Osteoarthritis (OA) is a progressive and complex joint disease that results from breakdown of articular cartilage and remodeling of underlying bone, which affects millions of Americans. While the expected lifetime of the load-bearing cartilage tissue should coincide with the lifespan of an individual, it has a limited ability to self-repair and the damage to the tissue can accumulate severely. One of the major challenges in OA treatment is its long asymptomatic period. Symptoms usually become noticeable when the disease is reaching advanced stages, and currently there is no effective intervention for early stages of OA. This may be due to lack of a reliable diagnostic method for detecting early OA. While OA is a degenerative joint disorder that may lead to gross cartilage loss and morphological damage to other joint tissues, a lot of subclinical, subtle biochemical changes occur in the early stages of OA progression. The degradation of collagen type II matrix in articular cartilage extracellular matrix (ECM) network may corelate with the progression of cartilage OA. During onset of OA, with the loss of collagen crosslinks, the collagen matrix in cartilage ECM becomes more disorganized and the cartilage can become susceptible to disorder and thus aggravate the degeneration. Raman spectroscopy has been utilized in studies of components of connective tissues, such as OA, and cartilage degradation. Although studies have demonstrated the potential of applying Raman for diagnosing cartilage degeneration, the analysis of Raman spectrum obtained from articular cartilage is rather complicated and so far there is no generally accepted quantitative analysis for diagnosing early stages of OA. The first stage of this doctoral study aims to extend the capability of Raman spectroscopy to quantitatively characterize collagen network in articular cartilage, to investigate the possible correlation with the degeneration of OA. The first part of this doctoral dissertation is focused on developing a novel, non-destructive, quantitative diagnostic modality, based on Raman spectroscopy that has potential to detect changes in biochemical composition of articular cartilage. The study is focused on the basic research associated with quantification of crosslink density and kinetics of the crosslinking process. A theoretical and computational framework for characterization of collagen crosslinks has been established and applied onto two models, 2-dimensional collagen type I thin films, and immature bovine, proteoglycan depleted, articular cartilage. Glutaraldehyde solution has been applied onto the model as a convenient method to introduce various levels of collagen crosslinks. Refractive error is a problem with focusing light accurately onto the retina due to the shape or other misfunctioning of the eye, rather than on the retina for the normal vision. The most common types of refractive errors are near-sightedness, far-sightedness, astigmatism, and presbyopia. Refractive errors have become a growing public health problem worldwide. Its incidence has doubled over the last 50 years in the United States and Europe. It is even more significant issue in some East Asian countries, where its prevalence reaches 70 to 90%. Most affected individuals use spectacles or contact lenses, which generally provides adequate refractive error correction. However, both are subject to limitations. Glasses do not work well in the rain and mist may form on them following changes in temperature or humidity. Contact lenses improve the field of vision and acuity, but many people find their presence on ocular surfaces intolerable. Over the last two to three decades, refractive surgery for the permanent correction of vision has thus emerged as an attractive choice for many patients. However, such a surgery is an invasive procedure that may compromise corneal structure, and postsurgical complications have been reported. In the second stage of this doctoral work, a novel, non-invasive femtosecond laser collagen crosslinks manipulation method is studied. This laser collagen crosslinking treatment is applied on corneal tissue for vision correction. Two examples of the laser treatment on an ex vivo porcine eyes model are proposed in the study: corneal flattening, which is used to correct refractive errors due to myopia, and corneal steepening, which is used to treat hyperopia. The effective refractive power is used to evaluate the effectiveness of the two different treatments. The depth of the crosslinked region in the cornea is assessed by two-photon autofluorescence (TPF) imaging. TPF imaging can be used to visualize changes induced in the cornea, because collagen is a primary extracellular source of nonlinear emissions. The safety of the proposed treatment methods is examined by haematoxylin and eosin (H&E) stained histological sections of corneas. The ex vivo porcine corneas are also cultured for one week after treatment, to determine whether crosslink density remains stable, and to check for degradation in the crosslinked layers of the stromal matrix, and further prove the safety of the proposed laser treatment method through the evaluation of cell viability after one week of treatment. An in vivo rabbit animal model, widely used as a model for the correction of refractive errors, is further utilized to demonstrate the stability and safety of the induced changes. The effective refractive power of live rabbits is assessed 24 h, seven days, and then weekly up to three months after the laser crosslinking treatment. The safety of the laser treatment is first evaluated by histology staining, and further confirmed by in vivo confocal laser scanning microscopy. This laser treatment approach could expand the pool of patients eligible for permanent vision correction, while simultaneously eliminating the adverse effects associated with current forms of surgery. Furthermore, the approach described is also suitable for the treatment of other disease for collagenous tissues. The last chapter of in this doctoral dissertation have discussed the results of applying this laser treatment techniques for the treatment of progressive OA. Finally, in a preliminary study, the proposed femtosecond laser treatment modality developed for corneal tissue has been applied onto articular cartilage towards slowing down or retarding progression of early osteoarthritis. We hypothesize that degradation of the articular cartilage extracellular matrix can be slowed down or reversed in the collagen network crosslinked with a femtosecond laser. We further theorize that the crosslinking mechanism introduced in the corneal tissue, which relies on laser ionization and dissociation of the tissue interstitial water to produce of refractive oxygen species, can increase crosslink density of collagen network in an articular cartilage. In the study, the treatment has been applied onto devitalized and live immature bovine cartilage explants, as well as cartilage plugs obtained from OA afflicted human cadaver joints. The preliminary results have shown that the proposed treatment has potential to enhance tissue mechanical properties, and increase wear resistance, an important factor in slowing down the progression of OA. Furthermore, preliminary imaging of live/dead stained tissue has shown that the laser treatment has minimal adverse effects up to two weeks after the laser irradiation.

Tightly focused femtosecond laser pulses are widely used in the biomedical field due to their nonlinear multiphoton precision and minimal thermal side effects. Below the threshold of optical breakdown, light energy contributes to photochemical reactions that introduce more chemical bonding in the form of collagen crosslinking (CxL) in extracellular matrices of transparent tissues such as corneal stroma. Previously, based on the principles of ultrafast laser-tissue interaction, a novel collagen CxL method relying on low-density plasma (LDP) generating reactive oxygen species (ROS) was proposed and applied to cornea tissue for vision correction by the Vukelic Group and extended to articular cartilage tissue for early osteoarthritis treatment in collaboration with Musculoskeletal Biomechanics Research Laboratory. Despite the efficiency and safety of the procedure, LDP was elusive and challenging to control due to its potential dependence on a cascade of intertwining factors such as ultrafast laser wavelength, power, pulse duration, repetition rate, and ionization resonance. This thesis has two aims: the first is to investigate the photochemical laser-tissue interaction with femtosecond nanojoule energy pulses, and the second is to develop robust and practical laser parameter envelopes for treating corneal ectatic diseases and osteoarthritis. Chapter 2 proposes a corneal epithelium-stromal level wound healing treatment. Relying on the interaction between reactive oxygen species (ROS) created by low-density plasma (LDP) therapy and inflammatory cytokines, epithelium recovery is accelerated on in vivo rabbit corneas. Chapters 3 to 5 focus on photochemical reaction-based morphological correction and biomechanical enhancement for corneal diseases such as keratoconus and astigmatism. A wavelength-independent, nonenzymatic CxL technique based on oxygen-independent, pentose-mediated glycation and ROS acceleration is developed; collagen CxL efficiency is tested through autofluorescence microscopy and nanoindentation. Subsequently, the combined effects of simultaneous external mechanical loading and nonenzymatic collagen CxL, achieved by both traditional CxL that involves soaking eyes with riboflavin solution, a photosensitizer, and then activating it with ultraviolet A light (UVA-Riboflavin-CxL) and new ROS catalyzed glycation CxL (ROS-Glycation-CxL) techniques, are investigated on ex vivo rabbit corneas. Through X-Ray Diffraction, permanent adjustments to the ultrastructure of collagen fibril packing are observed, ultimately contributing to refractive power changes in corneal topography. Furthermore, with the addition of melanin application that increases absorption and ionization efficiency, a robust method for generating plasma and reactive oxygen species (ROS) is proposed and implemented on ex vivo corneas to address ectatic diseases. Chapter 6 discusses the effect of plasma-guided laser collagen CxL on articular cartilages’ compressive equilibrium modulus and chondrocyte viability. Stemming from the melanin-assisted protocol and ultrafast pulses' high peak power, a plasma spark-mediated laser treatment is hypothesized to biomechanically enhance both bovine and human articular cartilage superficial zone for the treatment of osteoarthritis. Chapter 7 concludes this thesis and proposes future directions.

Osteoarthritis (OA) of the knee, a musculoskeletal disease characterized by degenerations in multiple joint tissues including the articular cartilage and subchondral bone, is a major clinical challenge worldwide that currently has no cure. Traumatic knee injuries such as anterior cruciate ligament (ACL) tear predispose subjects to early onset of post-traumatic OA (PTOA), necessitating the development of effective disease modifying therapies as total knee replacement surgeries have a limited lifetime. Significant knowledge gap remains in the pathogenesis of OA, while recent evidence suggests the important role of subchondral bone microstructure and mechanics in OA development. Subchondral bone is composed of the subchondral bone plate, a thin layer of cortical lamella, and the subchondral trabecular bone, composed of individual plate-like and rod-like trabeculae. These trabecular plates and rods determine the microstructure and mechanics of trabecular bone entirely and can be quantitatively analyzed using individual trabecula segmentation (ITS). Recent application of ITS showed that changes in the plate-and-rod microstructure of subchondral trabecular bone precede cartilage damage and are implicated to play a role in disease pathogenesis. Studies presented in this thesis aim to provide a deeper understanding of subchondral bone in knee OA scientifically and clinically, which may ultimately be used to improve diagnosis, prevention and treatment of this prevalent and disabling disease. In the first study, we comprehensively quantified microstructural and tissue biomechanical properties of the subchondral bone and articular cartilage in human knee specimens with advanced OA and control knees without OA. We found reduced tissue modulus in trabecular plates and rods in regions with moderate OA, where cartilage is still intact, that persisted in severe OA regions, where cartilage is severely damaged. These observations suggest that tissue biomechanical changes in the subchondral trabecular bone may precede cartilage damage in OA development. Furthermore, we found strong correlations between structural and mechanical parameters of the cartilage and subchondral bone in CT knees, suggesting cross-talk at the tissue level. This coupling persisted in moderate OA regions but disappeared in severe OA regions, suggesting that loss of tissue crosstalk may be an additional indicator of disease progression. In the second study, we quantified subchondral bone microstructural changes after ACL tear in vivo in human subjects using the second-generation high resolution peripheral quantitative computed tomography (HR-pQCT). We examined short-term longitudinal changes during the acute phase (~18 days to ~141 days) after injury, as well as long-term adaptations (~5 years post injury) in the injured knee relative to the contralateral knee in a cross-sectional cohort. We found subchondral bone loss within 1 month from injury that primarily targeted trabecular rods, especially at the distal femur. We also found increased spatial heterogeneity in subchondral trabecular microstructure within the injured knees compared to the contralateral knees in the long-term after injury. These findings indicate that ACL tear results in both short-term and long-term microstructural adaptations in the subchondral bone. ITS based on HR-pQCT knee scans may be a valuable tool to monitor disease progression in vivo. Finally, we quantified subchondral bone microstructural changes after ACL-transection in a canine model of PTOA and investigated the effects of bisphosphonate and NSAID treatment on subchondral bone changes and OA progression. Studies were conducted in skeletally-mature and juvenile animals to investigate the effect of injury age. We found that subchondral bone adaptations after surgery and treatment effects depended on skeletal maturity of animals. In mature animals, changes in the microstructure of trabecular plates and rods occurred 1-month post-op and persisted until 8-months post-op. Bisphosphonate treatment attenuated these microstructural changes and cartilage degeneration while NSAID treatment did not. In juvenile animals that have not reached skeletal maturity, transient changes in trabecular plate and rod microstructure occurred at 3-months post-op but disappeared by 9-months post-op. Neither bisphosphonate nor NSAID treatment attenuated bone microstructural changes or cartilage damages. These findings suggest that age and skeletal maturity at time of injury may need to be considered as additional factors in studying PTOA progression and developing preventative treatments. Taken together, these studies highlight the importance of microstructural and tissue biomechanical changes of subchondral bone in the development of OA. In vivo quantification of subchondral bone using advanced imaging modalities enable longitudinal monitoring of disease progression. Therapeutic agents targeting subchondral bone changes after traumatic injury may be effective preventative strategies for PTOA.

Post-traumatic osteoarthritis (PTOA) of the knee is a complication resulting from direct injury to the joint, such as anterior cruciate ligament and meniscus tears, and accounts for approximately 12% of all OA cases. The economic and clinical impact of PTOA is also greater than idiopathic OA, as patients are younger and often more active, requiring treatments for symptomatic OA over a greater fraction of their lifetime. A common strategy to manage pain and inflammation associated with PTOA is the intraarticular administration of corticosteroids. However, these injections are limited due to the requirement of high-doses imposed by synovial joint clearance rates and their resulting systemic side effects. In addition, currently used broad-spectrum corticosteroids are palliative and not curative, stemming from incomplete knowledge of specific mechanisms that drive cartilage degeneration and other joint pathologies. Thus, most patients with PTOA eventually undergo surgical procedures such as osteochondral graft transplantation for focal defects and in more severe cases, total knee arthroplasty. As such, the studies presented in this dissertation (i) offer specific insights into mechanisms by which traumatic injury can drive joint degeneration and (ii) present novel strategies to modulate joint responses to pathological factors by leveraging sustained drug-delivery platforms. In Part I, mechanistic assessments of human cartilage and synovium responses to insults are conducted to identify novel pathways that may lead to impaired joint homeostasis. First, a direct consequence of traumatic injury, hemarthrosis, is explored as a potential contributor to the development of PTOA specifically through contributions by red blood cells. We demonstrate for the first time the differential roles of erythrocytes in their intact and lysed states through measures of oxidative stress and changes to metabolomic profiles in the context of ferroptosis. Furthermore, we demonstrate the therapeutic potential of Ferrostatin-1, a lipophilic radical scavenger in inhibiting pathological changes to cartilage and its crosstalk with the neighboring synovium in an in vitro model of hemophilic arthropathy. Second, a strategy to prevent an indirect consequence of traumatic injury, arthrofibrosis, is presented in an in vitro model of joint contraction. Fibrosis and the presence of hyperplastic synovium are implicated in the progression of OA through pathological shifts in tissue composition as well as secreted factors that promote cartilage degeneration and the maintenance of a pro-inflammatory joint environment. A type I transforming growth factor beta-1 receptor inhibitor, SB-431542, is encapsulated in polymeric microspheres for the prophylactic treatment of arthrofibrosis through sustained low-dose drug delivery to circumvent the challenges associated with resident joint clearance rates. Utilizing human-based in vitro models of cartilage and synovium pathology, we present novel mechanisms and therapeutic strategies to prevent pathological changes following traumatic joint injury that may contribute to the development of PTOA. In Part II, the sustained delivery platform introduced in Part I is extended to the treatment of PTOA. Osteochondral graft transplantation is currently the clinical gold standard for large focal cartilage lesions. However, allograft procedures are limited due to the lack of available donor tissues and autografts are associated with complications due to donor-site morbidity. In both cases, grafts are subject to failure, potentially in part due to the continual presence of pro-inflammatory factors following surgical procedure. In this section, we present cellular agarose hydrogels embedded with dexamethasone-releasing microspheres that are integrated with a titanium base as a functional tissue-engineered alternative to native osteochondral allografts. These allogenic tissue-engineered grafts were assessed in an in vivo preclinical canine model in their ability to maintain clinical function and to modulate the inflammatory response over the course of 12 months. We successfully demonstrated the feasibility of using engineered grafts by comparing clinical measures of range of motion, function, lameness, and pain, as well as modified cartilage graft scores, against native osteochondral allograft controls. In addition, improvements in the histopathological scoring of neighboring synovial and meniscal tissues indicate the therapeutic capacity of dexamethasone released from within the joint to modulate the inflammatory response up to one-year post-implantation. Taken together, the studies presented in this dissertation identify novel mechanisms behind pathological changes to the cartilage and synovium that may contribute to the development of PTOA following injury. Potential therapeutic targets, inhibitory compounds, and delivery strategies are also assessed using human-based in vitro models of disease and further validated in an in vivo canine model through a clinically relevant timeframe. Ultimately, we demonstrate for the first time, the use of dual-function tissue-engineered grafts in a weight-bearing region of the knee joint to circumvent limitations associated with the clinical gold standard for the treatment of large focal cartilage defects.