Orientador: Bruno Watanabe Minto / Resumo: O objetivo da pesquisa foi avaliar a eficácia do Decanoato de Nandrolona (DN) em fraturas do rádio após indução da não união óssea por defeito segmentar. Foram utilizados 47 coelhos, da raça Nova Zelândia Branco, com peso médio de 3,8 kg, divididos aleatoriamente em grupos de 8 animais. Os grupos Controle Machos (GCM) e Controle Fêmeas (GCF) foram submetidos à eutanásia aos 45 dias, os grupos Nandrolona Machos (GNM) e Nandrolona Fêmeas (GNF) tratados com aplicações semanais de DN na dose de 10mg/kg, via intramuscular (IM) durante 4 semanas. Os Grupos Placebo Machos (GPM) e Grupo Placebo Fêmeas (GPF), tratados com aplicações semanais de NaCl 0,9% (IM), durante o mesmo período. Foi realizado exame radiográfico após a cirurgia e aos 15, 30, 45, 60, 75 e 90 dias. Os animais dos grupos GNM, GNF, GPM e GPF foram submetidos à eutanásia aos 90 dias. Realizou-se exame de densitometria óssea do rádio dissecado após a eutanásia em todos os grupos, bem como análise histológica. Para as variáveis não-paramétricas utilizou-se o teste de Kruskal-Wallis. Para as variáveis paramétricas foi utilizado o teste Shapiro-Wilk, ambos a 5% de significância. Radiograficamente, os grupos tratados com DN apresentaram média de escore superior aos demais grupos na formação de calo ósseo. Na análise densitométrica, também foi observada uma melhora significativa ao parâmetro de composição mineral óssea (BMC) nos grupos GNM e GNF. Os machos apresentaram maiores médias para os parâmetros conteúdo mineral ósse... (Resumo completo, clicar acesso eletrônico abaixo) / Mestre

Most of the load-bearing demand placed on the human body is transduced by skeletal tissue, and the capacity of the skeleton to articulate in various opposing directions is essential for body movement and locomotion. Consequently, cartilage and bone defects due to trauma, disease, and developmental abnormalities result in disabling pain and immobility for millions of people worldwide. A novel way of promoting cartilage and bone regeneration is through the incorporation of either primary cells or multipotent progenitor cells in a three-dimensional (3D) biomaterial scaffold, and/or the addition of exogenous growth and differentiation factors. The first part of this study reports a protocol for using freshly isolated mature chondrocytes seeded in a 3D hydrogel biomaterial scaffold, developed to explore mechanotransduction of engineered cartilage constructs cultured in a designed bioreactor. The bioreactor was designed to allow the application of physiological mechanical forces (compression and fluid flow), as well as a non-invasive/non-destructive method for analyzing regenerating tissue in real time through ultrasound transducers and a computerized monitoring system. In the second part of this study, an engineered immortalized osteoprecursor cell line, designated OPC1 (osteoblastic precursor cell line 1), was used as a culture model system for exploring the effects of exogenous growth and differentiation factors, mainly vitamin D, on early bone development. OPC1 was previously designed to provide a consistent reproducible culture system for direct comparisons of engineered bone constructs, evaluating bone development and cell/biomaterial interactions, and for investigating putative bone differentiating factors. One of the objectives of this research effort was to explore tissue development and regeneration by culturing OPC1 in the presence of vitamin D metabolites vitaD3 and 1,25OH2D3, while assaying the concomitant biological response. Results indicate that OPC1 is capable of metabolizing the parental metabolite vitaD3, and thus 25OHD3, to the active vitamin D form 1,25OH2D3. The metabolism of vita3 resulted in an anti-proliferative and pro-differentiative influence on OPC-1. These results support the hypothesis that extra-endocrine synthesis of 1,25OH2D3 functions in a tissue specific manner to regulate growth and differentiation, in addition to the classic calcimic actions of the vitamin D endocrine pathway. Understanding the influence of vitamin D on bone development will have significant implications on healthy aging, including the susceptibility to skeletal disorders involved in development and aging, such as osteoarthritis (OA) and osteoporosis.

Indiana University-Purdue University Indianapolis (IUPUI) / Enhancing osteoblast proliferation, survival, and extracellular matrix protein secretion are potential therapeutic approaches to treat bone fractures and diseases such as osteoporosis. BENS is a traditional medicine used in many countries such as India for thousands of years to treat many diseases including bone diseases. In this study, molecular, cell-based and in vivo approaches were utilized to investigate the effects of BENS on bone and cartilage regeneration. An osteosarcoma cell line (MG63) was incubated in serum free media with and without 0.8 mg/ml of BENS. BENS significantly increased cell survival up to 30 days and these cells retained their ability to proliferate in fresh media with serum. After adding BENS, there were statistically significant decreases in the expression of both anti-apoptotic and pro-apoptotic proteins. An in vivo non-critical size segmental bone defect Xenopus system was used to evaluate the ability of BENS to enhance cartilage formation. After a small segment of the anterior hemisection of the tarsus bone was excised, the frogs were divided into three groups and given subcutaneous injections of either phosphate-buffered saline or BENS once daily for 30 days and then bone/cartilage formation evaluated. The total cartilage area/total section area was significantly increased (2.6 fold) in the BENS treated samples. In an osteoporotic rat model, the anabolic properties of BENS on bone mass were assessed by histomorphometric analyses. Ovariectomized (OVX) rats received daily intraperitoneal injections for 4 weeks. Bone formation rates (BFRs) for the cortical periosteal bone surface of the midshaft tibia were 383.2, 223.9, 308.8, 304.9, and 370.9 µm3/µm2/year, and for the trabecular surface were 82.2, 113, 212.1, 157, and 165 µm3/µm2/year for the sham, OVX, PTH, 3 mg/kg BENS, and 30 mg/kg BENS groups, respectively. BENS increased both trabecular and cortical BFRs. It generated better results on cortical periosteal bone surface than did PTH. Taken together, these findings suggest that BENS promotes osteoblast survival due to its effects on altering the balance between pro-apoptotic and anti-apoptotic proteins. In addition, in vivo studies revealed that BENS enhanced cartilage formation in Xenopus and BFRs in rats. Therefore, BENS may possess anabolic bone/cartilage properties.

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External stresses surrounding bone can stimulate heat shock proteins (HSPs), which are involved in anti-apoptosis, cell proliferation, and differentiation. In vitro stress modulation and HSP induction may be critical factors for enhancing bone regeneration. We investigated whether applying individual or combinatorial stress conditioning (thermal, tensile, and biochemical) and effective HSP modulation could induce in vitro responses in preosteoblasts indicating mitogenic/osteogenic/angiogenic/anti-osteoclastic effects. A preosteoblast cell line (MC3T3-E1) was exposed to conditioning protocols utilizing thermal stress applied with a water bath, tensile stress using a Flexcellâ„¢ bioreactor, and biochemical stress with the addition of growth factors (GFs) (i.e. transforming growth factor-beta 1 (TGF-β1) and bone morphogenetic protein-2 (BMP-2)). Furthermore, the role of HSP70 in osteogenesis under normal conditions and in response to heat was investigated by transfecting preosteoblasts with HSP70 small interfering RNA alone or in combination with thermal stress and measuring cellular response. Heating at 44°C (for 8 minutes) rapidly induced osteocalcin (OCN), osteopontin (OPN), osteoprotegerin (OPG), vascular endothelial growth factors (VEGF), and cyclooxygenase 2 (COX-2) mRNA at 8 hour post-heating (PH). The addition of GFs with heating induced OPG and VEGF genes more than heating or GF addition alone. OPN, OCN, and OPG secretions increased with the addition of GFs. However, matrix metalloproteinase-9 (MMP-9) secretion was inhibited by heating, with more significant declines associates with GF inclusion. Equibiaxial tension (5%, 0.2 Hz, 10 seconds tension/10 seconds rest, 6 days) with GFs enhanced proliferation than tension or GF addition alone. MMP-9 secretion decreased in response to tension alone or more with GFs. Tension (1-5%, 24 hours) with GFs induced prostaglandin E synthase 2 (PGES-2), OPG, and VEGF genes more than tension or GFs alone. Combinatorial conditioning with thermal stress (44°C, 8 minutes) and tension (3%, 0.2 Hz, 10 seconds tension/10 seconds rest, 4 hours for HSP gene and 24 hours for VEGF secretion and MMP-9 gene) induced HSP27 and HSP70, secretion of VEGF (protein), and suppression of MMP-9 (gene) more than heating or tension alone. HSP70 silencing followed by heating (44°C, 8 minutes) enhanced expression of HSP27. Mitogenic activity was inhibited by heating with more significant decrease occurring by heating and HSP70 silencing. At 10 hours PH, TGF-β1, MMP-9, and ALP mRNA decreased in response to heating and HSP70 silencing. At 48 hours PH, heating following HSP70-silencing induced VEGF secretion significantly. In conclusion, effective application of individual or combinatorial conditioning utilizing heating, tension, and GFs could be beneficial as a bone healing-strategy by rapidly inducing stress proteins (HSPs), angiogenic factor (e.g. VEGF), anti-osteoclastogenic cytokines (e.g. OPG), and bone matrix proteins (e.g. OPN and OCN) with anti-resorptive activity by inhibiting MMP-9. / Ph. D.

Inflammatory processes are infamous for their destructive effects on tissues and joints in a variety of diseases. Within the body, inflammation is a highly regulated biological response whose purpose is to promote tissue regeneration following injury. However, in certain settings, inflammation persists and leads to progressive tissue destruction. This thesis focused on modulating inflammatory signaling in both contexts. Part I investigated the effects of a model pro-inflammatory cytokine, tumor necrosis factor-alpha (TNF-α), on the in vitro osteogenic differentiation of mesenchymal stem cells (MSCs). In contrast, Part II describes the development and in vivo evaluation of the first intra-articular controlled release system for the temporomandibular joint (TMJ), which silences inflammatory signaling and thus mitigates the painful joint damage seen in inflammatory TMJ disease. The following specific aims were addressed: (1) to determine the concentration of TNF-α that enhances in vitro osteogenic differentiation of MSCs; (2) to determine the temporal pattern of TNF-α delivery that enhances in vitro osteogenic differentiation of MSCs; (3) to determine the impact of bone-like extracellular matrix (ECM) on the concentration and temporal pattern of TNF-α delivery that enhances in vitro osteogenic differentiation of MSCs; (4) to evaluate the biocompatibility of intra-articular microparticles in the rat TMJ; (5) to develop a microparticle-based formulation for sustained release of a model anti-inflammatory small interfering ribonucleic acid (siRNA); and (6) to evaluate the therapeutic efficacy of intra-articular microparticles delivering siRNA in an animal model of TMJ inflammation. These studies led to the development of powerful strategies to rationally control inflammation to promote bone regeneration and mitigate joint damage in the setting of disease, both of which will ultimately improve the quality and specificity of therapies available in modern medicine. / Only volume 2 has been digitized.