CELLULAR BASIS OF CRANIOFACIAL BONE DISORDERS

正常的骨骼发育和重塑需要复杂的体液，细胞- 细胞和细胞-基质相互作用。 体液免疫和 本地信号是必要的，以允许适当的发展，维护 矿物质平衡，确保机械强度和支持 造血 现在有充分的证据表明，成骨细胞可以 协调破骨细胞发育并通过以下途径调节破骨细胞活性 旁分泌信号 有消息称 破骨细胞自身合成并释放因子， 骨重建 因此，我们假设破骨细胞源性 信号可能作为破骨细胞发育的自分泌效应物起作用 和活动，从而影响正常和病理 破骨细胞生成和破骨细胞介导的骨吸收。 我们有 破骨细胞产生小的促炎分子， 趋化因子，如IL-8和GRO α，这些分子可以作用于 调节破骨细胞前体的募集、发育和活性。 因此，为了进一步研究破骨细胞自分泌 我们提出以下具体目标：1）确定一个轮廓 人类和小鼠OC产生的趋化因子，并确定哪些表现出 自分泌对OC功能的影响（骨吸收、运动、游离 自由基产生、TRAP、组织蛋白酶K和碳酸酐酶II 表达式）。 选择性趋化因子对这类趋化因子的调节 将分析已知的OC功能调节剂。这里将包括 使用选择性趋化因子和趋化因子受体的研究 拮抗剂、趋化因子中和抗体和小鼠OC样细胞 从IL-8受体敲除获得的骨髓体外形成 小鼠 2)识别和表征趋化因子受体的概况 在OC上表达作为OC生理学和病理生理学的函数。 作为这一目标的一部分，我们将开始阐明细胞内信号 参与OC活性的趋化因子调节的转导途径。 3)研究趋化因子在OC前体募集中的潜在作用 以及体外、体内和卵内的分化。4.检查空间 趋化因子和趋化因子受体表达的时间方面， 原位杂交检测正常和病理骨组织， 免疫组化 包括小鼠IL-8受体敲除 问题研究 所有上述研究都将使用体内和体外的组合。 体外方法和模型系统，包括小鼠颅骨 用于组织形态计量学研究的注射模型， 原位杂交和免疫组织化学分析，分离的人 以及禽类OCs、人类OCs样细胞、小鼠OCs样细胞发育 模型和从IL-8受体敲除小鼠获得的细胞。 奥... 趋化因子的产生，mRNA的稳态水平和调节将是 通过RT-PCP、RNA酶保护试验、趋化因子ELISA和原位 杂交技术 破骨细胞的发育和活性将是 基于功能、生物化学和分子标记进行评估， 破骨细胞表型，包括骨吸收、破骨细胞抗原 抗酒石酸酸性磷酸酶活性和降钙素的表达 受体水平 这些研究预计将揭示新的方面， 正常的骨重建机制，如牙齿萌出， 有可能深入了解骨骼病理学，如牙周病 疾病、植入物松动、骨关节炎、其他炎症性骨骼 疾病和骨质疏松症。

Normal bone development and remodeling require complex humoral, cell- cell, and cell-matrix interactions. The integration of humoral and local signals is necessary to permit proper development, maintain mineral homeostasis, insure mechanical strength, and support haematopoeisis. There is now good evidence that osteoblasts can orchestrate osteoclast development and modulate osteoclast activity via paracrine signals. Information is just beginning to emerge that osteoclasts themselves synthesize and release factors that may influence bone remodeling. We therefore hypothesize that osteoclast-derived signals may function as autocrine effectors of osteoclast development and activity and therefore influence normal and pathological osteoclastogenesis and osteoclast-mediated bone resorption. We have shown that osteoclasts produce small proinflammatory molecules known as chemokines, such as IL-8 and GRO alpha and that such molecules can act to modulate osteoclast precursor recruitment, development, and activity. Therefore, to further investigate and define osteoclast autocrine regulation we propose the following specific aims: 1) Identify a profile of chemokines produced by human and mouse OC and determine which exhibit autocrine effects on OC function (bone resorption, motility, free radical production, TRAP, cathepsin K, and carbonic anhydrase II expression). The regulation of this subset of chemokines by select known modulators of OC function will be analyzed. Included here will be studies employing selective chemokine and chemokine receptor antagonists, chemokine neutralizing antibodies, and mouse OC-like cells formed in vitro from bone marrow obtained from IL-8 receptor knockout mice. 2) Identify and characterize the profile of chemokine receptors expressed on OC as a function of OC physiology, and pathophysiology. As part of this aim we will begin to elucidate the intracellular signal transduction pathways involved in chemokine modulation of OC activity. 3) Examine the potential role of chemokines in OC precursor recruitment and differentiation in vitro, in vivo, and in ovo. 4. Examine spatial and temporal aspects of chemokine and chemokine receptor expression in normal and pathological bone tissue by in situ hybridization and immunohistochemistry. Included here are mouse IL-8 receptor knockout studies. All of the above studies will use a combination of in vivo and in vitro approaches, and model systems including the mouse calvarial injection model for histomorphometric studies, human tissue sections for in situ hybridization and immunohistochemical analysis, isolated human and avian Ocs, human Oc-like cells, the mouse Oc-like cell developmental model, and cells obtained from an IL-8 receptor knockout mouse. Oc- chemokine production, mRNA steady state levels and regulation will be assessed by RT-PCP, RNAse protection assay, chemokine ELISA, and in situ hybridization techniques. Osteoclast development and activity will be evaluated based on functional, biochemical and molecular markers of the osteoclast phenotype, including bone resorption, osteoclast antigen expression tartarate-resistant acid phosphatase activity, and calcitonin receptor levels. Such studies are anticipated to reveal new aspects of normal bone remodeling mechanisms such as tooth eruption and have potential to lend insight into skeletal pathologies such as periodontal disease, implant loosening, osteoarthritis, other inflammatory skeletal disorders, and osteoporosis.