This study examines the possible correlations between vertebral osteoporosis, spondylolysis, spondylolisthesis, Schmorl’s nodes, vertebral osteoarthritis, osteophytosis, and laminal spurs. Further, this study examines the effects of sex, age, ancestry, and occupation on the vertebral pathologies. A total of 238 individuals (54 African Americans and 184 randomly selected European Americans) from the William M. Bass Donated Skeletal Collection at the University of Tennessee, Knoxville, were analyzed. Vertebral pathologies and anomalies were assessed using visual morphometric scoring methods outlined in previous research. It is hypothesized that positive correlations exist between osteoporosis and other vertebral pathologies and a positive correlation exists between vertebral pathologies and strenuous occupations. It is also hypothesized that there is a difference in the prevalence of vertebral pathologies between European American and African American ancestries due to African Americans generally showing higher bone mineral density than European Americans (Aloia 2008). The results of this research demonstrate numerous relationships: females are correlated with more severe osteoarthritis, osteoporosis, and spondylolisthesis, while males correlate with Schmorl’s nodes; European Americans are correlated with osteoporosis, osteoarthritis, osteophytosis, and Schmorl’s nodes, while African Americans are correlated with laminal spurs; individuals 40 years or older are correlated with osteoporosis, osteoarthritis, Schmorl’s nodes, and laminal spurs; and lastly, labor intensive occupations (i.e., construction worker) are correlated with osteoarthritis, osteophytosis, and Schmorl’s nodes, all with p-values less than 0.05. The majority of the pathological conditions also correlate with each other, for example, osteoporosis and osteoarthritis. This research demonstrates how pathological conditions correlate with sociodemographic risk factors and with other pathological conditions, which can help with the identification process of skeletal remains in archaeological and forensic contexts.

A Osteoartrite é o resultado da degeneração da cartilagem articular, sendo atualmente considerada uma condição comum e de causa multifatorial que afeta milhões de pessoas anualmente e uma das principais causas de dor e incapacidade funcional. Os joelhos são uma das articulações mais afetadas devido à sobrecarga, que constitui o principal mecanismo ativador ou determinante para o desenvolvimento da doença. Há diminuição importante da amplitude de movimento e da força muscular que acarreta em uma limitação funcional e alterações posturais interferindo nas atividades de vida diária. O objetivo principal desta pesquisa foi caracterizar as alterações posturais e a atividade eletromiográfica em pacientes com osteoartrite de joelho. Este estudo envolveu 30 mulheres voluntárias divididas em dois grupos: Grupo controle (GC) composto por 15 idosas (66,0±4,5 anos, IMC de 25,4±2,29 Kg/m2) e um grupo composto de 15 idosas com diagnóstico radiográfico de OA de joelhos Grupo osteoartrite (GO) (67,0±5,8 anos, IMC de 26,2±2,98 Kg/m2) bilateralmente, sendo um joelho sintomático e um assintomático. Para a análise postural foi utilizado o programa SAPO® na qual foram realizadas fotografias no plano sagital e frontal. Através do programa foram mensurados o ângulo Q e o ângulo do joelho bilateralmente. Foi realizada a avaliação eletromiográfica na CIMV bilateralmente do vasto lateral e medial. A avaliação na CIMV foi realizada com os sujeitos sentados na maca (joelhos em 90º de flexão). Foi solicitado ao sujeito realizar uma extensão de joelho ativa contra a resistência durante cinco segundos, repetindo esse processo por três vezes. Para a análise estatística foram utilizados teste de Shapiro-Wilk para normalidade, teste Levene para homocedasticidade e o teste de correlação de Spearman. As comparações entre os grupos foram realizadas por meio de duas ANOVAs one-way. No GO os sujeitos apresentaram uma capacidade funcional muito grave (IFL = 12), e de gravidade moderada na avaliação radiológica (escala de Kellgren = 2), no GC os sujeitos não possuíam dor. No GO o ângulo Q foi de 19,9º para o joelho com OA e de 19,1º para o assintomático (p=0,732). No GC obteve-se um ângulo Q de 19,8º (p=0,955) entre os dois joelhos. Na distância intercondilar o GC foi de 2,9 cm enquanto que no GO foi de 5,3 cm (p=0,168) e na distância intermaleolar o GC foi de 7,2 cm e no GO foi de 12,3 cm (p=0,156). Houve diferença estatística nos valores nas RMS do vasto lateral e medial nos joelhos controle (VL=545,2±40V / VM=456,9±45V) em relação aos joelhos sintomáticos (VL=338,6±54V / VM=291,7±40V) (p=0,001) para ambos os músculos. Já nas comparações entre os joelhos controle (VL=545,2±40V / VM=456,9±45V) e assintomático (VL=540,9±19V / VM=443,8±18V) não foram observados diferenças estatísticas (p=0,430 para VL e p=0,956 para VM). Nas comparações entre os joelhos sintomáticos (VL=338,6±54V / VM=291,7±40V) e assintomáticos (VL=540,9±19V / VM=443,8±18V) foram observadas as diferenças estatísticas significativas nos dois músculos analisados (p=0,001 para ambos). Ocorreu um predomínio de joelhos valgos nos dois grupos sem significância estatística. Na eletromiografia de superfície as portadoras de osteoartrite de joelhos apresentaram alterações na atividade muscular como diminuição da atividade muscular em VL e VM na CIMV em comparação ao GC e GO assintomáticos. / Osteoarthritis (OA) is the result of articular cartilage degeneration and is currently considered a common condition with multifactorial causes that affects millions of individuals annually and is one of the principal causes of pain and functional incapacity. The knees are one of the articulations most affected due to the overload that constitutes the principal activator or determinant mechanism for the development of the disease. An in important reduction in movement amplitude and muscular strength occurs that provokes functional limitation and postural alterations, interfering in daily life activities. The main objective of this research was to characterize the postural alterations and electromyographic activity in patients with OA in the knee. This study involved 30 female volunteers, divided into two groups: Control group (CG), composed of 15 elderly women (66.0±4.5 years-old, BMI 25.4±2.29 Kg/m2); and the Osteoarthritis group (OG), composed of 15 elderly women (67.0±5.8 yearsold, BMI 26.2±2.98 Kg/m2), with bilateral radiographic diagnosis of OA of the knees, one symptomatic and one asymptomatic. The SAPO® program was used for postural analysis, based on sagital and frontal plane photographs, which measured the Q angle and angle of the knees bilaterally. Electromyographic evaluation was conducted bilaterally to determine the maximum voluntary isometric contraction (MVIC) of the vastus lateralis and medialis. MVIC was performed with the subjects seated on a bed with the knees at 90º flexion. The subject was asked to perform active extension of the knee against resistance for 5 seconds, repeating this process three times. For statistical analysis, the Shapiro-Wilk test was used for normality, the Levene test for homocedasticity and the Spearman correlation test. Group comparisons were performed by one-way ANOVA. In the OG, the subjects presented severely affected functional capacity (LFI=12) and moderately severe radiological evaluations (Kellgren scale=2), while the CG presented no pain. In the OG, the Q angle was 19.9º for the OA knee and 19.1º for the asymptomatic knee (p=0.732), while in the CG, a Q angle of 19.8º (p=0.955) was obtained for both knees. The intercondylar distance was 2.9 cm for the CG and 5.3 cm for the OG (p=0.168), while the intermalleolar distance was 2.9 cm for the CG and 7.2 cm for the OG (p=0,156). Significant differences occurred in RMS values for the vastus lateralis and medialis in the control knees (VL=545,2±40V / VM=456,9±45V) compared to the symptomatic knees (VL=338,6±54V / VM=291,7±40V) (p=0.001) for both muscles; however, no statistical differences were observed between the control (VL=545,2±40V / VM=456,9±45V) and asymptomatic knees (VL=540,9±19V / VM=443,8±18V). Comparisons between symptomatic (VL=338,6±54V / VM=291,7±40V) and asymptomatic knees (VL=540,9±19V / VM=443,8±18V) showed statistically significant differences in both muscles analyzed (p=0.001 for both). Predominance occurred for valgus knees in both groups with no statistical significance. In the surface electromyography, patients with OA in the knees presented alterations in muscular activity, including diminished MVIC muscular activity in the VL and VM compared to the CG and OG asymptomatic knee.

Osteoarthritis (OA) currently affects 41 million Americans, and knee OA (KOA) alone causes the highest risk of mobility disability of any medical condition in people 65 years and older. There are no current treatments to reverse the degenerative changes of KOA, and research is aimed at finding biomarkers of KOA progression to aid in the development of effective therapies. Osteophytes are a hallmark feature of KOA and may act as a biomarker of joint space loss and pain progression. MR imaging, which is an accurate and non-invasive method to monitor KOA disease status, may aid in clarifying the role of osteophytes in KOA, especially using semi-automated quantitative software methods to accurately and efficiently calculate osteophyte volume in longitudinal studies. This study investigated the association of osteophyte volume change with joint space narrowing and pain progression in a randomized sample of 505 subjects from the FNIH OA Biomarker Consortium Project, a case-control study based on a larger longitudinal study of patients with KOA. We also aimed to further validate a software method that measured osteophyte volume in MRI. We found a moderate and significant association with osteophyte volume and joint space narrowing, but no significant association with pain progression. The software was further validated as responsive and efficient method to measure KOA osteophyte volume change.

Base teórica: A osteoartrite é uma doença crônica cuja principal característica é a degradação progressiva da cartilagem articular. Além do acometimento articular, frequentemente, os pacientes com osteoartrite apresentam fraqueza e atrofia dos músculos periarticulares. Apesar disso, os mecanismos moleculares envolvidos na perda muscular relacionada à osteoartrite não são conhecidos. Os principais mecanismos já estudados, em outras condições, estão relacionados ao aumento da degradação e à redução da síntese de proteínas musculares e a déficits na ativação das células-satélite, responsáveis pela regeneração muscular. A miostatina, um importante regulador negativo do crescimento da massa muscular, estimula o aumento da degradação e a redução da síntese de proteínas musculares. Por outro lado, MyoD e miogenina, são marcadores de proliferação e de diferenciação de células-satélite, respectivamente. Objetivos: Investigar os mecanismos moleculares envolvidos na perda muscular em um modelo animal de osteoartrite induzida por transecção do ligamento cruzado anterior em ratas. Métodos: Ratas Wistar fêmeas foram alocadas em dois grupos: OA (submetidas à cirurgia de transecção do ligamento cruzado anterior do joelho direito) e SHAM (submetidas à cirurgia fictícia do joelho direito). Durante o período experimental de 12 semanas foram avaliados, semanalmente, o peso corporal e a locomoção exploratória espontânea. Após a eutanásia, foram coletadas as articulações do joelho direito para confirmação do desenvolvimento da doença. Os músculos gastrocnêmio, tibial-anterior e sóleo, da pata posterior direita, foram dissecados, pesados e congelados. O músculo gastrocnêmio foi utilizado para a avaliação da atrofia muscular, através da análise da área seccional da miofibra, e para análise da expressão proteica de miostatina, MyoD e miogenina. Resultados: A locomoção exploratória espontânea, o peso corporal e o peso dos músculos gastrocnêmio, tibial-anterior e sóleo não apresentaram diferença significativa entre os grupos OA e SHAM. A histopatologia da articulação do joelho confirmou o desenvolvimento da doença nos animais do grupo OA. A área do músculo gastrocnêmio demonstrou redução de aproximadamente 10% no grupo OA, em comparação com o grupo SHAM. O grupo OA apresentou aumento na expressão proteica de miostatina e redução na expressão proteica de miogenina. A expressão proteica de MyoD não apresentou diferença entre os grupos. Conclusão: A atrofia do músculo gastrocnêmio presente na osteoartrite induzida por transecção do ligamento cruzado anterior envolve aumento na expressão de miostatina e redução na expressão de miogenina. Nesse modelo, a perda muscular pode estar relacionada à proteólise induzida pelos níveis aumentados de miostatina e ao déficit na diferenciação das células-satélite devido à redução na expressão de miogenina. / Background: Osteoarthritis is a chronic joint disease primarily characterized by cartilage loss. In addition to joint impairment, patients with osteoarthritis often suffer from weakness and atrophy of the periarticular muscles. However, the molecular mechanisms involved in osteoarthritis-related muscle wasting are not known. The main mechanisms studied, in other conditions, are related to increased degradation and reduced synthesis of muscle protein and to deficits in the activation of satellitecells, which are responsible for muscle regeneration. Myostatin, an important negative regulator of muscle growth, stimulates the increase of degradation and the reduction of synthesis of muscle protein. Moreover, MyoD and myogenin are markers of proliferation and differentiation of satellite-cells, respectively. Objective: To investigate the pathways involved in muscle wasting in a model of osteoarthritis induced by anterior cruciate ligament transection (ACL) in rats. Methods: Female Wistar rats were allocated into two groups: OA (submitted to the ACL transection) and SHAM (submitted to surgical procedures without ACL transection). The spontaneous exploratory locomotion and the body weight of animals were evaluated weekly. In the twelfth week after the induction of disease, animals were euthanized and the right knee joints were collected for further confirmation of the disease by histopathology. Gastrocnemius, tibialis-anterior and soleus muscles from right hind paw were dissected, weighed and frozen. Gastrocnemius was used for evaluation of muscle atrophy, by cross-sectional area measurement, and protein expression of myostatin, MyoD and myogenin. Results: Spontaneous exploratory locomotion, body weight and weight of muscles showed no difference between OA and SHAM groups. The histopathology of the knee joints confirmed the development of the disease in animals from OA group. Gastrocnemius area of animals from OA group had a reduction of about 10% compared to animals from SHAM group. Protein expression of myostatin was increased in animals from OA group, while myogenin expression was decreased. MyoD expression was similar in both OA and SHAM groups. Conclusion: Gastrocnemius atrophy in osteoarthritis induced by ACL transection involves increased protein expression of myostatin and decreased protein expression of myogenin. In this model, muscle wasting may be linked to myostatininduced proteolysis and to deficits in satellite-cell differentiation due to decreased expression of myogenin.

BACKGROUND: Osteoarthritis (OA) is the most common degenerative joint disease which affects the joint structures leading to disability. Studies in the last 20 years have documented the increased prevalence of knee pain and symptomatic knee OA. Similarly, of temporomandibular joint (TMJ) disorders OA is the most common. Lysyl oxidase like-2 (LOXL2) is a copper-dependent amine oxidase. previous studies showed that LOXL2 is elevated during mouse fracture healing. Our hypothesis that LOXL2 acts as a specific anabolic factor in chondrocytes METHODS: The activity of LOXL2 in human articular and TMJ chondrocytes was assessed by cell-based assays and RT-qPCR, and LOXL2-mediated activation of NF-κB and extracellular signal-related kinase (ERK) signaling pathways was measured by western blotting. To examine LOXL2-induced effect in vivo, we implanted Matrigel-imbedded human chondrocytes into nude mice and exposed them to exogenous LOXL2 for 6 weeks. We also examined if LOXL2 induces the proliferation of OA chondrocytes. RESULTS: LOXL2 staining was detected in damaged regions of human TMJ, hip and knee joints affected by OA. Stimulation with transforming growth factor (TGF)-β1 upregulated LOXL2 expression, while pro-inflammatory cytokines IL-1β and TNF-α downregulated LOXL2, in human chondrocytes. LOXL2 expression also inhibited IL-1β-induced phospho-NF-κB/p65 and TGF-β1-induced ERK1/2 phosphorylation. Matrigel constructs of human chondrocytes from the knee joint and TMJ implanted in nude mice showed anabolic responses after LOXL2 transduction, including increased expression of SOX9, ACAN, and COL2A1. We have found that LOXL2 does not induce the proliferation of human TMJ or knee OA chondrocytes. CONCLUSIONS: We showed that LOXL2 induces differentiation and attenuates OA related catabolic signaling pathways.

The glenohumeral joint has the largest range of motion in the body. This is due to its anatomy of the bony structure of the glenoid fossa providing a shallow socket with minimal constraint of the humeral head and the surrounding soft tissue structures serving as restraints to limit excessive humeral head translation. The bony and soft tissue structures function together with a delicate balance that when disrupted lead to several pathologies including degenerative osteoarthritis or glenohumeral instability, which are the focus of this research. For glenohumeral osteoarthritis, the gold standard treatment is total shoulder arthroplasty. Although the surgical success rate is reported at 95%, the long-term failure rate is as high as 30% and often caused by glenoid component failure. For glenohumeral instability, surgical capsular plication can significantly reduce recurrent dislocation rates, however, up to 70% of patients experience joint stiffness and a reduced range of motion. For these treatments, there is little consensus regarding what surgical parameters optimize functional recovery - consequently, several surgical techniques exist. Since long-term follow-ups are lacking and difficult to perform, basic science studies are needed to identify what surgical parameters are most likely to influence patient recovery. The objective of this research was to develop patient-specific computer models to create accurate representations of these pathologies and to investigate the effects of different surgical parameters in total shoulder arthroplasty and glenohumeral instability repair. A total shoulder arthroplasty computer model was developed to investigate the effect of surgical parameters of the glenoid implant component. An initial study performed a cadaveric validation of the methodology to simulate the reaming process for resurfacing the glenoid surface. This validated computer model was then used to investigate how the degree of correction of glenoid retroversion affects cement mantle stress and potential cement failure. The use of physiologic patient-specific bone models revealed that maintaining the cortical bone layer should take precedence over version correction when a high degree of glenoid deformity is encountered. A glenohumeral instability computer model was developed to investigate the effect of capsular repair on shoulder stability and joint range of motion. The computer model suggests that adding a plication of the posterior band of the inferior glenohumeral ligament offloads regions of high strain from the anterior region of the glenoid attachment site which may indicate a reduced risk of anterior capsular repair failure. An anisotropic hyperelastic material behavior was then incorporated to model the glenohumeral capsule by performing an inverse finite element analysis to obtain the optimized material parameters. The computer models developed in this research utilize radiographic patient images in order to replicate and investigate actual pathology. As a result, the studies performed provide a deeper understanding of the glenohumeral joint mechanics associated with the treatments of total shoulder arthroplasty and glenohumeral capsular plication. This information provides insight for the practicing shoulder surgeon in their pre-operative surgical planning to decide the optimal technique and approach for a patient with these challenging pathologies. Moreover, the methodologies developed for simulating these surgical techniques can have a wide application to advance the foundation of pre-surgical virtual simulation and provide critical data for computer aided surgical navigation of other joints and diseases.

Osteoarthritis is a joint disease associated with the irreversible breakdown of articular cartilage in joints, causing pain, impaired mobility, and reduced quality of life in over 27 million Americans and many more worldwide. The tolls by osteoarthritis (OA) on the workforce and healthcare system represent significant economic burdens. An attractive strategy for treating OA is cartilage tissue engineering (CTE). CTE strategies have been promising at producing cell-scaffold constructs at small sizes (3-5 mm in largest dimension), but OA often does not present symptoms until lesions reach 25 mm in diameter. Using bovine chondrocytes seeded in agarose, our lab has produced small CTE constructs with native cartilage levels of compressive stiffness and proteoglycan content. As construct dimensions are increased, however, the resulting tissue suffers from extreme heterogeneity of deposited matrix due to nutrient transport limitations. The ability to successfully scale up constructs to clinically relevant sizes is a major goal in CTE research. Another major and largely unresolved obstacle is the translation of successes from animal cell models to CTE systems with human cells, which is ultimately necessary for clinical treatment of OA. In this dissertation, experiments are placed forth which seek to address the nutrient limitations in large cartilage constructs and to help bridge the gap from animal cells to human cells for CTE. The growth of CTE constructs is limited by the poor availability of nutrients at construct centers due to consumption by cells at the construct periphery. The first series of studies in this dissertation sought to identify nutrients in culture media that are consumed by cells and are critical for matrix production, and to characterize their transport behavior. Among several candidate nutrients, glucose proved to be the most indispensable; little to no growth transpired in constructs when glucose fell below a critical threshold concentration. A subsequent study provided a system-specific glucose consumption rate. These parameters were informative for computational models of construct growth, which helped predict transport and growth phenomena in constructs and suggest improved culture techniques for later experiments. The cultivation of tissue constructs of increasing size presents logistical challenges, as the constructs’ requirements for nutrients, growth factors, and even sizes of culture vessels increase. The addition of nutrient channels to constructs to improve nutrient transport and tissue growth is a promising strategy, but more sophisticated casting and culture techniques are required for constructs with channels, particularly as construct size is increased. We first designed casting and culture devices for cylindrical 10 mm × 2.3 mm (diameter × height) constructs with 1 mm diameter nutrient channels. With information gleaned from computational models predicting glucose availability in constructs, we refined our culture system and demonstrated beneficial effects of nutrient channels on construct mechanical properties and extracellular matrix contents. This was the most successful instance to date of the use of nutrient channels in CTE, and is highly promising for channels’ ability to mitigate transport limitations in constructs. We next sought to optimize key parameters for culturing channeled constructs. The addition of channels is an optimization problem: greater numbers of closer-packed channels increase nutrient availability within the construct but simultaneously detract from the construct’s initial volume and cell population. Furthermore, we suspected that uneven swelling of 10 mm diameter constructs was a side effect of transient treatment with 10 ng/mL TGF-β, a highly effective and commonly-employed technique for elevating construct functional properties. By increasing channel densities in 10 mm diameter constructs, we identified a channel spacing that yielded optimal construct functional properties. In constructs with this channel spacing, reducing the TGF-β dosage by tenfold resulted in similar or elevated properties by constructs. These experiments supplied us with optimal parameters for further scaling up our constructs to clinically-relevant sizes, a practice that can be adapted for any CTE culture system for large constructs. The ability to treat severe OA by entirely resurfacing diseased joints with CTE would be highly desirable, yet this ability remains elusive, as efforts to grow constructs of such size have thus far been stymied by nutrient transport limitations. We scaled up our culture system for 10 mm diameter constructs, employing previously optimized culture conditions and channel spacing, and cultured articular surface-sized (40 mm diameter, 2.3 mm thick) constructs. These constructs were 100× the size of our small constructs, yet they still attained similar functional properties, reaching native cartilage levels of compressive stiffness and proteoglycan content. These are the largest CTE constructs to ever achieve such favorable properties. These results demonstrate that with nutrient channels, CTE constructs have the potential to replace entire joint surfaces that have been compromised by OA. Finally, we began to explore the feasibility of translating techniques from our bovine and canine model systems into human cells. We harvested adult human chondrocytes from expired osteochondral allografts and cast them in small (3 mm diameter) constructs, culturing the constructs under various conditions that have been previously successful for animal constructs. We observed similarities between human versus bovine and canine constructs, most notably that high initial cell seeding density led to marked increases in functional properties, in some cases approaching mechanical and biochemical properties of native human cartilage. Human constructs also exhibited poor GAG retention and long-term growth relative to animal constructs. By establishing successful techniques for human constructs in addition to identifying new challenges, we provided an in-depth characterization of human chondrocytes in agarose that is promising overall for eventual clinical translation. The body of work presented in this dissertation followed a methodical approach to scaling up CTE constructs to the sizes of entire joint surfaces, through experimentation with nutrient channels in constructs and with the support of predictive computational models. The principle behind nutrient channels is fundamental and therefore can be applied to CTE systems using other scaffold and cell types. By incrementally increasing the scale of bovine chondrocyte-laden constructs and by performing initial studies with small human CTE constructs, we have laid down groundwork for future studies seeking to grow articular surface-sized human engineered cartilage.

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.

A osteoartrite é a causa mais comum de claudicação em cavalos e, frequentemente está associada com a queda de desempenho e abandono precoce das atividades esportivas. Numerosos estudos têm investigado o potencial da função dos condroprotetores na desaceleração do processo degenerativo e no reparo da cartilagem articular. A finalidade deste estudo foi investigar o efeito da administração oral de glucosamina, condroitim sulfato e ácido hialurônico associados sobre a evolução clínica, radiológica, e glicosaminoglicanos (GAGs) da urina, do líquido sinovial e sérico de cavalos acometidos por osteoartrite. Foram utilizados seis equinos, com idades entre seis e doze anos, machos ou fêmeas, submetidos ao mesmo manejo nutricional. Estes animais receberam doses diárias de condroitim sulfato (CS) (2,8 g), ácido hialurônico (AH) (0,1g), glucosamina (3,1 g), via oral, por 25 dias. Exames clínicos e radiográficos foram realizados anteriormente e após o tratamento. Amostras de urina, líquido sinovial, e sangue foram coletados antes da primeira administração do suplemento; a cada 5 dias durante o tratamento e a cada 7 dias após o tratamento, durante 55 dias. Os glicosaminoglicanos urinários foram identificados por eletroforese em gel de agarose após cromatografia de troca iônica e quantificados por densitometria. A determinação de AH sérico foi realizada por ELISA. Após proteólise do líquido sinovial o CS e a AH foram igualmente identificados por eletroforese e quantificados por densitometria. No início do experimento, as concentrações médias urinárias de CS, DS, HS e GAGs totais (2,96 mg/l, 0,66 mg/l, 0,42 mg/l, 4,05 mg/l respectivamente) foram inferiores as médias urinárias, logo após o tratamento (5,38 mg/l, 1,30 mg/l, 0,96 mg/l, 7,64 mg/l respectivamente) (p<0,05) e similares após 30 dias (4,24 mg/l, 1,09 mg/l, 0,36 mg/l, 5,69 mg/l). A concentração sérica de AH aumentou após o tratamento e ao final do experimento em relação do início dos mesmos (10,61 ng/ml, 12,58 ng/ml, 21,08 ng/ml respectivamente) (p<0,05). O CS do líquido sinovial apresentou comportamento similar ao CS urinário (início: 76,13 µg/ml, final do tratamento: 93,05 µg/ml, final do experimento: 61,61µg/ml). Já o AH no líquido sinovial não sofreu alterações significativas (início: 440,07 µg/ml, final do tratamento: 351,15 µg/ml, final do experimento: 375,99 µg/ml) apesar da tendência a diminuição. Concluiu-se que a administração oral de glucosamina, CS e AH associados apresenta uma tendência ao aumento dos GAGs urinários e do CS do líquido sinovial imediatamente após o tratamento, com diminuição até 30 dias; aumento sérico do AH e manutenção da concentração de AH no líquido sinovial, com tendência a diminuição. / Osteoarthritis is the most common cause of lameness in horses, and often is frequently associated with poor performance and early end of sports activities. Numerous studies have investigated the potential function of chondroprotective drugs in slow down the degenerative process and helping to repair the joint cartilages. The purpose of this study was to investigate the effect of oral administration of glucosamine, chondroitin sulfate and hyaluronic acid associated with the clinical, radiological, and urinary glycosaminoglycans (GAGs), in the serum and synovial fluid of horses affected by osteoarthritis. We used six horses, aged between six and twelve years old, male or female, undergoing the same nutritional management. These animals received daily doses of condroitim sulfate (CS) (2.8 g), hyaluronic acid (HA) (0.1 g), glucosamine (3.1 g), oral route, for 25 days. Clinical and radiographic examinations were performed before and at the end of treatment. Samples of urine, synovial fluid and serum were collected before the first administration of the supplement, and every 5 days during the treatment and every 7 days after the end of the treatment, for a total of 55 days. The urinary glycosaminoglycans have been identified for agarose gel electrophoresis after ion-exchange chromatography on Q-Sepharose and quantified by densitometry. The determination of serum HA was performed by ELISA. After the sinovial liquid proteolysis the CS and the HA have been equally identified for eletrophoresis and quantified by densitometry. In the beginning of this study the avarage of urinary concentrations of CS, DS, HS and total GAGs (2.96 mg/l, 0.66 mg/l, 0.42 mg/l, 4.05 mg/l respectively) have been inferior the urinary averages, right after the treatment (5.38 mg/l, 1.30 mg/l, 0.96 mg/l, 7.64 mg/l respectively)(p< 0,05) and similar after 30 days (4.24 mg/l, 1.09 mg/l, 0.36 mg/l, 5.69 mg/l respectively). The HA serum concentration increased after treatment and the end of the experiment regarding the inicial values (10.61 ng/ml, 12.58 ng/ml, 21.08 ng/ml respectively) (p<0.05). The synovial fluid CS showed a similar behavior to the urinary CS (beginning: 76.13 µg/ml, end of treatment: 93.05 µg/ml, end of the experiment: 61.61 µg/ml). But the synovial fluid HA did not change significantly (beginning: 440.07 µg/ml, end of treatment: 351.15 µg/ml, end of the experiment: 375.99 µg/ml) despite the tendency to decrease. It was concluded that oral administration of glucosamine, CS and HA associated shows a tendency to increase the urinary GAGs and CS in the synovial fluid immediately after treatment, with reduction in 30 days; increase of serum HA and maintenance of the concentration of HA in synovial fluid, with a tendency to decrease.

Osteoporosis is a devastating condition characterized by decreased bone mass, and affects over 50% of the population over 50 years old. Progression of osteoporosis results in significantly heightened risk of fracture leading to loss of mobility, prolonged rehabilitation, and even mortality due to extended hospitalization. Current therapeutic options exist to combat low bone mass, but these treatments are being met with increasing concern as reports emerge of atypical fractures and necrosis. Thus, new therapeutic strategies are required. Bone is highly dynamic, and it has long been known that physical load is a potent stimulus of bone formation. Despite this, none of the current treatments for bone disease leverage the inherent mechanosensitivity of bone – the ability of bone cells to sense and respond to mechanical forces such as exercise. One potential therapeutic target is the primary cilium. Primary cilia are solitary antenna-like organelles, and over the last 20 years have been identified as a critical cellular mechanosensor. Primary cilia and cell mechanotransduction are critical to the function of numerous cells and tissues. Thus, understanding primary cilia-mediated mechanotransduction has potential applications in treating kidney and liver disease, atherosclerosis, osteoarthritis, and even certain cancers. Previous work from our group has demonstrated that disruption of the cilium impairs bone cell mechanosensitivity, resulting in abrogated whole bone adaptation in response to physical load. In this thesis we examine the potential of targeting the primary cilium to enhance bone cell mechanosensitivity and promote whole bone formation. First, we demonstrate the pharmacologically increasing primary cilia length significantly enhances cell mechanotransduction. Next, we expand our list of candidate compounds to manipulate ciliogenesis through the use of high-throughput drug screening. We developed an automated platform for culturing, staining, imaging, and analyzing nearly 7000 small molecules with known biologic activity, and classify them based on mechanism of action. One of these compounds is then used in a co-culture model to study the effects of manipulating osteocyte primary cilia-mediated mechanosensing on pro-osteogenic paracrine signaling to promote the activity of bone-forming osteoblasts and osteogenic differentiation of mesenchymal stem cells. Finally, we translate our in vitro findings into an in vivo model of load-induced bone formation using the same compound to enhance cell mechanotransduction. We demonstrate that we can sensitize bones to mechanical stimulation to enhance load-induced bone formation in healthy and osteoporotic animals, with minimal adverse effects. Together, this work demonstrates the therapeutic potential and viability of targeting primary cilia-mediated mechanotransduction for treating bone diseases.