微小颗粒骨移植骨细胞活性的实验研究

目的 观察微小颗粒骨在移植修复骨缺损过程中的骨细胞存活情况和生物活性. 方法 建立大鼠桡骨骨缺损模型,近交系DA大鼠88只,其中雄性大鼠28只,作为供体;雌性大鼠60只,作为受体.将受体随机分为块状骨组(n=56)、微小颗粒骨组(n=56)和空白对照组(n=4),取雄性大鼠髂骨为供体骨,分别制成直径为2mm的骨块和直径为300～500 μm的微小颗粒骨,植入骨缺损,于术后1 d、4 d、1周、2周、4周、6周、10周取材,采用原位杂交的方法观察受体内Y染色体性别决定基因(Sry)的表达情况,应用免疫组化法观察各组骨形态发生蛋白-2(BMP～2)、转化生长因子-β1(TGF-β1)、碱性磷酸酶(ALP)和I型胶原的表达情况. 结果 块状骨组在移植早期Srv的表达逐渐减少,至1周消失,4周后再次出现,并且随时间延长表达逐渐增多;微小颗粒骨组各时间段均有Sry的表达,在同一时间点,微小颗粒骨组Sry的阳性细胞数多于块状骨组(P<0.05),两种骨移植物中参与修复骨缺损的细胞类型不同.微小颗粒骨内和周围组织中BMP-2、TGF-β1、ALP和Ⅰ型胶原的阳性细胞数在术后2周内多于块状骨组(P<0.05). 结论 微小颗粒骨与块状骨修复骨缺损时均有供体骨细胞参与,但微小颗粒骨内有更多的骨细胞存活.微小颗粒骨内存活的骨细胞具有生物学活性,合成并分泌骨生长因子和骨基质蛋白,可以加速骨缺损的修复.     

An electron microscopic study of the changes observed in osteocytes under ischemic conditions

The purpose of this study was to observe the process of ischemia in osteocytes using light and electron microscopy and to compare the changes in these ischemic osteocytes with those in other types of osteocytes (i.e., degenerative osteocytes in physiological states, steroid-induced lipid-accumulating osteocytes) that have been previously reported. Five female Japanese white rabbits were used in this study. Osteochondral chips were taken from one side of the femoral condyle, covered with Millipore filters, and then inserted into the other side of the knee joint. These tissues were examined after 12 h and after 2, 5, 8, and 14 days of ischemia under both light and electron microscopy. Under light microscopy, osteocytes and lacunae were classified into four types: normal osteocyte, pyknotic osteocyte, pale osteocyte, and empty osteocyte lacuna. The number of each type of osteocyte (or lacuna) in a settled area was counted. The ratio of normal osteocytes decreased significantly (p less than 0.001) after the second day of ischemia. Pyknotic osteocytes increased at 12 h (p less than 0.01) and 2 days (p less than 0.001) of ischemia. On the fifth day of ischemia, the percentage of pale osteocytes reached a peak. This was followed by a gradual increase in the number of empty lacunae. On the fourteenth day of ischemia, empty lacunae constituted greater than 40% of the cell types. When viewed by electron microscopy, these necrotic osteocytes were similar to the degenerative osteocytes that have been observed in physiological states and apparently different from lipid-accumulating osteocytes. The results suggested that there could be at least two types of necrotic processes in osteocytes that eventually lead to cell death.     

Size and density of osteocyte lacunae in different regions of long bones

Summary Size and density of osteocyte lacunae were evaluated at different levels of long bones to investigate whether or not the proportion of bone tissue occupied by osteocytes changes in skeletal regions, characterized by clear-cut differences in bone turnover rates. Statistical analysis of the results shows that the mean cross-sectional area of osteocyte lacunae (C) is lowest in compact bone of diaphysis and metaphysis, highest in spongy bone of metaphysis and epiphysis. On the contrary, the mean surface of bone tissue surrounding each osteocyte (T=bidimensional osteocyte territory, indirectly calculated from the number of lacunae/mm² of bone) is largest in compact bone of diaphysis, smallest in metaphyseal spongiosa, and shows intermediate values in the cortex of metaphysis and in epiphyseal spongiosa. The proportion of bone tissue occupied by osteocyte lacunae (%C/T) appears to follow at different levels of long bones, the same pattern recorded for the data of bone turnover rate, by the tetracycline labeling technique: it is lowest in mid-diaphyses, highest in metaphyses, and intermediate in epiphyses. On the basis of these findings, it is suggested that the action exerted by osteocytes on the surrounding calcified matrix, whatever the function of these cells, is not uniform throughout the skeleton and is to some extent correlated with the activity of the other bone cells—osteoblasts and osteoclasts. The significance of some of the data reported is also discussed in relation to investigations of periosteocytic lacunar morphometry.     

The role of pressurized fluid in subchondral bone cyst growth

Pressurized fluid has been proposed to play an important role in subchondral bone cyst development. However, the exact mechanism remains speculative. We used an established computational mechanoregulated bone adaptation model to investigate two hypotheses: 1) pressurized fluid causes cyst growth through altered bone tissue loading conditions, 2) pressurized fluid causes cyst growth through osteocyte death. In a 2D finite element model of bone microarchitecture, a marrow cavity was filled with fluid to resemble a cyst. Subsequently, the fluid was pressurized, or osteocyte death was simulated, or both. Rather than increasing the load, which was the prevailing hypothesis, pressurized fluid decreased the load on the surrounding bone, thereby leading to net bone resorption and growth of the cavity. In this scenario an irregularly shaped cavity developed which became rounded and obtained a rim of sclerotic bone after removal of the pressurized fluid. This indicates that cyst development may occur in a step-wise manner. In the simulations of osteocyte death, cavity growth also occurred, and the cavity immediately obtained a rounded shape and a sclerotic rim. Combining both mechanisms increased the growth rate of the cavity. In conclusion, both stress-shielding by pressurized fluid, and osteocyte death may cause cyst growth. In vivo observations of pressurized cyst fluid, dead osteocytes, and different appearances of cysts similar to our simulation results support the idea that both mechanisms can simultaneously play a role in the development and growth of subchondral bone cysts.     

骨细胞网络结构的研究进展

骨细胞是成熟骨中最丰富的细胞,它在骨组织中的作用已达成共识,但其作用方式和原理仍未完全阐明。为更好认识骨细胞在骨组织中的作用,作者对骨细胞在骨组织中的网络结构进行了综述。     

氯离子通道5在模拟失重下磷代谢异常致骨丧失中作用的初步研究

目的：探讨氯离子通道5（chloride channel 5, CLC5）在模拟失重下磷代谢异常致骨丧失的作用机制,为失重性骨代谢异常的防治提供数据参考。方法①利用回转器对IDG?SW3骨细胞模拟失重,将骨细胞分为模拟失重组（MG组）和失重对照组（MG?CON组）,2 d后采用Real?time PCR检测牙本质基质蛋白1（dentinmatrix protein 1, DMP1）和CLC5在IDG?SW3骨细胞中的表达情况;②利用尾部悬吊法对小鼠模拟失重,将10只1月龄C57BL/6雌性小鼠随机分为悬尾组（SUS组）和空白对照组（CON组）,每组5只,4周后取两组小鼠胫骨制作石蜡切片进行CLC5免疫组织化学染色;③2月龄DMP1基因敲除鼠和DMP1转基因鼠各5只（体重为20~25g）,制备胫骨石蜡切片,进行CLC5免疫组织化学染色。结果 Real?time PCR检测结果显示2D回转后DMP1和CLC5在IDG?SW3骨细胞中的表达量均升高,差异均有统计学意义（均P＜0.05）;免疫组织化学染色显示CLC5在CON组和SUS组胫骨细胞胞质和基质中均有表达,且SUS组表达量明显高于CON组,差异有统计学意义（P＜0.05）;CLC5在DMP1基因敲除鼠和DMP1转基因鼠骨细胞中的表达也较CON组升高,差异均有统计学意义（均P＜0.05）。结论模拟失重下CLC5参与了DMP1对磷代谢的调控,但其具体作用机制有待于进一步研究。     

骨硬化蛋白:一个治疗骨质疏松的新靶点

高骨量疾病骨硬化症(sclerosteosis)和Van Buchem病由SOST基因表达缺失导致.由于SOST基因突变导致骨硬化蛋白不能表达或功能缺陷,可造成该病患者的过度骨形成,其作用机制与抑制Wnt信号转导通路相关.骨硬化蛋白通过与该通路共受体LRP5/6结合来阻断Wnt通路,进而对成骨细胞分化及矿化起抑制作用.由于骨硬化蛋白在抑制骨形成起关键作用且仅在骨组织表达,其单克隆抗体可用于治疗低骨量疾病如骨质疏松.     

An immunohistochemical and ultrastructural study of the pericellular matrix of uneroded hypertrophic chondrocytes in the mandibular condyle of aged c-src-deficient mice

OBJECTIVE: Previous studies indicate that hypertrophic chondrocytes can transdifferentiate or dedifferentiate and redifferentiate into bone cells during the endochondral bone formation. Mandibular condyle in aged c-src-deficient mice has incremental line-like striations consisting of cartilaginous and non-cartilaginous layers, and the former contains intact hypertrophic chondrocytes in uneroded lacunae. The purpose of this study is to determine the phenotype changes of uneroded hypertrophic chondrocytes. DESIGN: Immunohistochemical and ultrastructural examinations of the pericellular matrix of hypertrophic chondrocytes in the upper, middle, and lower regions of the mandibular condyle were conducted in aged c-src-deficient mice, using several antibodies of cartilage/bone marker proteins. RESULTS: Co-localisation of aggrecan, type I collagen, and dentin matrix protein-1 (DMP-1) or matrix extracellular phosphoprotein (MEPE) was detected in the pericellular matrix of the middle region. Ultrastructurally, granular substances in the pericellular matrix of the middle region were the remains of upper region chondrocytes, which were mixed with thick collagen fibrils. In the lower region, the width of the pericellular matrix and the amount of collagen fibrils were increased. Versican, type I collagen, DMP-1, and MEPE were detected in the osteocyte lacunae. Additionally, DMP-1 and MEPE were detected in the pericellular matrix of uneroded hypertrophic chondrocytes located in the lower, peripheral region of the mandibular condyle in younger c-src-deficient mice, but not in the aged wild-type mice. CONCLUSIONS: These results indicate that long-term survived, uneroded hypertrophic chondrocytes, at least in a part, acquire osteocytic characteristics.     

LPS刺激小鼠骨细胞RANKL/OPG和IL-6表达的实验研究

目的: 探讨LPS对体外培养的小鼠MLO-Y4细胞RANKL/OPG和IL-6表达的影响。方法: 以5 mg/L的LPS刺激细胞,用CCK-8法于(12、24、48 h)后检测细胞的增殖;以不同浓度(1、10、100、500、1000 μg/L)的LPS刺激细胞,分别在作用4 h和1.5 h后用RT-PCR检测细胞对RANKL/OPG和IL-6的相对表达;以100 μg/L的LPS刺激细胞,分别在作用(0.5、1、2、4、8 h)和(0.5、1、1.5、2、4 h)两种不同时间后用RT-PCR检测细胞对RANKL/OPG和IL-6的相对表达。结果: LPS对MLO-Y4细胞增殖无影响;100 μg/L的LPS能显著上调细胞对RANKL和IL-6的相对表达,与(500, 1000 μg/L)LPS的上调结果无统计学差别;除0.5 h外其余时间点LPS均上调细胞对RANKL和IL-6的相对表达,并分别在4 h和1.5 h达到峰值;所有样本LPS对细胞OPG的相对表达均无影响。结论: 一定浓度的 LPS上调了MLO-Y4 细胞对RANKL和IL-6的表达,而对OPG的表达无影响。     

Histochemical assessment on osteocytic osteolysis in lactating mice fed with a calcium-insufficient diet

ObjectivesThis study aimed to examine if feeding lactating mice a calcium-insufficient diet while simultaneously administering alendronate (ALN) could potentially induce osteocytic osteolysis.MethodsLactating mice were fed calcium (Ca)-insufficient diets with or without ALN administration, and then their femurs were examined for TRAP and ALP, and observed by Kossa staining and transmission electron microscopy (TEM). Mice that had been fed a Ca-insufficient diet were then fed a ⁴⁴Ca-containing diet, and their tibial sections were examined by isotope microscopy.ResultsMice fed a Ca-insufficient diet had a reduced number of TRAP-positive osteoclasts after ALN administration. ALN-treated, lactating mice fed a Ca-insufficient diet had enlarged lacunae in their cortical bones, and TEM imaging demonstrated expanded regions between osteocytes and lacunar walls. In ALN-treated lactating mice fed a Ca-insufficient diet, huge areas of demineralized bone matrix occurred, centered around blood vessels in the cortical bone. Isotope microscopy showed ⁴⁴Ca in the vicinity of the osteocytic lacunae, and in the broad, previously demineralized region around the blood vessels in the cortical bone of lactating mice fed a ⁴⁴Ca-sufficient diet.ConclusionsBone demineralization likely takes place in the periphery of the osteocytic lacunae and in the broad regions around the blood vessels of lactating mice when they are exposed to severely reduced serum Ca through a Ca-insufficient diet coupled with ALN administration.