Distribution of matrix metalloproteinases and their inhibitor, TIMP-1, in developing human osteophytic bone

Connective tissues synthesise and secrete a family of matrix metalloproteinases (MMPs) which are capable of degrading most components of the extracellular matrix. Animal studies suggest that the MMPs play a role in bone turnover. Using specific polyclonal antisera, immunohistochemistry was used to determine the patterns of synthesis and distribution of collagenase (MMP-1), stromelysin (MMP-3), gelatinase A (MMP-2) and gelatinase B (MMP-9) and of the tissue inhibitor of metalloproteinases-1 (TIMP-1) within developing human osteophytic bone. The different MMPs and TIMP showed distinct patterns of localisation. Collagenase expression was seen at sites of vascular invasion, in osteoblasts synthesising new matrix and in some osteoclasts at sites of resorption. Chondrocytes demonstrated variable levels of collagenase and stromelysin expression throughout the proliferative and hypertrophic regions, stromelysin showing both cell-associated and strong matrix staining. Intense gelatinase B expression was observed at sites of bone resorption in osteoclasts and mononuclear cells. Gelatinase A was only weakly expressed in the fibrocartilage adjacent to areas of endochondral ossification. There was widespread but variable expression of TIMP-1 throughout the fibrous tissue, cartilage and bone. These results indicate that MMPs play a role in the development of human bone from cartilage and fibrous tissue and are likely to have multiple functions.     

Evidence for cell-specific changes with age in expression of oestrogen receptor (ER) alpha and beta in bone fractures from men and women

Oestrogen is recognized as important for maintaining bone mass in men and women. Oestrogen receptor (ER) alpha and the recently described ER-beta are both expressed in bone cells, but have different affinities for oestrogen agonists and plant oestrogens, which could be important in developing treatments for bone loss in both men and women. It is unclear, however, which isoform predominates in bone; cell type and age may influence their relative expression. The present study has compared ER-alpha and ER-beta expression in serial sections of human fracture callus from males (n = 19, age range 5-72 years) and females (n = 15, age range 3-86 years) by indirect immunoperoxidase. Fracture callus was used as it can be readily obtained from individuals over a wide age range and contains a variety of bone cells. Antibody specificity was confirmed by western blotting and comparison of immunoreactivity in sections of breast tumour and benign prostate hyperplasia. No gender difference in ER expression was found in bone from individuals less than 40 years old. Proliferative chondrocytes were positive for both isoforms, but few larger hypertrophic cells were immunoreactive. ER-alpha and ER-beta were co-expressed in osteoclasts, suggesting that oestrogen may act directly on these cells. Osteoblasts, osteocytes, and mesenchymal cells also expressed both isoforms. In women over 40 years of age, however, relatively fewer biopsies contained osteocytes positive for ER-alpha and ER-beta. Likewise, the proportions of osteoblasts and mesenchymal cells expressing ER-beta were reduced but ER-alpha remained unaffected. In contrast, in men over 40 years, only the proportion of biopsies containing ER-beta-positive mesenchymal cells was lower. In these older men and women, ER-alpha and ER-beta expression was retained by the small proliferative chondrocytes. These results demonstrate that gender, age, and cell type are important determinants of ER isoform expression in skeletal cells.     

The skeleton as a unique environment for breast cancer cells

Bone is a favored location for several cancer metastases especially breast, prostate and myeloma. This review evaluates various properties of the skeleton that contribute to its successful colonization by breast cancer cells. The first consideration is the unique aspects of the vasculature of metaphyseal bone, which may account for the initial lodging of breast cancer cells in specific regions of the skeleton. Metasphyseal bone, found at the ends of long bone, in ribs and in vertebrae, is comprised of trabecular bone interspersed with marrow and a rich vasculature. The chemotactic factors that arise from bone marrow and bone cells are discussed in terms of cancer cell migration out of the vasculature and entry of cancer cells into the marrow cavity. Once the breast cancer cells have migrated into the metaphysis, they interact both directly and indirectly with bone cells and other cells in the marrow. As tumor growth progresses, functional bone cells are lost, most likely through apoptosis. This revised version was published online in July 2006 with corrections to the Cover Date.     

自体成骨细胞加同种异体骨复合移植的研究

目的：选择较理想的骨移植替代物。方法：抽取实验组家兔骨髓2mL分离出间质细胞，在特殊培养液中进行体外诱导．增殖成成骨细胞种植于同种异体骨表面进行培养4周后，将细胞和同种异体骨复合移植物植入实验兔节段性尺骨缺损中。另取12只家兔单纯植入同种异体骨作为对照组，观察16周。结果：经碱性磷酸酶染色阳性，Chfα1免疫组织化学染色阳性，Von Kossa染色显示骨小结，表明骨髓间质细胞在体外成功诱导成成骨细胞。动物实验表明植入体周同无炎性细胞浸润．伤口Ⅰ期愈合，术后7周开始出现骨痂，11周出现桥梁骨痂，术后16周达到骨性愈合。明显优于对照组。结论：此复合移植物生物相容性好，有较强的骨诱导作用，是比较理想的骨移植替代物。     

续断对成骨细胞增殖、分化、凋亡和细胞周期的影响

在动物实验的基础上,探讨续断对成骨细胞增殖、分化、凋亡、细胞周期和Ⅰ型前胶原αmRNA表达影响.根据文献方法培养大鼠成骨细胞株UMR-106/01,采用3H-Tdr法检测细胞增殖,PNPP法、RIA法、茜素红染色法以及定量RT-PCR法分别检测碱性磷酸酶活性、骨钙素活性、矿化结节形成和Ⅰ型胶原αmRNA的表达,从而了解续断对成骨细胞增殖、分化的影响;FCM法检测细胞周期.结果显示续断中、高剂量能显著促进成骨细胞的增殖、增加碱性磷酸酶的表达及矿化结节形成的数量,促进成骨细胞骨钙素和Ⅰ型前胶原mRNA的表达(P＜0.01,P＜0.05),和雌激素组比较差异无显著性.续断高剂量组细胞增殖率、S期细胞比率和细胞增殖指数等均显著高于空白对照组(P＜0.01,P＜0.05),和雌激素组比较差异也无显著性.细胞凋亡率和G0-G1期细胞比率显著低于空白对照组(P＜0.01,P＜0.05).表明续断能有效促进成骨细胞的分化、增殖,防止成骨细胞凋亡,可能是该药促进骨折愈合、防治骨质疏松的机制之一.     

流体剪切力通过下调p21促进 MC3T3-E1成骨细胞增殖

目的 探讨流体剪切力(fluid shear stress, FSS)对成骨细胞p21表达的影响,并明确p21在FSS诱导的成骨细胞增殖过程中的作用。方法 对成骨细胞加载不同时间(0、15、30、45、60 min)、1.2 Pa FSS。用CCK-8实验、EdU实验检测成骨细胞增殖活性。用siRNA p21或pcDNA p21转染成骨细胞,并用Western blotting检测转染效果。Western blotting检测不同干预条件下p21、cyclin D1、CDK4的表达变化。结果 加载1.2 Pa FSS后,p21表达显著下调,且加载45 min后表达水平最低。加载FSS和下调p21表达都显著增强成骨细胞增殖,并增加cyclin D1、CDK4表达。而上调p21表达后,加载FSS不再具有增强成骨细胞增殖和增加cyclin D1、CDK4表达的作用。结论 1.2 Pa FSS能够下调成骨细胞p21表达,在加载45 min时下调最为明显。p21下调对成骨细胞增殖具有促进作用,且FSS通过下调p21促进成骨细胞增殖。     

染料木素对大鼠成骨细胞骨钙素表达的影响

目的:探讨染料木素体外对大鼠成骨细胞骨钙素表达的影响.方法:用改良的组织块法分离培养新生大鼠颅骨成骨细胞,染料木素以不同浓度加入细胞培养体系,作用不同时间后,用放免法测定细胞骨钙素(BGP)的水平.结果:染料木素在1×10-9mol/L 范围内48h和72h,1×10-8mol/L及1×10-7mol/L 72h提高成骨细胞骨钙素的水平.结论:染料木素体外能提高成骨细胞骨钙素的表达.     

鸟巢蕨水提取液对兔成骨细胞的影响

目的研究鸟巢蕨水提取液对体外培养成骨细胞增殖及分化作用的影响,探讨其促进骨折愈合、防治骨质疏松的机制。方法将不同浓度的鸟巢蕨提取液（分别为0g/L、1.0g/L、1.2g/L、1.4g/L、1.5g/L、1.6g/L、2.0g/L,共7组）与成骨细胞共同体外培养,进行相应的干预。观察细胞形态,检测成骨细胞增殖与分化指标（包括Ⅰ型胶原蛋白、碱性磷酸酶、骨钙素量、钙盐沉积量和矿化结节）。结果与同一时间点的空白组比较,除第1天的1.0g/L组外,其余各组的成骨细胞增殖差异均具有统计学意义（P〈0.01）。与空白组比较,其余组别平均光密度值、碱性磷酸酶含量、骨钙素含量、钙沉积量差异均具有统计学意义（P〈0.01）。与空白组比较,除1.0g/L组外,其余组别矿化结节数差异均具有统计学意义（P〈0.01）。同一时间点1.6g/L组与2.0g/L组比较,成骨细胞增殖差异均无统计学意义（P〉0.05）;1.6g/L组与2.0g/L组比较,平均光密度值、碱性磷酸酶含量、骨钙素含量、矿化结节数差异无统计学意义（P〉0.05）,钙沉积量差异具有统计学意义（P〈0.01）。结论鸟巢蕨水提取液对成骨细胞的增殖分化具有促进作用,适宜浓度下具有促进成骨细胞增殖分化作用,过高浓度后作用达到饱和。