Using optogenetic recapitulation of physiological spatiotemporal gradients of TGF-β signaling in Aim 3, we formed stratified human cartilage integrated with subchondral bone substrate, towards in vitro engineering of native-like, zonally organized articular cartilage. Collectively, these studies established novel cartilage tissue engineering approaches which can be leveraged to alleviate the burden of joint disease.

A thesis submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfillment of the requirements for the degree of Doctor of Philosophy Johannesburg, 2019 / Background Evidence shows that the global prevalence of knee osteoarthritis (KOA) is high, with limited data on the management of the disease. The use of novel modalities to treat the condition is low due to poor understanding of their clinical effects. Therefore there are gaps in the knowledge on the prevalence and treatment modalities for patients diagnosed with KOA. Aim: The aim was threefold; (i) to determine the prevalence of KOA in South Africa aged 45yrs-75yrs; (ii) to determine the current management of KOA; and (iii) to determine the effect of Low Level Laser therapy (LLLT) on the structural and functional components related to KOA in a South African cohort, aged 45-75yrs. Methods: The methodology will be discussed in terms of the three specified objectives; (i) prevalence study data - a self-reported data collection sheet listing 19 relevant ICD 10 codes; completed by South African medical aid providers. (ii) The treatment paradigm study, which encompassed a deemed KOA management paradigm validated questionnaire sent electronically to 742 general, specialist and allied practitioners, identifying the incidence of KOA and deemed efficacy and compliance of various management tool. These practitioners were identified from a database of medical and allied practitioners in both the private and public sector of South Africa. The questionnaire consisted of two close ended questions indicating the incidence of KOA and bilateral KOA patients consulted at the practice; one choice question indicating the most suggested mode of therapy from a choice of pharmaceutical, surgical, homeopathic, physical exercise therapy and LLLT and finally, 3 Likert type scale questions on the deemed efficacy and compliance of the modes of therapy as stated above. (iii) The intervention study which was a randomized controlled trial (RCT) utilizing pre marked questionnaire sheets on 111 participants. Participants were randomized into one of three intervention groups; (1) exercise group (n=39), (2) LLLT group (n=40), and (3) combined exercise-LLLT group (n=32). Data on knee circumference, the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC), knee range of motion (ROM) and the one minute timed sit–to-stand test was used. These tests were done at four time points: (T1) baseline, (T2) post-12 session intervention, (T3) one month post intervention and (T4) three months post intervention. Results: The results will be discussed in terms of the three specified objectives; (i) The prevalence of KOA was reported as 17.5%, 28.0% and 38.5% in a South African population over 45yrs. (ii) Four hundred and thirteen clinicians completed the questionnaire, reporting a KOA patient intake of 53%. Pharmacology (36.3%) and physical exercise (35.3%) was the most common management protocols compared to surgical intervention, homeopathy and LLLT. Pharmacotherapy (73%) and physical exercise (92%) were observed as effective treatments. Seventy five percent of all practitioners responded with an answer of “no comment” when asked the deemed efficacy of LLLT. Practitioners viewed patients with KOA to have low compliance with physical exercise and pharmacotherapy (iii) the participant demographic included 86 females and 25 males, the average age reported was 61.8 ± 5.6yrs. At 12-week follow-up, knee circumference decreased significantly in all groups (p<0.05), the effect was highest in the LLLT group. All groups experienced improvements in the WOMAC pain scale, but the LLLT group showed the greatest improvement (p<0.05). Knee ROM values improved significantly across all three groups; however, the effect of the intervention was most significant (p<0.005) in the combined LLLT-exercise group. Physical functionality scores showed a greater improvement in the combined LLLTexercise group at all three data collection points. Conclusions: The estimated prevalence of KOA is 17-35% based on data collected from a specified South African cohort. Pharmacotherapy is a commonly suggested KOA management mode, whilst clinicians view physical exercise as effective. LLLT was not a known tool for the treatment of KOA. In addition to the improved functionality observed, pain was lowered significantly, particularly in the combined exercise-LLLT group. Study results have shown that LLLT used in isolation or in combination with physical exercise is an effective management tool. / MT 2020

Osteoarthritis is a highly prevalent, debilitating disease characterized by the wear and degradation of articular cartilage. While many surgical interventions exist, few are consistently effective and those that are effective are not necessarily suitable for all patients. The objective of this dissertation is to improve patient care through the development of a new surgical technique and through basic science studies which seek to better understand articular cartilage wear initiation. Four studies, which address this objective are summarized below. Osteochondral allograft transplantation provides a safe and effective treatment option for large cartilage defects, but its use is limited partly due to the difficulty of matching articular surface curvature between donor and recipient. We hypothesize that bendable osteochondral allografts may provide better curvature matching for patella transplants in the patellofemoral joint. The finite element study presented in Chapter 2 investigates patellofemoral joint congruence for unbent and bendable osteochondral allografts, at various flexion angles. Finite element models were created for 12 femur-patella osteochondral allograft pairings. Two grooves were cut into the bony substrate of each allograft, allowing the articular layer to bend. Patellofemoral joints with either unbent (OCA) or permanently bent (BOCA) allografts were articulated from 40 to 70 degrees flexion and contact area was calculated. OCAs and BOCAs were then shifted 6 mm distally toward the tibia (S-OCA, S-BOCA) to investigate the influence of proximal-distal alignment on congruence. On average, no significant difference in contact area was found between native patellofemoral joints and either OCAs or BOCAs (p > 0.25), indicating that both types of allografts restored native congruence. This result provides biomechanical support in favor of an emerging surgical procedure. S-BOCAs resulted in a significant increase in contact area relative to the remaining groups (p < 0.02). The fact that bendable osteochondral allografts produced equally good results implies that these bendable allografts may prove useful in future surgical procedures, with the possibility of transplanting them with a small distal shift. Surgeons who are reluctant to use osteochondral allografts for resurfacing patellae based on curvature matching capabilities may be more amenable to adopting bendable osteochondral allografts. The recent development of bendable osteochondral allografts provides the potential for improved osteoarthritis treatment for joints whose current treatment is unsatisfactory. One such joint is the carpometacarpal joint in the thumb. While the current standard of care for carpometacarpal osteoarthritis, ligament reconstruction and tendon interposition, can reduce pain in the joint, it does not restore full joint function and mobility. A proposed alternative includes using an osteochondral allograft harvested from the femoral trochlea in a donor knee, machining grooves in the bone to allow the allograft to bend, and replacing the trapezium with this bent osteochondral allograft [1,2]. Chapter 3 of this dissertation discusses adjustments to the original design of the bendable allograft and the design of a custom surgical tool to perform the proposed surgery. Specification changes of the allograft included an overall size reduction in order to better fit within the carpometacarpal joint, minimum bone thickness requirements to avoid bone cracking during the surgical procedure, and a reduction from three grooves to two grooves, which provided sufficient bending yet avoided fracture of the allograft. The surgical tool was designed to be a custom forceps device, whose primary features included (1) jaws with an angled face to match the angle of allograft bending and (2) insertion holes for the Kirschner wire and compression screws used to anchor the allograft in the bent position. These customizations allow the tool to be used to bend the allograft, fix it in the bent configuration, and place the allograft in its proper position in the hand during anchoring of the bent allograft to the native trapezium. The final two studies presented in this dissertation focus on furthering our current understanding of wear and structure-function relationships of articular cartilage. We hypothesize that cartilage wears due to fatigue failure in reciprocating compression instead of reciprocating friction. Chapter 4 compares reciprocating sliding of immature bovine articular cartilage against glass in two testing configurations: (1) a stationary contact area configuration (SCA), which results in static compression, interstitial fluid depressurization and increasing friction coefficient during reciprocating sliding, and (2) a migrating contact area configuration (MCA), which maintains fluid pressurization and low friction while producing reciprocating compressive loading during reciprocating sliding. Contact stress, sliding duration, and sliding distance were controlled to be similar between test groups. SCA tests exhibited an average friction coefficient of μ=0.084±0.032, while MCA tests exhibited a lower average friction coefficient of μ=0.020±0.008 (p<10^(-4)). Despite the lower friction, MCA cartilage samples exhibited clear surface damage with a significantly greater average surface deviation from a fitted plane after wear testing (R_q=0.125±0.095 mm) than cartilage samples slid in a SCA configuration (R_q=0.044±0.017 mm, p=0.002), which showed minimal signs of wear. Polarized light microscopy confirmed that delamination damage occurred between the superficial and middle zones of the articular cartilage in MCA samples. The greatest wear was observed in the group with lowest friction coefficient, subjected to cyclical instead of static compression, implying that friction is not the primary driver of cartilage wear. Delamination between superficial and middle zones imply the main mode of wear is fatigue failure under cyclical compression, not fatigue or abrasion due to reciprocating frictional sliding. The final study of this dissertation, presented in Chapter 5, investigates the importance of collagen fibril distribution in articular cartilage computational models. Finite element models were created to approximate a bovine humeral head and replicate previous experimental loading conditions [3]. Five different finite element analyses were run, each using a different fibril distribution model. Three of the models used two, four, or eight discrete fibril bundles, while two models used continuous fibril distributions with either isotropic or depth-dependent ellipsoidal distributions. Two primary findings arose from this investigation. The first was the discovery that as the fibril distribution became more isotropic, the strain throughout the tissue decreased, even though the contact area between the articular surface and rigid platen remained relatively equal across distribution models. This suggests that computational models which approximate the collagen fibrils with an isotropic distribution may be underestimating the strain through the depth of the tissue. The second primary finding was that in the discrete distribution model with two fibril bundles, which followed the classically described Benninghoff structure [4], the greatest magnitude of shear strain during compressive loading was observed in the middle zone. However, the highest magnitude of shear strain observed in the isotropic fibril distribution model occurred in the deep zone near the subchondral surface. The observed results suggest that the type of fibril distribution used to model collagen in articular cartilage plays a role in depth-dependent strain magnitude and strain distribution.

Thesis (MScPhysio)--Stellenbosch University, 2012. / Background: Evidence for the effectiveness of physiotherapeutic interventions in the management of knee osteoarthritis (OA) is synthesised in the current clinical guidelines (CGs), providing clinicians with readily accessible and interpretable practice guidelines. However, CGs are often not specific to the local context of the target users, therefore hindering successful implementation of evidence into clinical practice. Formulating succinct and composite recommendations by synthesising the current CGs reporting on the evidence-based (EB) management of knee OA may assure contextual relevance and facilitate implementation of evidence into clinical practice. In addition, multifaceted interventions, such as evidence-based practice (EBP) workshops, are also postulated to promote the implementation of guideline recommendations, thereby enhancing clinical outcomes. Objectives: The primary objectives of this study were to: 1) describe the range of EB physiotherapeutic interventions in the management of knee OA as documented in the current CGs; and 2) develop composite clinical recommendations for a specific group of users working in Jerusalem. A secondary study objective was to ascertain the effect of translating the knowledge through a specifically-designed EBP workshop on the uptake of knowledge and implementation of EBP into clinical practice by physiotherapists working in Jerusalem. The EBP workshop was aimed at educating physiotherapists about the EB physiotherapeutic techniques for knee OA management. Study design: Two studies were conducted. A systematic review (SR) into EB clinical guidelines was conducted to describe and synthesise the available evidence and formulate composite recommendations for knee OA. The results of the SR were used to design an EBP workshop aimed at educating physiotherapists about EB physiotherapeutic techniques for treating knee OA patients. A pre-post quasi-experimental design was then conducted to assess the effect of this EBP workshop on the uptake and implementation of EBP into clinical practice amongst public sector physiotherapists working in Jerusalem. Methodology for quasi experimental study: Physiotherapists who regularly treat knee OA patients were recruited from a list of members registered with the Palestinian Physiotherapy Association Jerusalem. A three-month retrospective audit (initial audit) of knee OA patients’ physiotherapy records kept by the participating physiotherapists was conducted to establish current management patterns. EB strategies for knee OA was presented to the participating physiotherapists during a one-day workshop. A second audit of physiotherapy records was conducted three months after the EBP workshop to establish changes in the selection of physiotherapeutic management techniques for knee OA. Results: The initial audit revealed that the participating physiotherapists utilized one high EB modality namely, exercises, as a core management strategy in knee OA, but did not frequently implement other high EB modalities such as self-management and weight-loss programs. Following the EBP workshop, a statistically significant increase (p=0.008) in the implementation of weight-loss and self-management strategies in the management of knee OA was noted. Conversely, a statistically significant decrease was noticed in using patellar taping (low EB modality) in the management of knee OA (p=0.04). No significant changes were noticed in the utilization of other physiotherapy modalities supported by weak or modest EB recommendations. Conclusion: The study concluded that physiotherapists inherently prescribed exercise as a core management strategy for knee OA. Modalities supported by modest levels of evidence were used as adjunct treatments. The EBP workshop facilitated the increased application of high EB modalities such as weight-loss and self-management programs. The results of this study illustrate that an EBP workshop may be effective in promoting the implementation of EB physiotherapeutic modalities in the management of knee OA. However, larger studies with longer follow-up periods are required. / No Afrikaans abstract available

Osteoarthritis, a degenerative joint disease that affects nearly 30 million Americans, is characterized by lesions of articular cartilage that often lead to severe pain and loss of joint function. The current economic burden of osteoarthritis is estimated to be approximately $190 billion, and with the prevalence of arthritis expected to rise due to the aging population, the associated costs are forecasted to increase. Debilitating osteoarthritis is managed clinically by the surgical implantation of a cartilage graft or cartilage cells to replace the damaged tissue; however, current repair methods often result in poor long-term outcomes due to inadequate integration of the graft with host cartilage and bone. Thus, there is a significant clinical need for approaches that enable functional connection of grafting devices to the host tissue. To address this challenge, the strategy described in this thesis is a versatile, cup-shaped fibrous scaffold system designed to promote the simultaneous integration of the cartilage graft with both the host cartilage and subchondral bone. This thesis is guided by the hypotheses that 1) graft integration with native cartilage can be strengthened by inducing chondrocyte migration to the graft-cartilage junction through chemotactic factor release from the walls of the cup, and 2) graft integration with host bone and the formation of calcified cartilage can be facilitated by pre-incorporation of calcium phosphate nanoparticles in the base of the cup. To test these hypotheses, a microfiber-based integration cup was designed with degradable, polymer-based walls that release insulin-like growth factor-1, which is well-established for inducing chondrocyte migration, and a base consisting of polymer with calcium deficient apatite nanoparticles. In the first aim of this thesis, the dose of insulin-like growth factor-1 in the cup walls was optimized to enhance the migration of cells from surrounding cartilage into the scaffold, and this design was tested in vitro to ensure that the scaffold supports chondrocyte viability, growth, and biosynthesis of a cartilage-like matrix. In the second aim of this thesis, the composition and dose of calcium phosphate in the base of the cup was optimized to support chondrocyte growth and the production of calcified cartilage-like tissue. Subsequently, in the third aim, the independently developed walls and base were joined into a scaffold that was tested in vitro and in vivo, using a simulated full thickness defect model, to examine its potential for clinical translation. Results from these studies demonstrate that the cup system can be implemented with autologous tissue and cell-based grafting strategies as well as with tissue engineered hydrogel grafts to promote integration with host tissue. Moreover, these investigations have yielded new insights into both chemical and structural parameters that direct chondrocyte migration and calcified cartilage formation. In summary, this thesis describes the design and optimization of a novel, multi-functional device for improving integration of cartilage grafts with host tissues. The impact of the studies in this thesis extends beyond cartilage integration, as the interface scaffold design criteria elucidated here are readily applicable to the formation of interfaces between other grafts and host tissues.

Articular cartilage is a hydrated soft tissue with a fibrous solid matrix characterized by high porosity and low permeability. It is the bearing material of diarthrodial joints, permitting motion and transmitting loads with extraordinarily low friction. This function may be disrupted pathologically by osteoarthritis, a disease where cartilage becomes weakened and eroded. Osteoarthritis creates pain during normal activities like walking or grasping, thus diminishing quality of life. The disease affects nine percent of Americans and is one of the leading causes of disability worldwide. There is presently no cure or prevention for osteoarthritis, only palliative treatments designed to help patients manage pain and regain mobility. New such treatments are developed in part by advancing the science of cartilage mechanics, structure and function, and this dissertation presents novel contributions toward this effort: Chapters 2, 3, and 4 enhance our knowledge of the structure-function relationships critical to our understanding of cartilage friction and load support. Whereas most prior theoretical and experimental studies have focused on the analysis of small cylindrical explants, or idealized joint geometries such as cylindrical or spherical articular layers, these chapters describe novel investigations performed on whole articular layers of the shoulder and knee joints. Insights from these investigations have a direct impact on our formulation of design objectives in cartilage tissue engineering, whose purpose is to grow constructs that reproduce the functional properties of native cartilage. The studies presented in this chapter are critical to ongoing tissue engineering studies in our laboratory, which has pioneered the development of anatomically sized cartilage constructs. Finally, Chapter 5 describes the development of a novel clinical treatment for thumb osteoarthritis that uses bent osteochondral allografts (living bone and cartilage from human donors) to replace the eroded thumb trapezial articular layer with a healthy and thick articular layer from another joint such as the knee. This highly promising treatment strategy overcomes the limitation of size mismatch between donor and recipient which had relegated osteochondral allograft surgery to a niche treatment. Like other fibrous tissues, cartilage exhibits tension-compression nonlinearity, meaning it can be 100 times stiffer in tension than in compression. Tension-compression nonlinearity allows compressive physiologic joint loads to be supported by tensile stress within the collagen fibers and elevated fluid pressure, effectively shielding the solid matrix from compressive load. According to theory, fluid load support derives directly from tension-compression nonlinearity. Fluid load support is also a dominant mechanism of cartilage lubrication. Because cartilage is 80 to 90% water, most of the contact traction on the porous cartilage surface takes the form of hydrostatic fluid pressure. Friction forces only occur upon solid-on-solid contact, so cartilage friction is nearly negligible, even for joint contact forces that may routinely exceed three or four times the body’s total weight. The dependence of friction on fluid load support is demonstrated by experiments that simultaneously measure interstitial fluid pressure and friction - a transient rise in friction occurs as pressure subsides and fluid drains from the tissue. These structure-function relationships have been identified over decades of research, mostly through small cartilage explant studies, which have supported hypothesized mechanisms under non-physiologic conditions. Therefore, in situ studies utilizing intact, naturally-congruent articular surfaces under physiologic loading conditions would significantly extend and validate these principles. For example, friction may rise nearly 100-fold after only 1 hour in cartilage explant experiments, yet there is no evidence that normal daily activities spanning 16 hours or more lead to cartilage damage. Can fluid load support sustain low friction under these relatively harsh conditions? To date, no study has examined this question, so Chapter 2 of this work addresses the hypothesis that the friction coefficient of diarthrodial joints can remain low over a full day of loading at physiologic speeds and load magnitudes. Another question that may be uniquely addressed by an in situ analysis is: What is the complete state of stress within naturally-congruent cartilage layers? A primary hypothesis for the initiation and progression of osteoarthritis is that the state of stress within articular cartilage may exceed a threshold beyond which the tissue is unable to repair itself. Since the complete stress tensor within a material is immeasurable, techniques such as finite element analysis must be used to examine the state of stress. Additionally, a theoretical framework such as mixture theory may be used to examine the stresses in the fluid and solid constituents of the tissue separately, making it possible to test theories of solid matrix damage. Chapter 3 of this work uses this strategy to examine the hypothesis that physiologic solid matrix stresses within anatomically-shaped, biphasic, tension-compression nonlinear cartilage layers are primarily tensile, despite the fact that the articular layers are loaded in compression. The proteoglycan content of articular cartilage gives the tissue an osmotic swelling pressure that is resisted by tensile stresses in the collagen fibrils, even in the absence of external loads. This charge effect may be additionally incorporated into a mixture theory finite element analysis to examine the role of osmotic swelling on the solid matrix stresses in a physiologic, in situ analysis. This capability has only been developed recently and is explored for the first time in Chapter 4. The final part of this work translates basic cartilage science into a clinical therapy for thumb joint osteoarthritis, a common site for this disease. The current gold-standard treatment for thumb joint osteoarthritis replaces the trapezium bone with a soft-tissue tendon autograft, relieving pain but significantly weakening hand strength. Living osteochondral allograft transplantation may provide a relatively straightforward treatment alternative, though this procedure has not been used for the thumb due to the inadequate availability of suitable allografts. The ideal allograft would have a relatively thick articular layer to provide sufficient compliance for promoting joint congruence with the mating metacarpal surface, and surface curvatures that match the saddle-shaped anatomy of the distal trapezial articular surface to reproduce the normal joint motions. A potential solution that would provide suitable trapezium osteochondral allografts for patients involves precisely machining and bending allografts from a lower extremity joint with thicker cartilage, such as the distal femoral surface of the knee, to match the shape and curvature of the trapezium. Such bent osteochondral allografts would provide all the desired benefits of the ideal arthroplasty. Chapter 5 outlines the development of this novel technology, including proof of concept and feasibility demonstrations, business strategy and market analysis.

Osteoarthritis is a debilitating and irreversible disease afflicting the synovial joints. It is characterized by pain and hindered mobility. Given that osteoarthritis has no cure, current treatments focus on pain management. Ultimately, however, a patient's pain and immobility necessitates joint replacement surgery. An attractive alternative to this treatment paradigm, tissue engineering is a promising strategy for resurfacing the osteoarthritis-afflicted cartilage surface with a biochemically and biomechanically similar tissue to the healthy native cartilage tissue. The most successful cartilage tissue engineered systems to date can repeatably grow constructs ~4 mm in diameter with native proteoglycan and compressive mechanical properties. Unfortunately, as symptomatic cartilage typically presents only once lesions span large regions of the joint (~25 mm in diameter), these small construct are of limited use in clinical practice. Numerous attempts to simply grow a construct large enough to span the size of an osteoarthritic lesion have shown that the growth of large engineered tissues develop heterogeneous properties, emphasizing the need for culture protocols to enhance tissue homogeneity and robustness. In particular, as nutrient limitations drive heterogeneous growth in engineered cartilage, developing strategies to improve nutrition throughout the construct are critical for clinical translation of the technology. To this end, our lab has successfully supplemented nutrient channels within large engineered cartilage constructs to improve the functional properties of developing tissue. However, it is unknown what the optimal nutrient channel spacing is for growing large cartilage constructs of anatomical scale. Additionally, the fundamental factors and mechanisms which drive tissue heterogeneity have not been implicated, making the results of channel-spacing optimizations difficult to translate across different systems. Computational models of growth, faithful to the physics and biology of engineered tissue growth, may serve as an insightful and efficient tool for optimally designing culture protocols and construct geometries to ensure homogeneous matrix deposition. Such computational tools, however, are not presently available, owing to the unresolved mechanical and biological growth phenomena within developing engineered cartilage. This dissertation seeks to develop and implement computational models for predicting the biochemical and biomechanical growth of engineered tissues and apply these models to optimizing tissue culture strategies. These models are developed in two stages: 1) based on our recent characterization of the nutrient demands of engineered cartilage, models are developed to simulate the spatial biochemical deposition of matrix within tissue constructs and, subsequently, 2) based on models of biochemical matrix deposition we develop models for simulating the mechanical growth of tissue constructs. To accomplish these tasks, we first develop models simulating glucose availability within large tissue constructs using system-specific modeling based on our recent characterization of the glucose demands of engineered cartilage. These models led to early insight that we had to enhance the supply of glucose within large tissue constructs to ensure maximal matrix synthesis throughout culture. Experimental validations confirmed that increasing glucose supply enhanced matrix deposition and growth in large tissue constructs. However, even despite the increased glucose supply, increasing the size of constructs demonstrated that severe matrix heterogeneities were still present. Subsequent nutrient characterization led to the finding that TGF-ß transport was significantly hindered within large tissue constructs. Incorporating the influence of glucose and TGF-ß into the computational model growth kinetics. Using both nutrients, models recreated the heterogeneous matrix deposition evident in our earlier work and could account for the role of cell seeding density and construct geometry on tissue growth. The insights gathered from this modeling analysis led to important changes in our culture protocols: we could reduce the dose of TGF-ß from 10 ng/mL to 1 ng/mL for constructs cultured with channels, saving considerable expense while still maintaining a high level of matrix synthesis throughout the construct. In the presence of sufficient nutrition, we witnessed an unprecedented level of matrix deposition and physical growth of the constructs. In fact, by using developmentally physiologic cell seeding densities (120 million cells/mL) and providing adequate nutrition, constructs physically grew to 9-times their originally cast size. Despite such encouraging growth, tissue function properties plateaued at sub-physiologic levels. For insight into the connection between matrix deposition and tissue mechanics, we extended the computational growth models to consider the mechanisms underlying physical growth. Interestingly, we found that a large matrix synthesis mismatch between proteoglycans and collagen gave rise to the excessive tissue swelling. Computational models of this matrix synthesis mismatch predicted the high tissue swelling displayed experimentally only when a damage-able collagen fiber material was implemented. Together, the experimental and modeling evidence suggested a new mechanism of cartilage growth: the high proteoglycan deposition creates a swelling pressure within the nascent tissue which outcompetes the restraining force of newly deposited collagens; this rapid tissue swelling disrupts a functional collagen network from forming. Subsequent analysis suggested that the disruption of the collagen network prevented the formation of collagen crosslinks, stymieing the development of native functional properties. Based on this insight into the mechanisms of cartilage growth, we developed a culture systems to improve tissue functional properties. Modeling analysis indicated two novel routes for improving tissue mechanics: either through 1) reducing the swelling response (synthesis and deposition) of proteoglycans or 2) enhancing and reinforcing the newly synthesized collagen to prevent disruptions brought on by tissue swelling. We developed a cage culture system for resisting the swelling pressure of deposited proteoglycans and reenforcing the deposition of new collagens. Using this cage system, we grew tissue constructs with enhanced functional properties using two separate scaffold systems – agarose and a cartilage-derived matrix hydrogel – suggesting this mechanism of growth is fundamental to engineered cartilage development. This work has generated a novel paradigm towards engineering cartilage constructs using biomimetic strategies. Performing simulations with the validated, computational growth models allowed anatomically-sized cartilage constructs to grow into the largest, homogeneous cartilage constructs grown to date. Models presented a new level of insight into the nutrient demands of developing tissues, allowing for the first time the successful development of large tissue constructs grown with developmentally physiologic cell seeding densities. In this way, tissue constructs growth followed a biomimetic approach, based on the high cell densities and cartilage canals and vasculature present during fetal cartilage development. Adequate nutrition led to high levels of tissue growth not previously experienced in vitro, a result of adequately nourishing primary chondrocytes, a cell type which preferentially deposits proteoglycans over collagen. We therefore developed a cage-based growth system to resist the proteoglycan-induced tissue swelling in a manner similar to the fetal development of cartilage where the resident cells synthesize more collagens than proteoglycans. Together, the use of nutrient growth models, high cell seeding densities, and culture systems to strengthen the collagen-framework of de novo cartilage proved beneficial for engineering anatomically-sized cartilage constructs. The fundamental mechanisms identified here are likely to be universal across a number of engineered cartilage systems and will be adapted to more clinically-relevant cell sources in future our work.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

Osteoarthritis is a degenerative joint disease characterized by painful, progressive articular cartilage lesions that deteriorate joint function. It remains leading cause of disability in the United States, affecting nearly 30 million Americans with increasing prevalence in the aging population, which has resulted in an annual economic burden of $128 billion. Symptomatic, full thickness cartilage injuries often require surgical intervention, because the tissue is predominantly avascular and thus has a limited self-healing capacity. However, clinical management strategies including matrix-induced autologous chondrocyte implantation and osteochondral grafting are inadequate in the long-term due to poor integration of cartilage grafts with surrounding host cartilage and subchondral bone. In addition to physical congruence between graft and host cartilage, a structural or chemically functional barrier that limits osseous invasion into the cartilage compartment is critical in order to maintain the integrity of repaired cartilage. Given these significant clinical challenges, the objective of this thesis is to establish design principles for homotypic and heterotypic tissue integration via a cup-shaped fibrous scaffold system that encapsulates cartilage grafts (autologous or engineered), and integrates them simultaneously with host cartilage and bone at their respective interfaces. Additionally, to facilitate clinical translation of the scaffold cup, an innovative “green electrospinning” method is developed using FDA Q3C Class 3 solvents with minimal manufacturing impact on the environment. It is hypothesized that, to fuse cartilage grafts with host cartilage, the walls of the envisioned cup can direct cell migration directly to the graft-host cartilage interface via chemotactic agent delivery, where scaffold electroactivity will encourage cells to deposit a structurally contiguous neocartilage matrix. At the boundary between the graft and underlying bone, the scaffold cup base will mimic the topography and ceramic chemistry of the native osteochondral interface while preventing bone vasculature from growing upwards into the cartilage, guided by the hypothesis that this will enable the formation of a calcified cartilage interface layer that merges the graft and subchondral bone. To test these hypotheses, this thesis began with green electrospinning the scaffold cup walls incorporated with insulin-like growth factor 1 (IGF-1), a well-established chondrocyte chemoattractant that induced cell migration from cartilage autografts towards resulting fibers. Additionally, the walls contained an optimized dose of graphite nanoparticles to impart electroactivity to the fibers. Mimicking the fixed charge density of cartilage in this way promoted chondrocyte proliferation and biosynthesis of a hyaline cartilage-like matrix in vitro, with selective regulation of proteoglycans (biglycan and decorin) and downregulation of collagen type I compared to a graphite-free fiber control. Moreover, the graphite fibers sequestered IGF-1, sustaining release of the growth factor and improving functional graft-cartilage shear integration strength in vitro. In a full thickness defect osteochondral construct repaired with the scaffold cup and implanted subcutaneously in rat dorsi, localized IGF-1 delivery promoted graft-host cartilage interface matrix elaboration with significantly greater integration strength measured with graphite in the cup walls. For integration with subchondral bone, design criteria for the scaffold cup base were set by quantitatively mapping the compositional and morphometric characteristics of healthy and osteoarthritic human osteochondral tissues, and evaluating FEBio simulations of calcified cartilage and polymer-ceramic composite fibers in silico. These analyses established the need for an interdigitating mesh topography and ceramic particle incorporation, which minimize shear and distribute loading across the fibers, respectively, recapitulating the osteochondral interface’s force gradient from cartilage to bone in order to functionally integrate the tissues. Thus, the dose of calcium deficient apatite (CDA) nanoparticles, which capture the high calcium-phosphate ratio and semi-crystalline atomic structure of native bone mineral, was optimized to promote deep zone chondrocyte growth and biosynthesis of a calcified cartilage matrix in vitro. Moreover, CDA enhanced remodeling of the interface in vivo, with undulating fibers preventing osseous upgrowth. Taken together, these findings delineate the importance of strategic biomimicry in scaffold design, specifically with regards to interface regeneration and cartilage integration. The proposed approach is unique in that it utilized cell homing and an electroactive substrate to mimic properties of the cartilage matrix, with a strategy for simultaneous graft integration with host cartilage and bone. Moreover, the cup design is readily adaptable to current cartilage repair techniques including press-fit autografting and cell-based graft implantation, as well as emerging tissue engineered grafting strategies. Beyond cartilage repair, the scaffold design criteria established in this thesis are broadly applicable to integrating other complex tissue systems, and may inform the regeneration of critical soft-soft (muscle-tendon) and soft-hard (tendon- or ligament-bone) interfaces in the musculoskeletal system.