脱钙骨基质诱导成骨活性的因素分析（文献综述）

本文对影响脱钙骨基质诱导成骨活性的主要因素作了详细的阐述，认为只要在脱钙骨基质制备过程中对这些因素加以注意，则可以较好地发挥其诱导成骨活性。     

骨诱导性载体复合骨形态发生蛋白的双重成骨作用

本文用猪骨基质明胶（ｐＢＭＧ）复合猪骨形态发生蛋白（ｐＢＭＰ）植入小鼠肌肉内，以期达到ｐＢＭＧ和ｐＢＭＰ双重诱导成骨的叠加作用，并与单独植入的明胶海绵、ｐＢＭＧ、ｐＢＭＰ和无机骨－ｐＢＭＰ（ＡＯＢ－ｐＢＭＰ）的成骨效应相比较。结果显示，含有ｐＢＭＰ的各组术后４天有明显的间充质细胞化，新骨在１０天左右开始形成。ＡＬＰ的活性高峰出现在术后２１～２８天，组间钙、磷含量差异具有高度显著性（Ｐ＜０．０１），明胶海绵组最低，而ｐＢＭＧ－ｐＢＭＰ组含量最高。各组的钙、磷相关系数为高度正相关。作者讨论了ｐＢＭＧ复合ｐＢＭＰ后的双重成骨作用及其优越性，认为ＡＬＰ主要由诱导性新骨成熟期的成骨细胞和骨细胞所产生，新骨中的钙、磷含量因呈高度正相关，两者均可作为新骨定量的指标，但以钙含量为主。     

脱钙骨基质中BMP-2含量与其体内/外成骨活性的相关性研究

目的 探讨脱钙骨基质(demineralized bone matrix,DBM)中BMP-2含量与其体内/外成骨活性的相关性,以期选择一种简易、便捷的评价DBM成骨活性的方法.方法 取9具人尸体左侧股骨中段组织,采用动态脱钙工艺制备DBM (S1～ S9),同时制备灭活DBM(对照).分别采用蛋白酶抑制剂法、胶原酶法...     

重组合异种骨在小鼠体内诱导成骨活性的剂量依赖性研究

本文是本所重组合异种骨（ＲＢＸ）的系列研究之一。采用碱性磷酸酶和钙量测定，成骨量微机图像分析，对牛骨形态发生蛋白含量不同的ＲＢＸ诱导成骨活性进行了研究。结果提示了ｂＢＭＰ含量不同的ＲＢＸ诱导面骨能力的差异及其下限，并认为实验采用的成骨定量方法，是较可靠易行的指标。     

异种骨基质明胶骨诱导活性的实验研究

按Urist方法制备了人、兔、猪和羊四种骨基质明胶,并分别将其植入25只成年SD大鼠的胸部和股部肌肉内。在植入手术的2、4、6和8周后取标本,通过光学显微镜观察各种骨基质明胶在肌肉内的免疫排斥反应和诱导成骨性。结果表明,异种骨基质明胶(BMG)具有一定的诱导成骨能力,但在BMG周围亦存在程度不同的排斥反应。如能进一步改进制作方法,提高其诱导成骨活性,降低BMG的抗原性,异种BMG将具有重要的临床应用价值。     

一种新型生物活性人工骨的制备及成骨活性的研究

目的：研制CPC/BMP复合人工骨,检测其成骨活性。方法：制备CPC/BMP及CPC骨块,扫描电子显微镜观察表面结构。用小鼠肌袋植入实验观察材料的成骨活性。结果：BMP在CPC中呈微球状均匀分布。CPC植入小鼠肌袋内不能诱导,CPC/BMP植入后1周有软骨细胞出现,2周有编织骨,4周以后小梁骨生成,16周出现成熟的板层骨。同时材料出现降解迹象。有机质含量、碱性磷酸酶浓度在CPC/BMP组出现升高,扫描电镜结果同样证实有新骨形成。结论：CPC/BMP生物活性人工骨可异位诱导成骨,可望成为新型的骨缺损修复材料。     

制备高诱导成骨活性的脱钙骨基质

1前言 骨缺损一直都是困扰骨科医师的一大难题。人们对其的关注和研究由来已久。几个世纪以来，人们尝试了众多不同的修复方法并公认自体骨移植是治疗骨缺损的有效方法。但是，自体骨的数量有限且患者还须经受另一次的额外手术打击，就整体而言，有其难以克服的一些缺点。因此，广大医师及科研工作者长期致力于寻找一种合适的材料来替代自体骨。     

BMP及其诱导成骨的分子生物学基础

ＢＭＰ及其诱导成骨的分子生物学基础谭祖键李起鸿自１９６５年Ｕｒｉｓｔ报道了用脱钙骨基质（ＤＢＭ）植入肌肉诱导间充质细胞分化形成新骨以来，这一研究得到迅速发展。ＤＢＭ经进一步分离提纯得到一种蛋白质物质，被命名为骨形态发生蛋白（ＢＭＰ）。目前对ＢＭＰ的生...     

珍珠层成骨活性物质的研究进展

骨质疏松症是骨代谢异常导致的骨量减少及骨微结构改变,骨脆性增加后继发骨折的一类全身性骨病,近年来已成为影响老年生活质量的主要因素之一。目前,其治疗药物在疗效及长期应用上存在问题,而珍珠层含有成骨活性物质,具有应用于骨质疏松症及其他骨病的潜力。该文就珍珠层活性成分的特点、提取方法及成骨作用研究进展进行综述。     

In vivo osteogenic activity of isolated human bone cells

Human bone cells were obtained as the outgrowth from cancellous bone fragments pretreated with collagenase and DNase. The osteogenic potential of cells in primary culture was assessed upon intramuscular transplantation into young mice pretreated with cortisone. Transplants were recovered after 2 weeks and examined by light microscopy. Of 34 transplants, 6 showed evidence of osteogenesis and 12 the production of unmineralized matrix. Only cells were observed in the other transplants. In an attempt to find a biochemical marker for osteogenic cells we have assayed medium osteocalcin and alkaline phosphatase activity levels in cultures before transplantation. No correlation was found between the level of expression of the two osteoblast markers and the osteogenic potential of the cells.     

The anatomy of bone sialoprotein immunoreactive sites in bone as revealed by combined ultrastructural histochemistry and immunohistochemistry

Bone sialoprotein was immunolocalized at the EM level in thin Lowicryl K4M sections of rat bone. Because of the unconventional EM morphology of the bone matrix seen in thin demineralized acrylate sections, the pattern of immunolabeling was compared with detailed structural images of demineralized bone obtained using an en bloc treatment of tissue samples with the cationic electron dye, Malachite Green (MG), which provides stabilization and retention of anionic material throughout specimen processing. A system of structures corresponding to the sites of bone sialoprotein (BSP) immunoreactivity, as seen in Lowicryl K4M thin sections, could be readily identified in the MG-treated, expoxy thin sections. This system includes the cement lines, and aggregates of similar material within mineralized bone and mineralizing osteoid. The virtual identity of BSP distribution with the arrangement of the MG-visualized material indicates that a BSP-enriched, noncollagenous phase can be demonstrated using different, unrelated tissue preparation and imaging protocols for EM. Besides improving our understanding of the distribution of bone sialoprotein in bone, these data assign a previously unrecognized structural dimension to noncollagenous material in the bone matrix.     

Ultrastructural immunolocalization of bone sialoprotein in guinea-pig osteoarthritis

In diarthrodial joints, bone and cartilage are structurally and functionally inseparable as shown in osteoarthritis (OA), where subchondral bone changes are integral in the disease process. By ultrastructural immunohistochemistry using polyclonal antibodies against guinea-pig bone sialoprotein (BSP), we investigated the distribution of this matrix protein at the osteocartilaginous interface in Hartley guinea-pig knees at different stages of primary osteoarthritis. Between 6 and 12 months they developed moderate osteoarthritic changes predominantly in the medial condyle, progressing to severe OA at 30 months. In all age groups BSP labeling was concentrated to the osteocartilaginous interface at a 1 μm narrow zone at the interface. In the medial osteoarthritic condyle, BSP was increased as compared with the lateral nonosteoarthritic condyle, but only at 30 months, when cartilage fibrillation correlated to BSP. Our observations suggest that altered BSP abundance may be a potential bone marker for late stage OA, while early events in bone cannot be monitored. BSP is expressed early in osteogenesis and may have a role in biological mineralization and growth. Since a sharp zone of intense BSP labeling remains at a remarkably constant level throughout life in guinea-pigs, BSP may have an important structural and/or regulating role at the interface. The protein may act as an anchor of calcified articular cartilage to subchondral bone or by regulating mineralization at the osteocartilaginous interface.     

Affinity of bone sialoprotein and several other bone and dentin acidic proteins to collagen fibrils

Summary Bone and dentin contain several kinds of mineral-binding proteins and cell-attachment proteins. The authors examined the affinity of these proteins to type I collagen, a major matrix protein of the tissue. Bone sialoprotein (BSP), bone Gla protein (BGP), bone small proteoglycan II (PG II), osteonectin (ON), and dentin phosphophoryn (DPP) were labeled with fluorescein isothiocyanate and incubated with reconstituted type I collagen fibril. DPP, BGP, BSP, and PG II were absorbed significantly to the collagen fibril at physiological ionic strength with dissociation constants of 10^-6–10^-7 M. BSP and PG II enhanced the fibrillogenesis of collagen. These acidic proteins can affect the surface properties of collagen fibril, and BSP, having the cell-attachment sequence Arg-Gly-Asp, possibly mediates interaction between collagen fibril and cells.     

The effect of the atelocollagen matrix and bFGF on bone regeneration in defects of the calvaria of rats

The purpose of this study was to evaluate the effect of the atelocollagen matrix and basic fibroblast growth factor (bFGF) on bone regeneration in a bone defect of the calvaria. The subjects were 10-week-old male Wister rats weighing 350 g. Two kinds of atelocollagen matrices, which were either strongly cross-linked or weakly cross-linked, were placed in defects created in the rat cranium. Two doses of bFGF, either 30 μg or 50 μg, were injected into the weakly cross-linked atelocollagen matrix. Both the bone mineral content in the defects and the area of bone regeneration in the median sections of the defects were measured 2, 4, and 8 weeks after surgical treatment. Our findings suggested that the weakly cross-linked atelocollagen matrix tends to demonstrate a scaffold of bone regeneration, and an appropriate dosage of bFGF may inhibit bone absorption to maintain the volume of the regenerated bone.     

Restoration of bone defect and enhancement of bone ingrowth using partially demineralized bone matrix and marrow stromal cells

PURPOSE: This study aimed to investigate the capability of combining marrow stromal cells (MSC) and partially demineralized bone matrix (PDBM) to fill bone defect and enhance bone ingrowth using a canine non-weight-bearing gap model. METHODS: Custom-made implants with 3mm gap between the porous surface and the host bone were used. The implants were inserted into the distal femurs of 25 mongrel dogs and the gaps were randomly assigned to be filled with culture-expanded autologous MSC-loaded PDBM, autograft, fresh-frozen allograft, PDBM alone, or nothing as controls. Histomorphometry using backscattered scanning electron microscopic examination, and mechanical push-out test were performed at 6 months after surgery. RESULTS: Histomorphometry showed that amounts of bone regeneration in the gap and bone ingrowth into the porous-coated surface in the MSC-loaded PDBM-treated group were comparable to those of autograft-treated group and were significantly greater than those of allograft-treated, PDBM-treated, or non-grafted groups. Mechanical test showed the same differences. CONCLUSION: The results of this study showed that combining PDBM and autologous culture-expanded MSC restored bone stock and enhanced bone ingrowth into the porous-coated area in a canine non-weight-bearing gap model. This combination may provide an option for reconstructing bone defect when we perform a cementless revision arthroplasty.     

Recombinant human bone morphogenetic protein-2 potentiates the in vivo osteogenic ability of marrow/hydroxyapatite composites

A composite of marrow mesenchymal stem cells (MSCs) and porous hydroxyapatite (HA) has bone-forming capability. To promote the capability, we added recombinant human bone morphogenetic protein-2 (BMP) to the composite. The bone formation was assessed by rat subcutaneous implantation of 4 different kinds of implants, i.e., HA alone, BMP/HA composites, MSCs/HA composites, and the composites containing BMP (MSCs/BMP/HA). Both HA and the BMP/HA composites did not show bone formation at any time after implantation. The MSCs/HA composites showed moderate bone formation at 4 weeks and extensive bone formation at 8 weeks. The MSCs/BMP/HA composites showed obvious bone formation together with active osteoblasts at 2 weeks and more bone formation at 4 and 8 weeks. The MSCs/BMP/HA composites demonstrated high alkaline phosphatase and osteocalcin expression at both the protein and gene levels. These results indicate that the combination of MSCs, porous HA, and BMP synergistically enhances osteogenic potential and provides a rational basis for their clinical application in bone reconstruction surgery.     

Osteoblast-like cell adhesion to bone sialoprotein peptides.

A number of studies have suggested that biomimetic peptides can be used in the design of a new generation of prosthetic implants to promote the successful biointegration of the implant materials. In the current study, the in vitro bioactivities of several peptides representing RGD (Arg-Gly-Asp)-containing sequences of bone sialoprotein (BSP) toward an osteoblast-like cell line (MC3T3-E1) were examined to provide insight into the molecular basis of BSP's interaction with bone cells. BSP residues 283-288, 281-290, 278-293 and 278-302 were coated on polystyrene surfaces in 96-well non-tissue (untreated) culture plates, and their osteoblast adhesive properties compared to intact BSP and fibronectin as positive controls. BSP peptides 278-302 and 278-293 were found to be the most potent in their adhesive activity, increasing the number of adherent cells to 350% of control levels at an added concentration of 1 microM. Since these two peptides were equivalent in potency, it is suggested that the region 294-302 beyond the RGD domain is not necessary for cell binding. In comparison, peptides 283-288 and 281-290 were only active at concentrations greater than 200 microM. 50-70% of the peptide-stimulated adhesion was inhibited by the pretreatment of cell suspensions with solution phase RGD, suggesting that a portion of the peptides' adhesive effects was specific and integrin-mediated, although other non-RGD flanking regions were probably also involved in the mechanism of adhesion. Importantly, a modified BSP peptide, in which an aspartic acid residue at position 288 of the RGD sequence was replaced by a glutamic acid residue to form RGE, was completely inactive as a cell adhesion stimulus at concentrations up to 200 microM. Thus, despite the potential role of non-RGD flanking regions, an intact RGD tripeptide was essential for all of the adhesive activity of the BSP peptides.     

血清骨唾液酸蛋白的生物变异性研究

目的　骨唾液酸蛋白（Ｂｏｎｅ ｓｉａｌｏｐｒｏｔｅｉｎ,简称ＢＳＰ）是由成骨细胞分泌的一种非胶原蛋白质,反映骨转换和骨形成的指标。最近研究认为：血清ＢＳＰ浓度可反映破骨细胞活性和骨吸收过程,也可能是一个骨吸收指标。本实验测定了血清ＢＳＰ在人体内的生物变异性。方法　采用ＲＩＡ 法测定了２９０例不同性别和年龄的正常人血清ＢＳＰ的正常值,血清ＢＳＰ的天－天变异性及２４ ｈ 生物周期。　结果　在儿童组血清ＢＳＰ正常水平明显高于成人组,其最高值在新生儿期和青春期。绝经后妇女其血清浓度比绝经前妇女水平明显升高（Ｐ＜ ０．０５）。血清ＢＳＰ在每天同一时间的波动范围在 ７．３％ 至１７．７％ （平均１１．７％ ）。２４ ｈ 内有一个明显的生物周期性变化,表现为峰值在凌晨４～８ 时,然后逐渐下降直到午后１４时为最低。其生物周期的最大波幅为±２０％ ,（平均血浓度为１０．５ ｎｇ／ｍ ｌ）。结论　血清ＢＳＰ反映了骨转换的生理变化与年龄有明显相关性,其血清水平２４ ｈ 内有一个明显的生物周期,而天与天之间变异性较小。