流体切应力促进组织工程骨成骨和血管化的研究进展

流体切应力在组织工程学中得到越来越多的重视。通过模拟流体切应力作用于骨组织后对感应细胞（成骨细胞和血管内皮细胞）产生刺激,达到骨量增多和快速血管化的目的。通过阐述流体切应力对成骨细胞和血管内皮细胞的作用机制,为促进组织工程骨的成骨和血管化提供思路和依据。     

不同比例共培养内皮细胞和成骨细胞的成骨及成血管效应*☆

背景：近年来研究显示内皮细胞和成骨细胞共培养修复骨组织的过程中存在相互促进的作用。 目的：观察不同比例下内皮细胞和成骨细胞共培养对其成血管和成骨作用的影响。 方法：分离培养人脐静脉内皮细胞和成骨细胞MG63,内皮细胞和成骨细胞按照1∶0,8∶1,4∶1,1∶1,1∶4,1∶8,      0∶1在24孔板中进行共培养,内皮细胞和成骨细胞分别单纯单层培养作对照。在第7,14,21天时观察细胞生长状态、血管内皮生长因子表达水平及碱性磷酸酶活性。 结果与结论：各组细胞在21 d中生长良好,第14天1∶8组可见成骨细胞开始集落式生长,第21天1∶4组钙化染色结节要明显强于1∶8组,而单纯成骨细胞组并未出现钙化结节。1∶4组碱性磷酸酶含量在3个时间点均明显增高(P < 0.05)且第7天时血管内皮生长因子开始增高,直到第21天时达到高峰。说明内皮细胞和成骨细胞共培养比例为4∶1时,成血管作用较强,而比例为1∶4时成骨作用更强。      

重组人骨形态发生蛋白2与碱性成纤维细胞生长因子联合促进兔骨髓基质细胞血管内皮细胞生长因子表达及其成骨潜能

背景：了解在体外或体内环境下,应用重组人骨形态发生蛋白2和碱性成纤维细胞生长因子,能否达到既加强成骨又促进血管内皮细胞生长因子表达的双重作用。 目的：观察兔骨髓基质细胞经重组人骨形态发生蛋白2与碱性成纤维细胞生长因子刺激后,其血管内皮细胞生长因子表达及成骨潜能的变化。 方法：取兔双侧股骨骨髓基质细胞,采用骨髓基质细胞体外培养技术,单独或联合以重组人骨形态发生蛋白2、碱性成纤维细胞生长因子刺激细胞。细胞培养5 d后,进行细胞形态、增殖情况、碱性磷酸酶活性、成骨结节、血管内皮细胞生长因子阳性细胞率等项目的检测。 结果与结论：联合先后应用重组人骨形态发生蛋白2、碱性成纤维细胞生长因子在细胞计数、碱性磷酸酶活性、矿化面积百分率、血管内皮细胞生长因子阳性细胞率4个检测项目上优于同时应用重组人骨形态发生蛋白2或碱性成纤维细胞生长因子以及单独应用重组人骨形态发生蛋白2或碱性成纤维细胞生长因子。结果表明合理的联合使用重组人骨形态发生蛋白2和碱性成纤维细胞生长因子,不仅可促进骨髓基质细胞的快速增殖及向成骨细胞转化,还可促进促血管内皮细胞增生的重要介质血管内皮细胞生长因子的表达。     

骨髓微环境中微血管内皮细胞作用的研究进展

骨生长和骨重建依赖于骨内微环境中成骨细胞、破骨细胞以及其他细胞之间的相互作用。正常情况下，成人骨骼系统具有精细的调节功能，使骨形成和骨吸收过程处于平衡，但在老化或各种病理状态下，这种平衡状态可能就会打破。现已认识到起源于器官组织微循环系统的血管内皮细胞在形态和功能上是不同于起源于作为通道血管的血管内皮细胞。不同器官和组织起源的微血管内皮细胞在形态、基因、表型和功能方面都有差别，这些差别与细胞所起源的器官和组织的功能特性密切相关。近年来研究表明骨内微环境中存在一个复杂的由多种细胞组成的调控网络。而骨髓微血管内皮细胞可能是这个网络中的重要一员。现就骨髓微血管内皮细胞在骨微环境中的作用综述。     

骨髓间充质干细胞成骨分化中血管内皮生长因子和骨形态发生蛋白2的表达

背景：有研究表明血管内皮生长因子可促进内皮细胞增殖、分化和迁移,而骨髓间充质干细胞经诱导分化后细胞自身的血管内皮细胞生长因子及骨形态发生蛋白2如何表达,却鲜见报道。 目的：观察兔骨髓间充质干细胞成骨诱导分化过程中血管内皮细胞生长因子和骨形态发生蛋白2的表达。 方法：取第3代新西兰大白兔骨髓间充质干细胞,1 d后将细胞分为两组：对照组不更换培养基;诱导组加入成骨诱导培养基进行成骨诱导,流式细胞仪检测其表面抗原。RT-PCR和Western-Blot检测细胞成骨分化过程中血管内皮细胞生长因子和骨形态发生蛋白2的表达。 结果与结论：兔骨髓间充质干细胞成骨诱导后,细胞形态由长梭形逐渐变为短梭形,多角形,最后成集落层叠生长;在诱导过程中,碱性磷酸酶活性逐渐增高,与对照组比较有显著性差异(P < 0.05);诱导7 d时Ⅰ型胶原免疫细胞化学染色、14 时骨钙素免疫细胞化学染色、21 d时钙化结节染色阳性;RT-PCR和Western-Blot结果显示两组均有骨形态发生蛋白2和血管内皮细胞生长因子表达,诱导后第7,14,21天表达比较,差异有显著性意义(P < 0.05),第7天,两者表达最强。说明兔骨髓间充质干细胞分离、提取操作简便易行,且有较强的生长增殖能力,可诱导分化为成骨细胞。其在成骨诱导过程中,骨形态发生蛋白2和血管内皮生长因子表达均先增强后减弱,血管内皮细胞生长因子表达可能对血管生成有促进作用。     

骨髓间充质干细胞经不同时间成骨细胞诱导后细胞性质比较

目的 探讨骨髓间充质干细胞经成骨细胞诱导不同时间后的细胞性质。方法 成人骨髓分离单个核细胞（MNC）,体外培养获得间充质干细胞（MSC）,传至第4代后加入地塞米松（DEX）、抗坏血酸和β-甘油磷酸,诱导成骨分化。诱导7d和14d后的细胞与正常成人成骨细胞分别加入不同浓度骨形态发生蛋白-2（BMP-2）和DEX,48h后检测细胞增殖、碱性磷酸酶（ALP）活性和骨钙素（OCN）的含量。结果 BMP-2和DEX对诱导7d后的细胞有明显的刺激增殖和ALP活性的作用;对诱导14d后的细胞,BMP-2和DEX都不能刺激细胞增殖和ALP活性,但OCN含量明显增加。正常成人成骨细胞对BMP-2和DEX作用的反应与诱导14d的成骨样细胞基本一致,但其OCN绝对含量明显高于MSC来源的成骨细胞。结论 骨髓MSC向成骨诱导7d和14d后细胞分化程度不同。诱导时间在MSC的成骨分化中是一个非常关键的因素,但其机制和最佳诱导时间尚需进一步研究。诱导14d后的MSC的成骨活性低于正常成人成骨细胞。     

VEGF通过上调CCN2促HUVEC迁移和血管生成

目的 探讨血管内皮生长因子(VEGF)诱导的成骨细胞中结缔组织生长因子(CTGF/CCN2)对人脐静脉血管内皮细胞(HUVECs)的影响.方法 用Real time PCR法及ELISA法检测VEGF诱导成骨细胞(OSE)中CCN2含量;制备成骨细胞(OSE)上清液;将细胞分为control组、OSE组和VEGF-OSE组(n=3).用小干扰RNA (siRNA)转染法抑制成骨细胞中CCN2的表达;Transwell法检测内皮细胞迁移;Matrigel实验检测管样结构形成能力.结果 VEGF呈时间和剂量依赖性上调成骨细胞中CCN2 mRNA和蛋白的表达;CCN2可促进内皮细胞的迁移和管样结构形成(P＜0.05),当CCN2被siRNA基因沉默或者加入CCN2抗体后,CCN2对内皮细胞迁移和管样结构形成的促进作用均受到明显抑制(P＜0.05).结论 VEGF通过上调成骨细胞中CCN2的表达,促内皮细胞(HUVECs)的迁移和血管生成.     

血管内皮生长因子165/骨形态发生蛋白改善缺氧复氧状态下成骨细胞损伤

背景：研究发现,血管内皮生长因子165和骨形态发生蛋白两种因子在缺氧复氧过程中相互作用,通过调节细胞内信号通路的活化,参与骨细胞损伤的修复过程。目的：进一步探究血管内皮生长因子165/骨形态发生蛋白与缺氧复氧成骨细胞损伤的关系分析。方法：取成骨细胞,建立缺氧复氧损伤模型,建模前后Real-Time PCR法和免疫印迹法检测血管内皮生长因子165、骨形态发生蛋白2的mRNA及蛋白表达。分别给予建模后成骨细胞不同质量浓度(10,20,40ng/mL)血管内皮生长因子165或骨形态发生蛋白2处理12,24,36,48,72 h,CCK-8法检测细胞增殖,DAPI检测细胞凋亡。结果与结论：(1)与建模前相比,建模后成骨细胞中血管内皮生长因子165、骨形态发生蛋白2的mRNA和蛋白表达量显著降低(P <0.05);(2)成骨细胞增殖率随着血管内皮生长因子165质量浓度的升高而明显升高(P <0.05);成骨细胞凋亡率随着血管内皮生长因子165质量浓度的升高而明显降低(P <0.05);(3)成骨细胞增殖率随着骨形态发生蛋白2质量浓度的升高而明显升高(P <0.05);成骨...     

兔骨髓间充质干细胞定向诱导分化为脂肪细胞及成骨细胞的研究

用密度梯度离心和贴壁法分离和纯化兔骨髓间充干细胞，建立诱导兔MSCs向脂肪细胞及成骨细胞表型转化的方法及条件。在成脂诱导剂或成骨诱导剂作用下，对原代和第2代兔MSCs进行成脂和成骨诱导培养，并鉴定成脂及成骨表型。结果表明：原代及第2代兔MSCs均有一定的成脂、成骨能力，且第2代细胞的分化能力较原代低。在诱导培养条件下，原代及第2代兔MSCs均能分化，成脂诱导21d，75％的兔MSCs转化为脂肪细胞；成骨诱导21d，75％的兔MSCs转化为成骨细胞。兔MSCs在适当的诱导条件下可快速分化为脂肪细胞或成骨细胞。     

脉冲电磁场对体外培养成骨细胞的影响

脉冲电磁场影响体外培养的成骨样细胞,促使其增殖、分化、并加速骨样组织形成,对成骨细胞形态学上也有一定影响,本文综述近年来国内外在电磁场对成骨细胞作用方面的一些研究进展.     

Identification and location of bone-forming cells within cartilage canals on their course into the secondary ossification centre

Osteoblasts and osteocytes derive from the same precursors, and osteocytes are terminally differentiated osteoblasts. These two cell types are distinguishable by their morphology, localization and levels of expression of various bone cell-specific markers. In the present study on the chicken femur we investigated the properties of the mesenchymal cells within cartilage canals on their course into the secondary ossification centre (SOC). We examined several developmental stages after hatching by means of light microscopy, electron microscopy, immunohistochemistry and in situ hybridization. Cartilage canals appeared as extensions of the perichondrium into the developing distal epiphysis and they were arranged in a complex network. Within the epiphysis an SOC was formed and cartilage canals penetrated into it. In addition, they were successively incorporated into the SOC during its growth in the radial direction. Thus, the canals provided this centre with mesenchymal cells and vessels. It should be emphasized that regression of cartilage canals could never be observed in the growing bone. Outside the SOC the mesenchymal cells of the canals expressed type I collagen and periostin and thus these cells had the characteristics of preosteoblasts. Periostin was also expressed by numerous chondrocytes. Within the SOC the synthesis of periostin was down-regulated and the majority of osteoblasts were periostin negative. Furthermore, osteocytes did not secret this protein. Tissue-non-specific alkaline phosphatase (TNAP) staining was only detectable where matrix vesicles were present. These vesicles were found around the blind end of cartilage canals within the SOC where newly formed osteoid started to mineralize. The vesicles originated from osteoblasts as well as from late osteoblasts/preosteocytes and thus TNAP was only expressed by these cells. Our results provide evidence that the mesenchymal cells of cartilage canals express various bone cell-specific markers depending on their position. We suggest that these cells differentiate from preosteoblasts into osteocytes on their course into the SOC and consider that cartilage canals are essential for normal bone development within the epiphysis. Furthermore, we propose that the expression of periostin by preosteoblasts and several chondrocytes is required for adhesion of these cells to the extracellular matrix.     

Buried alive: how osteoblasts become osteocytes.

During osteogenesis, osteoblasts lay down osteoid and transform into osteocytes embedded in mineralized bone matrix. Despite the fact that osteocytes are the most abundant cellular component of bone, little is known about the process of osteoblast-to-osteocyte transformation. What is known is that osteoblasts undergo a number of changes during this transformation, yet retain their connections to preosteoblasts and osteocytes. This review explores the osteoblast-to-osteocyte transformation during intramembranous ossification from both morphological and molecular perspectives. We investigate how these data support five schemes that describe how an osteoblast could become entrapped in the bone matrix (in mammals) and suggest one of the five scenarios that best fits as a model. Those osteoblasts on the bone surface that are destined for burial and destined to become osteocytes slow down matrix production compared to neighbouring osteoblasts, which continue to produce bone matrix. That is, cells that continue to produce matrix actively bury cells producing less or no new bone matrix (passive burial). We summarize which morphological and molecular changes could be used as characters (or markers) to follow the transformation process.     

Histological identification of osteocytes in the allegedly acellular bone of the sea breams Acanthopagrus australis,Pagrus auratus and Rhabdosargus sarba (Sparidae,Perciformes, Teleostei)

The bone of advanced teleost fishes such as those of the family Sparidae is said to lack osteocytes or to be acellular. Acellularity has been determined by apparent lack of osteocyte lacunae. This study questions the validity of this criterion. Scanning electron and light microscopy of paraffin and resin sections were used to show that the sides of sea bream mandibles consist of laminar parallel-fibred bone that we call tubular bone, because it contains tubules, and localised regions of Sharpey fibre bone. Osteocytes lie along the walls of tubules that also contain collagen fibril bundles (T-fibres), or in the lumens of tubules that do not contain T-fibres. We show that the osteocytes are derived from osteoblasts. The T-fibre system is different from other fibre systems that have been described. The tubules enclose wide T-fibres (lenticular in cross-section, maximum width about 8 μm) that taper at their ends and continue as thin T-fibres (round in crosssection, about 2 μm wide). The T-fibres originate in the periosteum. In mature tubular bone, spaces of increasing size develop around the osteocytes. Osteocytes are released from the bone matrix and become postosteocytes or bone-lining cells. Secondary bone lines the largest spaces. In Sharpey fibre bone, small osteocytes in small lacunae (about 2 μm wide) are found in columns parallel to the Sharpey fibres. Large osteocytes are found in large round spaces and are much larger than comparable osteocytes in lacunae in the bone of the salmon Salmo salar. We conclude that an absence of visible or conventional osteocyte lacunae does not mean that the cells themselves are absent. There are cells and two types of collagen fibre bundle in the tubules. The cells are osteocytes derived from osteoblasts, and these osteocytes apparently resorb bone with the result that large amounts of bone are destroyed. “Acellular” tubular and Sharpey fibre bone are types of cellular bone that differ from each other and from conventional cellular bone.     

Ultrastructure of osteoid osteoma.

The ultrastructure in five cases of osteoid osteoma is described. The osteoblasts generally had a morphology similar to that of normal osteoblasts with a few differences. They contained irregular indented nuclei, glycogen particles, abundant fine intracytoplasmic fibrils, and rare iron containing lysosomes. In several osteoblasts in two cases there were atypical mitochondria with a lobulated or "honeycomb" appearance. These atypical mitochondria were also observed in two osteoclasts; otherwise the cells resembled normal osteoclasts. Other cells present in osteoid osteoma besides osteocytes included osteoprogenitor cells resembling Scott type A and B cells and cells in transitional stages of differentiation. The osteoblasts most likely originated from Scott type A cells of preosteoblasts. The areas of mineralized matrix conformed to the morphology of coarse woven bone. Varying amounts of osteoid were noted. In two cases the osteoid contained, in addition to collagen, fine granular material, which probably represents polysaccharides. No nerve fibers were identified in the material studied. It is thought that osteoid osteoma is a benign neoplastic lesion. A case of osteoblastoma was studied for comparison; the osteoblastoma cells were found to have basically the same morphology as the cells in osteoid osteoma, including the atypical mitochondria. Our ultrastructural observations support the idea that osteoid osteoma and osteoblastoma are closely related lesions.     

Endothelin-1 localization in bone cells and vascular endothelial cells in rat bone marrow

Endothelin-1 (ET-1) localization in bone cells and associated vascular endothelial cells in metaphyseal bone marrow of the rat femur was examined by a biotin-streptoavidin-horseradish peroxidase method in paraffin sections and by indirect immunogold techniques in post-embedded ultrathin sections. Mouse anti-ET-1 monoclonal antibody was used as the primary antibody. In metaphyseal bone marrow, intense immunostaining was observed over osteoclasts, osteoblasts, young osteocytes, and vascular endothelial cells. But bone and cartilage matrices and chondrocytes in the proliferating zone were negative for immunoreaction. At the subcellular level, specific immunogold labeling was localized along plasma membranes and in the cytoplasm including those of ruffled borders and clear zones of osteoclasts. Some colloidal gold particles were also detectable within pale vacuoles of osteoclasts. Immunoreactivity was also found along the plasma membranes, cisterns of rough-surfaced endoplasmic reticulum, mitochondria, and cytoplasmic matrices of osteoblasts, but was less intense than that of osteoclasts. In endothelial cells of blood capillaries in close proximity to bone cells, intense immunolabeling occurred over the cytoplasm. None of the cases examined showed accumulation of immunogold particles in the secretion granules of these cells. © 1993 Wiley-Liss, Inc.     

人胎儿股骨发生过程中血管内皮生长因子的表达

目的探讨血管内皮生长因子(VEGF)与人胚胎骨发生及发育的关系。方法用免疫组化技术对11~36 W人胎儿股骨中血管内皮生长因子的分布和表达情况进行了研究。结果VEGF阳性反应主要见于成骨细胞、破骨细胞及新生骨细胞中,血管内皮细胞及骨髓细胞也呈阳性反应;骺软骨钙化区肥大的软骨细胞为VEGF阳性反应,但其它区域的软骨细胞中无VEGF表达。结论VEGF在胚胎发生时期在血管内皮细胞和成骨细胞的表达,说明VEGF在人胎儿骨发生及发育过程中发挥重要作用。     

Recent progress in studies on osteocytes--osteocytes and mechanical stress

Although osteocytes are of the most abundant cells in bone, our knowledge about the role of osteocytes in bone metabolism is still poor compared with that about osteoblasts and osteoclasts, both being on the surface of bone. Osteocytes are terminally differentiated bone-forming cells. During bone formation, some of the osteoblasts lining the surface of bone are incorporated into the newly formed osteoid matrix and become osteocytes, while the other osteoblasts remain on the surface as lining cells. During this transition from osteoblasts to osteocytes, the cells lose numerous osteoblastic phenotypes and acquire osteocytic characteristics such as high expression of osteocalcin and particularly their specific morphology. Osteocytes are connected with each other in bone and with osteoblasts on the bone surface through canaliculi, forming cellular networks; and gap-junctions present at the contact sites mediate their intercellular communication. Several roles of osteocytes in bone have been proposed so far. Of them, based on the morphological characteristics of osteocytes, sensation of mechanical stress loaded onto bone is suspected to be one of their functions. One of the mechanical stresses on bone is fluid shear stress. Between the osteocyte's plasma membrane and the bone matrix is the periosteocytic space. This space exists both in the lacunae and in the canaliculi, and it is filled with extracellular fluid (ECF). Application of mechanical stress to bone locally deforms the tissue. This periodical deformation subsequently causes an increase in the flow of ECF in the periosteocytic space, resulting in shear stress on the surface of the osteocytes. Experimental studies demonstrated that bone cells were equivalently or more sensitive to the fluid shear stress than epithelial cells. Osteocytic cells cultured enhanced expression of prostaglandin (PG) G/H synthase-2 (COX-2) mRNA in response to shear stress. PGE2 is a potent regulator of proliferation and function of osteoblasts and osteoclasts. Therefore, a metabolic response by osteoblasts and osteoclasts lining the bone surface may be caused by PGE2 produced by osteocytes in response to shear stress when the prostanoid reaches the surface through the canaliculi. In conclusion, osteocytes play an important role in sensing extracellular mechanical stress, and the mechanical signals mediated by osteocytes may regulate the overall metabolism of cells in bone tissue.     

Histological evidence of the altered distribution of osteocytes and bone matrix synthesis in klotho-deficient mice

Mice homozygous for klotho gene deletion are well established aging models as they mimic certain aspects of human senescence e.g. osteoporosis. Induced senescence may affect cellular functions and alter the histological properties of the extracellular matrices. The present study examined the histological and ultrastructural features of osteocytes and the surrounding bone matrix in klotho-deficient mice. As expected, osteoblasts showed a flattened shape with a weak immunoreactivity for alkaline phosphatase, and the bone matrix contained many empty osteocytic lacunae. The walls of both normal and empty lacunae were intensely immunopositive for osteopontin and dentin matrix protein-1, but featured an inconsistent immunoreactivity for osteocalcin and type I collagen. Not surprisingly, TUNEL-positivity, indicative of apoptosis, was found in many osteoblasts, osteocytes, and bone marrow cells of the klotho-deficient mice. In transmission electron microscopy, an amorphous matrix containing non-collagenous organic materials was recognizable around osteoblasts and in the osteocytic lacunae. Some osteoblasts on the bone surface featured these amorphous materials in vacuoles associated with their trans-Golgi network, indicating that, under klotho-deficient conditions, they synthesize and secrete the non-collagenous structures. Some osteocytes displayed pyknosis or degenerative traits. Thus, our findings provide histological evidence that klotho gene deletion influences the spatial distribution of osteocytes and the synthesis of bone matrix proteins in addition to the accelerated aging of bone cells.     

576个椎体孤立性微骨痂形成的病理研究

从２４例尸体解剖所得完整的脊柱标本共５７６个椎体，检查了微骨痂的存在，数量及分布，描述了它的组织学特点，初步探讨了它的发病机理，组成微骨痂的细胞来源于骨小梁表面的骨母细胞及邻近的骨髓网织细胞，早期为幼稚的波纹骨，以后演变为成熟的板层骨。微骨痂的形成与所受应力有关，受力越大微骨痂数量越多，腰椎是微骨痂形成最多的部位、微骨痂的形成与年龄有关，微骨痂数量在５０岁以后有随着年龄增加而增多的趋势，可能与不同     

A study of intercellular relationships between trabecular bone and marrow stromal cells in the murine femoral metaphysis

The cellular relationship between the substantia spongiosa of bone (cancellous or trabecular bone) and the haematopoietic bone marrow in the femoral metaphysis of C57BL/6NJCL mice was studied by transmission electron microscopy (TEM). Special attention was directed to intercellular junctions between osteocytes, osteoblasts, and bone marrow reticular cells. These were gap junctions and adhesive devices of simple architecture referred to as primitive junctions or zonula adherens-like junctions. Gap junctions were observed between osteocytes (within the trabeculae) and osteoblasts (at the trabecular surface) and between osteoblasts and marrow reticular cells. Gap junctions were also observed between the same cell type within each of these categories. These junctions involved the plasmalemmal membranes of adjacent cell bodies and of processes. Primitive cell junctions had a similar cellular distribution. Quantitative analysis of the cell types covering or positioned around the trabecular bones and of gap junctions between these and other cells was carried out by TEM. It was found that osteoblasts were the most numerous cell type, occupying 31% of the total of each cell type positively identified around the trabeculae (31%), while pre-osteoblasts, (flattened bone marrow reticular cells) took up 26%. These data emphasise the intimate relationship of the various mesenchymal cells based on processes and intercellular junctions, and point to an anatomical and probably functional integration of trabeculae and marrow. The functional significance and putative regulatory activity of this unit are discussed.