脱脂程序对异体松质骨成骨能力影响的实验研究

[目的]研究脱脂程序对同种异体松质骨移植后成骨能力的影响.[方法]取Lewis大鼠长骨干骺端作为移植骨块,随机分4组分别采用4种不同程序进行处理,检测处理后各组骨块内总脂肪和总蛋白含量.取Fisher 344大鼠作为骨移植受体,根据移植骨块不同随机分为4组,行股骨下干骺端骨缺损填充术.于取材前14d和4 d对受体大鼠行双荧光标记;术后8周时取出标本,不脱钙切片后行骨组织形态计量学分析.对各组的骨矿化速率(MAR)同相应骨块总脂肪含量行相关分析.[结果]超声清洗结合化学方法能较彻底去除骨块内脂肪成份.术后8周时取出标本不脱钙骨切片荧光显微镜下观察,均可见双色平行荧光带.影像分析系统测定骨矿化速率示D组最大,同其他各组比差异均有统计学意义(P＜0.05).骨矿化速率同总脂肪含量呈负相关.[结论]脱脂程序可提高异体松质骨移植后的成骨能力.超声清洗结合化学方法可较彻底清除骨内的脂肪成分和有效提高其移植后的成骨能力,可作为一种异体骨的有效处理方法.     

延时脱钙和骨基质几何形态对其成骨能力的影响

目的：研究延时脱钙和骨的几何形状对骨基质成骨能力的影响。方法：切取鼠股骨干分成5组。骨干24h组，骨干48h组（对照组），骨干48B组和骨干72h组，分别用0.6NHC1脱钙24，48或72h，或颗粒48B组的骨碾磨成125-800μm的颗粒，50只Wistar鼠作为受体，术后6周取标本测定重量，干燥后的重量，钙含量，碱性磷酸酶含量和组织学观察。结果：颗粒48H组的干燥重量高于控制组，二者有统计学差别。结论：骨延时脱钙不影响其成骨能力。125-800μm的颗粒状鼠骨基质成骨能力优于块状骨基质。     

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。     

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。     

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。     

局部注射脱钙骨基质,骨基质胶治疗骨不连及骨迟延愈合临床观察

局部注射ＤＢＭ、ＢＭＧ治疗骨不连、骨迟延愈合是一种改变临床传统手术的新疗法，此种方法可在门诊施行无须住院。作者报告１９８７年６月～１９９３年３月随访６个月以上的５４例。结果：骨不连组临床愈合率８８％，骨迟延愈合组９１．３％。ＤＢＭ组诱导成骨成功率９６％，ＢＭＧ组９５．６５％，临床满意。结果证实：局部注射ＤＢＭ、ＢＭＧ和手术植入一样符合诱导成骨的理论和原则。作者认为是一种简易方便、行之有效的可靠治疗方法。     

骨蛋白强化脱钙骨基质板块修复犬长骨节段性骨缺损

目的 :研究骨蛋白 (boneprotein ,BP)强化脱钙骨基质 (demineralizedbonematrix ,DBM) (BP/DBM )板块在修复节段性骨缺损中的作用。方法 :在犬双侧桡骨中段各做一 1 5cm的骨膜骨缺损 ,分别植入板块状BP/DBM ,DBM ,自体髂骨块及留置空白 ,观察时间为 4个月。结果 :BP/DBM植入组有 3例完全骨愈合 (3 / 5 ) ,自体骨移植组只有 3例部分骨愈合 ,单纯DBM组及空白组未见骨愈合。生物力学测试 :术后 4个月BP/DBM组新生骨极限压缩强度值最高 ,已达到正常桡骨组织的 48%。BP/DBM组新生骨为成熟的板状骨。结论 :BP/DBM板块可促进节段性骨缺损的修复。     

脱钙异体骨关节移植诱导成骨和成软骨作用的研究

评价脱钙异体骨关移植诱导成骨和成软骨的作用。方法：将２４只成年雄性新西兰兔随机分成两组。．除Ｉ组左侧桡骨作为正常对照外，余均截去桡骨近段２０ｍｍ，包括骨膜和关节软骨，分别移植脱钙异体骨关节和不移植。术后２－２４周做Ｘ线检查，组织学和检查和生物力学试验。     

成骨细胞特异性钙黏蛋白涂布脱钙骨基质材料对BMSCs黏附及成骨分化能力的影响

目的 探讨成骨细胞特异性钙黏蛋白(cadhefin ectodomain Ⅱ,Cad-Ⅱ)涂布于同种异体脱钙骨基质材料(freeze-dried demineralized bone matrix,FDBM)对兔BMSCs黏附、增殖及成骨分化能力的影响.方法 取4周龄日本大耳白兔10只,体重0.61～0.88 kg,雌雄不限,常规分离培养BMSCs.取第2代BMSCs(细胞密度1×106个/mL)分别与经Cad-Ⅱ修饰的FDBM(实验组)和未经Cad-Ⅱ修饰的FDBM(对照组)复合,行MTT检测细胞增殖能力,细胞黏附实验检测细胞上架率和上架数,倒置相差显微镜、扫描电镜及HE染色观察细胞生长情况.另取第2代BMSCs(细胞密度5×105个/mL)分别与经Cad-Ⅱ修饰的FDBM(实验组)和未经Cad-Ⅱ修饰的FDBM(对照组)复合培养,行ALP活性检测和骨钙素免疫组织化学染色观察细胞成骨分化情况.结果 MTT检测发现BMSCs在经Cad-Ⅱ修饰的支架材料上能正常增殖,但与对照组比较差异无统计学意义(P>0.05).实验组细胞上架率为87.41%±5.19%,明显高于对照组35.56%±0.75%(P<0.01);对照组每片材料细胞上架数平均为2.6×104个,实验组平均高达5.0×105个,明显多于对照组(P<0.05).倒置相差显微镜、扫描电镜及HE染色观察均显示实验组支架上黏附细胞数量明显多于对照组.成骨诱导培养7 d,实验组和对照组细胞均可表达骨钙素,具有成骨细胞表型;培养14 d,实验组和对照组ALP活性分别为29.33±1.53和18.31±1.32,骨钙素免疫组织化学染色阳性率分别为83%±7%和56%±7%,两组比较差异均有统计学意义(P<0.01);与同组7、21 d比较差异有统计学意义(P<0.01),7 d与21 d组间及组内比较差异均无统计学意义(P>0.05).结论 Cad-Ⅱ修饰的FDBM对BMSCs增殖无明显促进作用,但能提高细胞黏附性,并促进BMSCs向成骨细胞分化.     

脱钙人牙基质骨诱导的研究

我们通过选用本院口腔科拔除的各类人牙为材料,制取脱钙人牙基质,在纯种日本长耳大白兔股部肌肉群内植入后.通过X线,组织切片,电子显微镜等方式观察其异位诱导成骨作用.     

四种载体吸附自体骨髓细胞移植成骨作用的实验研究

目的　比较骨基质海绵 ,羟基磷灰石 ,明胶海绵及海螵蛸结合自体骨髓细胞在兔肌肉中的成骨作用。方法　应用骨基质海绵 ,羟基磷灰石 ,明胶海绵及海螵蛸结合自体骨髓细胞经过孵育后 ,植入兔股部肌肉内 ,分别于不同时间通过X线及组织学观察 ,计算机图像分析 ,评价各不同组织的成骨情况。结果　 12周时 ,骨基质海绵组成骨能力最强 ,羟基磷灰石组次之 ,海螵蛸组成骨能力最弱 ,明胶海绵组无成骨能力。结论　骨基质海绵吸附自体骨髓是修复骨折及骨缺损的较好材料。     

几种制备因素对同种骨移植物的生物力学特性的影响

本文研究了超声清洗,冷冻干燥和Co~(60)r射线辐照灭菌几项制备因素对同种骨移植物的生物力学特性的影响。结果表明,实验组和对照组之间无显著性差异,即这些制备因素对同种骨移植物的生物力学特性无影响。     

Mechanical Stimulation of Bone Formation is Normal in the SAMP6 Mouse

With aging, the skeleton may have diminished responsiveness to mechanical stimulation. The senescence-accelerated mouse SAMP6 has many features of senile osteoporosis and is thus a useful model to examine how the osteoporotic skeleton responds to mechanical loading. We performed in vivo tibial bending on 4-month-old SAMP6 (osteoporotic) and SAMR1 (control) mice. Loading was applied daily (60 cycles/day, 5 days/week) for 2 weeks at peak force levels that produced estimated endocortical strains of 1,000 and 2,000 με. In a separate group of mice, sham bending was applied. Comparisons were made between right (loaded) and left (nonloaded) tibiae. Tibial bone marrow cells were cultured under osteogenic conditions and stained for alkaline phosphatase (ALP) and alizarin red (ALIZ) at 14 and 28 days, respectively. Tibiae were then embedded in plastic and sectioned, and endocortical bone formation was assessed based on calcein labels. Tibial bending did not alter the osteogenic potential of the marrow as there were no significant differences in ALP or ALIZ staining between loaded and nonloaded bones. Tibial bending activated the formation of endocortical bone in both SAMP6 and SAMR1 mice, whereas sham bending did not elicit an endocortical response. Both groups of mice exhibited bending strain-dependent increases in bone formation rate. We found little evidence of diminished responsiveness to loading in the SAMP6 skeleton. In conclusion, the ability of the SAMP6 mouse to respond normally to an anabolic mechanical stimulus distinguishes it from chronologically aged animals. This finding highlights a limitation of the SAMP6 mouse as a model of senile osteoporosis. Presented in part at the 2004 meeting of the American Society of Bone and Mineral Research and the 2005 meeting of the Orthopaedic Research Society.     

Influence of Factors Regulating Bone Formation and Remodeling on Bone Quality in Osteonecrosis of the Femoral Head

Osteonecrosis of the femoral head (ONFH) usually affects young individuals and has a major impact on lifestyle. Notably, the pathogenetic mechanisms of osteonecrosis are unresolved and no effective treatment exists. The objective of this study was to assess the gene expression levels of factors regulating bone formation and remodeling (bone morphogenetic protein [BMP]-2, BMP-7, Runx2, osteocalcin, osteoprotegerin [OPG]) in patients with ONFH and to compare them to those of patients with primary osteoarthritis (OA). The cellular and macromolecular composition of the bone matrix was assessed by osteocalcin immunohistochemistry, and the three-dimensional organization of trabecular bone was characterized by micro-computed tomographic analysis. Our results demonstrate that gene expression of BMP-2, BMP-7, and Runx2 is elevated in patients with ONFH. We observed increased extracellular osteocalcin deposition, presumably caused by a higher number of osteoblasts in concordance with increased activity of Runx2. Constant gene expression level of OPG implies an unchanged osteoclast differentiation rate in ONFH bone. We found no significant change in bone volume, connectivity, and structural model index; further, no significant differences were detected for trabecular properties in ONFH bone. In conclusion, we have shown increased gene expression of factors regulating bone formation and remodeling in the femoral head and/or neck of patients with ONFH. Further, we observed an increase in osteocalcin immunoreactivity and osteoblast/osteocyte cell number, while no significant changes in trabecular microarchitecture were detected. This study increases our understanding of the pathophysiology and repair process following ONFH and might help in the development of new treatment strategies in the future.     

Stimulation of Bone Formation by Recombinant Fibroblast Growth Factor-2 in Callotasis Bone Lengthening of Rabbits

Bone lengthening by callotasis is one of the most useful methods not only for the treatment of short extremities but also for extensive bone defects; however, the procedure takes a long time especially for the consolidation of the distracted callus. In this study, effects of a single local injection of recombinant human fibroblast growth factor-2 (FGF-2 or basic FGF) on callotasis bone lengthening were examined in rabbits. Ten days after the osteotomy at the middle of the tibia and the installment of an external fixator, the osteotomized site was distracted at a rate of 1.4 mm/day for 7 days, resulting in 9.8 mm lengthening. On the final day of distraction, 200 μg of FGF-2 in 150 μl of saline solution or vehicle alone was injected into the center of the distracted callus. Injection of FGF-2 increased bone formation at the distracted callus radiologically and histologically. A significant effect on bone mineral content (BMC) at the callus was observed as early as 2 weeks, and FGF-2 increased the BMC about twofold at 5 weeks after a normal remodeling process. We conclude that the callotasis method in combination with FGF-2 injection at the consolidation step could be clinically beneficial to shorten the bone lengthening period. Received: 6 May 1997 / Accepted: 1 November 1998     

Bone Marrow Mesenchymal Stem Cells Form Ectopic Woven Bone In Vivo Through Endochondral Bone Formation

Abstract:  Autologous vascularized bone grafts, allografts, and biocompatible artificial bone substitutes each have their shortcomings. Bones regenerated using recombinant human bone morphogenetic proteins, demineralized bone powder, or combinations of these are generally small and do not meet the need. The current trend is to use tissue engineering approaches with bone marrow mesenchymal stem cells (MSCs) to generate bones of a desired size and shape. A suspension of osteogenically induced MSCs (CD11a−, CD29+, CD44+) was added to 2% alginate, gelled by mixing this combination with calcium sulfate (CaSO₄ 0.2 g/mL), and injected into the subcutaneous pocket in the dorsal aspect of nude mice. Cells of various concentrations (0, 10, 50, and 70 million/mL) were used. These implanted constructs were harvested at predetermined times up to 30 weeks for histology. The doubling time of bovine MSCs is 3.75 ± 1.96 days and the proliferation is rapid. Histological evaluation revealed signs of endochondrosis with woven bone deposition. The equilibrium modulus increased with time in vivo, though less than that of normal tissue. Implants seeded with 70 million cells/mL for 6 months resulted in the best formation of equilibrium modulus. This approach has several advantages: (i) obtaining MSCs is associated with low donor morbidity; (ii) MSCs proliferate rapidly in vitro, and a large number of viable cells can be obtained; and (iii) the MSC/alginate constructs can develop into bone-like nodules with high cell viability. Such a system may be useful in large-scale production of bony implants or in the repair of bony defects. The fact that endochondral bone formation led to woven bone suggests its potential feasibility in regional cell therapy.     

Photocrosslinkable Chitosan Hydrogel Can Prevent Bone Formation in Both Rat Skull and Fibula Bone Defects

UV light irradiation to a photocrosslinkable chitosan (Az-CH-LA) resulted in an insoluble and flexible hydrogel within 30 s. The purpose of this study was to evaluate the ability of the photocrosslinkable chitosan to inhibit bone formation in the bone defects. A 5-mm-diameter defect was made in the rat calvarium, and then photocrosslinkable chitosan was implanted and irradiated with UV for 30 s. Furthermore, a 2-mm defect was made in the fibula of a rat hind leg, and then photocrosslinkable chitosan was implanted and irradiated with UV. Bone formations in the rat skull and fibula defects with photocrosslinkable chitosan hydrogel were significantly prevented for 8 weeks. Thus, the chitosan hydrogel has an inhibitory effect on bone formation.     

Aminobisphosphonates Cause Osteoblast Apoptosis and Inhibit Bone Nodule Formation In Vitro

Bisphosphonates are widely used for the treatment of bone diseases associated with increased osteoclastic bone resorption. Bisphosphonates are known to inhibit biochemical markers of bone formation in vivo, but it is unclear to what extent this is a consequence of osteoclast inhibition or a direct inhibitory effect on cells of the osteoblast lineage. In order to investigate this issue, we studied the effects of various bisphosphonates on osteoblast growth and differentiation in vitro. The aminobisphosphonates pamidronate and alendronate inhibited osteoblast growth, caused osteoblast apoptosis, and inhibited protein prenylation in osteoblasts in a dose-dependent manner over the concentration range 20-100 microM. Further studies showed that alendronate in a dose of 0.1 mg/kg inhibited protein prenylation in calvarial osteoblasts in vivo, indicating that alendronate can be taken up by osteoblasts in sufficient amounts to inhibit protein prenylation at clinically relevant doses. Pamidronate and alendronate inhibited bone nodule formation at concentrations 10-fold lower than those required to inhibit osteoblast growth. These effects were not observed with non-nitrogen-containing bisphosphonates or with other inhibitors of protein prenylation and were only partially reversed by cotreatment with a fourfold molar excess of ss-glycerol phosphate. We conclude that aminobisphosphonates cause osteoblast apoptosis in vitro at micromolar concentrations and inhibit osteoblast differentiation at nanomolar concentrations by mechanisms that are independent of effects on protein prenylation and may be due in part to inhibition of mineralization. While these results need to be interpreted with caution because of uncertainty about the concentrations of bisphosphonates that osteoblasts are exposed to in vivo, our studies clearly demonstrate that bisphosphonates exert strong inhibitory effects on cells of the osteoblast lineage at similar concentrations to those that cause osteoclast inhibition. This raises the possibility that inhibition of bone formation by bisphosphonates may be due in part to a direct inhibitory effect on cells of the osteoblast lineage.