非血管化游离骨移植中细胞活力的实验研究

目的 :探讨非血管化游离骨块中细胞存活能力及数量与离体时间的关系。方法 :取狗非血管化游离髂骨块 ,在离体 2 5～ 15 0min的不同时间点 ,消化骨块获得细胞 ,进行活细胞计数。对消化获得细胞体外培养2 4h后 ,再进行活细胞计数 ,分析细胞存活与离体时间的关系。结果 :在离体 2 5～ 15 0min时间内 ,骨块细胞的存活率从 91.96%降至 9.5 2 %。统计学检验 ,2 5min组与 70min以前组之间没有统计上的差别 ,P >0 .0 5 ;2 5min组与 70min及其以后组之间有明显差别 ,P <0 .0 1。培养 2 4h后 ,以上各时间点的细胞存活率则从88.73 %到 3 .88% ,组间差别与刚离体时相同 ,但 70min后的各组活细胞率明显下降。结论 :非血管化狗游离髂骨块的离体时间对其中的细胞存活有明显影响 ,离体 70min可能是其关键点 ;在非血管化游离骨移植中 ,尽量缩短骨块的离体时间 ,有利于保存更多的活细胞     

Normal bone physiology,remodelling and its hormonal regulation

The skeleton has structural and locomotor functions, and is a mineral reservoir. Bone turnover by osteoclasts and osteoblasts is a lifelong process, incorporating growth, modelling and remodelling to repair microdamage and access the mineral reservoir. Signalling between bone cells is essential for the co-ordination of these processes. Osteoblasts regulate osteoclast activity through the RANK/RANK ligand/OPG system, and osteocytes regulate osteoblast activity through sclerostin secretion. If resorption and formation are balanced there is no net change in bone mass after each remodelling cycle, but with ageing and some disease states resorption exceeds formation, leading to negative bone balance, decreased bone mass and loss of microstructural integrity. The rate of remodelling is determined by factors including mechanical loading and endocrine influences. The most important endocrine regulator of bone turnover is oestrogen, but other hormones regulating bone metabolism include IGF-1, PTH and gut and adipocyte hormones.     

Elevated circulating sclerostin correlates with advanced disease features and abnormal bone remodeling in symptomatic myeloma: reduction post-bortezomib monotherapy

Sclerostin is a Wingless and Int-1 inhibitor, which is produced by osteocytes and inhibits osteoblast-driven bone formation. Sclerostin is implicated in the pathogenesis of bone loss in metabolic bone disorders but there is no information for its effect on multiple myeloma (MM)-related osteolytic disease. We evaluated circulating sclerostin in 157 newly diagnosed patients with symptomatic myeloma, in 25 with relapsed myeloma who received bortezomib monotherapy, in 21 patients with monoclonal gammopathy of undetermined significance (MGUS), and in 21 healthy controls. Patients with active myeloma had elevated circulating sclerostin compared to MGUS patients and controls (p < 0.01). MM patients who presented with fractures at diagnosis (n = 34) had very high levels of circulating sclerostin compared with all others (p < 0.01), whereas sclerostin correlated negatively with bone specific alkaline phosphatase (a bone formation marker; r = -0.541, p < 0.0001) and positively with C-telopeptide of collagen type-1 (a bone resorption marker; r = 0.524, p < 0.0001). Patients with International Staging System (ISS)-3 disease had higher circulating sclerostin compared to ISS-1 and ISS-2 MM (p = 0.001). Furthermore, patients with high sclerostin (upper quartile, n = 40) had a median survival of 27 months versus 98 months of all others (p = 0.031). Relapsed MM patients had higher levels of circulating sclerostin even compared to newly diagnosed patients (p < 0.01). Bortezomib monotherapy resulted in a reduction of sclerostin by almost 50% in both responders and non-responders. These results suggest that patients with active myeloma have elevated circulating sclerostin, which correlated with advanced disease features including severe bone disease. Our study indicates sclerostin as a possible target for the development of novel therapies to enhance osteoblast function in myeloma.     

大鼠去卵巢后不同时期骨微结构和力学性能的变化特征

目的揭示大鼠在去卵巢后不同时期腰椎松质骨微结构退变的变化特征，探讨骨整体力学性能下降的同时可能存在的各种适应性代偿性变化。方法50只7月龄SD大鼠随机分为基线、去卵巢组（OVX组）和假手术组（SHAM）。基线组10只，其余每组均20只。实验开始时先将基线组10只处死，OVX组和SHAM组分别在手术后3周、15周各处死10只，留取动脉血清及腰椎标本，骨微结构、力学和生化指标的测定。结果去卵巢后3周时OVX组表观骨密度、骨体积分数、骨小梁厚度和骨小梁数量均较SHAM和基线降低（P〈0．05），各向异性度较基线下降（P〈0．05）而与SHAM组无统计学差异。骨小梁面积密度、骨小梁间隔和结构模型指数均较SHAM和基线组增加（P〈0．05）。去卵巢后3周时OVX组最大应力、弹性模量、血清TRAP-5b和骨细胞密度均低于基线（P〈0．05）而与SHAM组无统计学差异。去卵巢后15周时OVX组表观骨密度、骨体积分数、骨小梁数量和联接密度、最大应力、TRAP-Sb和骨细胞密度均较SHAM和基线组降低（P〈0.05），骨小梁结构模型指数、骨小梁间隔和各向异性度均较SHAM和基线组增加（P〈0．05），骨小梁面积密度和厚度均与SHAM和基线组无统计学差异。结论大鼠去卵巢后腰椎骨量快速丢失，骨微结构逐渐退变，而血清TRAP-5b水平下降及骨细胞密度、骨小梁各向异性度和厚度的适应性增加，可能在一定程度上代偿骨力学性能的下降，有利于维持骨结构的完整性。     

骨细胞的功能：生物学研究和机理探讨

骨细胞（osteocyte）是在矿化骨基质内名副其实的骨的细胞（bonecell）。通过细胞突触，骨细胞彼此相连，很可能也与骨髓中某些细胞相接，从而形成三维细胞网络。从骨的超微结构看，骨细胞处于很理想的位置，可以担当骨生理的重要调节因子。然而，由于骨细胞处于矿化骨基质深层，对其功能了解甚少。但随着新技术方法的应用，就有可能对这种成骨细胞系中的终末分化细胞进行深入研究，人们也由此对骨细胞的兴趣大增。骨细胞被认为是骨的机械应力感受器，它有可能参与骨重建。骨细胞能分泌具有调节成骨细胞功能的硬骨素（sclerostin）。一种能降低肾脏磷回吸收的激素——调磷因子FGF23也主要源白骨细胞。随着我们对骨细胞了解的深入，清楚显示，骨细胞不仅能影响局部骨转换活性，而且对全身矿物质的体内平衡也有多种功能。因此，对骨细胞的研究，可以为代谢性骨病的治疗探索新的途径。     

Human Primary Osteocyte Differentiation in a 3D Culture System

Studies on primary osteocytes, which compose >90–95% of bone cells, embedded throughout the mineralized matrix, are a major challenge because of their difficult accessibility and the very rare models available in vitro. We engineered a 3D culture method of primary human osteoblast differentiation into osteocytes. These 3D‐differentiated osteocytes were compared with 2D‐cultured cells and with human microdissected cortical osteocytes obtained from bone cryosections. Human primary osteoblasts were seeded either within the interspace of calibrated biphasic calcium phosphate particles or on plastic culture dishes and cultured for 4 wk in the absence of differentiation factors. Osteocyte differentiation was assessed by histological and immunohistological analysis after paraffin embedding of culture after various times, as well as by quantitative RT‐PCR analysis of a panel of osteoblast and osteocyte markers after nucleic acid extraction. Histological analysis showed, after only 1 wk, the presence of an osteoid matrix including many lacunae in which the cells were individually embedded, exhibiting characteristics of osteocyte‐like cells. Real‐time PCR expression of a set of bone‐related genes confirmed their osteocyte phenotype. Comparison with plastic‐cultured cells and mature osteocytes microdissected from human cortical bone allowed to assess their maturation stage as osteoid‐osteocytes. This model of primary osteocyte differentiation is a new tool to gain insights into the biology of osteocytes. It should be a suitable method to study the osteoblast‐osteocyte differentiation pathway, the osteocyte interaction with the other bone cells, and orchestration of bone remodeling transmitted by mechanical loading and shear stress. It should be used in important cancer research areas such as the cross‐talk of osteocytes with tumor cells in bone metastasis, because it has been recently shown that gene expression in osteocytes is strongly affected by cancer cells of different origin. It could also be a very efficient tool for drug testing and bone tissue engineering applications.     

Slow Resorption of Anorganic Bovine Bone by Osteoclasts in Maxillary Sinus Augmentation

Purpose: Different biomaterials have been suggested for guided bone regeneration (GBR). These might show the ideal properties to let a new bone formation in the grafted area. Among these ideal features, it is essential their controlled resorption in order to be replaced for new vital bone. Bovine bone has been used widely as a good biomaterial for GBR, however there is still an interesting controversy about its resorbable capacity. In this sense, the objective of this study was to examine the behavior of anorganic bovine bone (ABB) in long‐term maxillary sinus graft healing and study its relationship with morphological and morphometrical variables. Materials and Methods: Seventeen maxillary sinus augmentation procedures were performed in patients. Bone cores were obtained from implant receptor sites at 6 months, 3 years, and 7 years of implant placement for histological, morphometric, and immunohistochemical (tartrate resistant acid phosphatase [TRAP]/cathepsin K/CD68) studies. Results: The percentages of bone, ABB particles, connective tissue, osteocytes, and osteoblasts in maxillary sinus grafts were similar at 6 months, 3 years, and 7 years. A progressive and significant decrease was detected in osteoclasts (p = .05, Kruskal‐Wallis test), TRAP and cathepsin K expression (p = .014 and p = .021, respectively), and osteoid lines (p = .038). Conclusion: According to these data, a decrease in osteoclasts over time may, partially, explain the ABB persistence observed in core biopsies. Further studies with more cases and different graft maturation times are required to elucidate the resorption rates and cell events underlying these phenomena.     

载荷作用下骨细胞分泌因子对成骨细胞和破骨细胞的调节

骨细胞是骨组织主要的力学感受及转导细胞,它们通过众多突触结构相互连接,形成庞大的骨稳态细胞调控网络,联系着成骨细胞、破骨细胞等骨基质表面细胞。骨细胞通过旁分泌途径影响成骨细胞骨形成和破骨细胞骨吸收来调节骨代谢,维持骨更新。针对骨细胞在受到力学刺激后分泌或释放的一些信号分子或蛋白因子对成骨细胞和破骨细胞生长分化的影响,本文综述近年来关于受力学刺激的骨细胞如何与成骨/破骨细胞进行通讯,为骨细胞生物力学研究提供新思路。     

成骨细胞和骨细胞的力学信号转导

背景: 骨骼具有功能适应性的特点,骨骼细胞是力学信号敏感细胞,但细胞的力学信号转导功能是如何实现的,对骨骼如何调控仍不明确。 目的：了解成骨细胞和骨细胞的力学信号转导途径,为利用力学信号改善骨骼功能提供理论依据。 方法：应用计算机检索PubMed数据库2000-01/2011-03相关文献。英文检索词为“osteoblast,osteocyte,bone cells,mechanical stress”, 根据纳入标准共69篇文章进行综述,以此对骨骼细胞力学信号转导相关内容进行总结。 结果与结论：骨骼具有功能适应性的特点,骨骼细胞是力学信号敏感细胞,但细胞的力学信号转导功能是如何实现的,对骨骼具有怎样的调控仍不明确。研究表明,由于骨骼的结构特点和细胞位置,成骨细胞和骨细胞是最重要的力学敏感性细胞。力学信号在骨骼内的转导过程分为4个阶段：①力学偶联。②生化偶联。③信号的传递。④效应性细胞的反应。通过这4个阶段的作用,作用在骨骼上的应力信号转导为生物化学信号,并影响细胞的功能,最终导致骨骼组织出现相应的结构变化以适应应力环境的需要。对于力学信号在骨髓间充质干细胞中的调控机制还有待继续深入探索。