骨诱导性磷酸钙陶瓷支架在脂肪组织中构建体内组织工程骨

背景：作为体内组织工程骨的构建场所,非骨组织的选择至关重要。早期研究大多选用肌肉作为异位骨移植物的构建区域,但其位置较深,可利用面积小、手术操作复杂,不利于临床推广。 目的：采用体内骨组织工程的方法,探索骨诱导性磷酸钙陶瓷支架在不同非骨组织中构建骨移植物的可行性。 方法：选取家犬的背部肌肉组织和脂肪组织为构建区,分别植入骨诱导性磷酸钙陶瓷支架以构建体内组织工程骨移植物。于移植后4,8,12,16周取样进行单光子计算机断层扫描及组织学检测,观察其构建过程,比较各个观测时间内,不同构建区的骨移植物中新骨组织的形成情况,评价不同非骨构建区域对体内骨组织骨移植物形成的影响。 结果与结论：骨诱导性磷酸钙陶瓷支架在肌肉组织和脂肪两处非骨组织中均可形成体内组织工程骨移植物,在构建初期,肌肉组中新骨形成的时间比脂肪组早,骨量也较多,但随着构建时间的延长,两组的新生骨量差异逐渐减小。提示,应用体内骨组织工程的方法在肌肉和脂肪组织均可构建出具有生命活性的自体骨移植物。与肌肉组织比较,脂肪组织面积宽广,位置浅表,因此在脂肪组织中构建体内组织工程骨移植物更有临床应用前景。     

成骨诱导脂肪基质细胞在骨组织工程体内成骨中的作用

背景：以往研究认为,经过成骨诱导后的脂肪基质细胞通过转化为成骨细胞分泌骨基质进而修复骨缺损,然而并没有明确结论证实。 目的：将经过体外成骨诱导的脂肪基质细胞复合支架材料分别植入骨缺损区和非骨区,根据是否成骨,验证经过成骨诱导后的脂肪基质细胞是否转化为成骨细胞。 方法：取12月龄犬背部皮下脂肪,经胶原酶消化法获得单个核细胞,将培养的第3代细胞与双相磷酸钙陶瓷形成复合物。在犬下颌骨两侧制备长20 mm、高10 mm的箱状缺损,拔除术区牙齿,将细胞支架复合物植入一侧术区,空白侧留作对照;另外在犬背部皮下肌肉区植入细胞支架复合物及骨诱导性磷酸钙陶瓷材料,术后6周及12周经组织学检测骨缺损修复情况。 结果与结论：脂肪基质细胞复合双相磷酸钙陶瓷在骨缺损区成骨,在肌肉区未形成新骨;骨诱导性磷酸钙陶瓷在肌肉区形成新骨。提示成骨诱导并不能将脂肪基质细胞转化为成骨细胞,其确切机制有待进一步研究。     

脂肪基质细胞构建组织工程骨修复下颌骨缺损的实验研究

背景：以往实验认为,只有经过成骨诱导后的脂肪基质细胞才能作为骨组织工程的种子细胞。然而成骨诱导周期过程复杂,延长了细胞体外培养时间和花费。 目的：探讨未经过成骨诱导的犬脂肪基质细胞作为种子细胞,利用组织工程技术修复犬下颌骨缺损的可行性。 方法：取12个月龄犬背部皮下脂肪,经胶原酶消化法获得单个核细胞,将培养的第3代细胞与双相磷酸钙陶瓷形成支架复合物。在犬下颌骨两侧制备长20 mm、高10 mm的箱状缺损,拔除缺损区牙齿,分别植入细胞支架复合物和单纯双相磷酸钙陶瓷支架,不进行干预的区域作为空白对照。植入后4周及8周经组织学检测骨缺损修复情况。 结果与结论：支架植入后4周,部分支架材料降解,缺损区形成新生骨,双相磷酸钙陶瓷组成骨量明显少于细胞支架复合物组,形成少量新骨及部分新生血管。8周时,两组形成更多的新骨,广泛分布于骨缺损区域,但双相磷酸钙陶瓷组仍明显少于细胞支架复合物组,差异有显著性意义(P < 0.01)。提示脂肪基质细胞复合双相磷酸钙陶瓷可在体内成骨,不经过体外成骨诱导的脂肪基质细胞作为种子细胞,利用组织工程技术可修复下颌骨缺损。     

体内组织工程骨的BMP4 mRNA表达

比较磷酸钙陶瓷构建的体内组织工程骨和自然再生骨的BMP4 mRNA表达.制备Φ5 mm×8 mm的骨诱导性磷酸钙陶瓷20粒并分别植入5只狗的竖直肌内,同时,拔除狗的一颗磨牙形成自然再生骨模型.术后1、2、4、12及24周,取出体内组织工程骨及自然再生骨.提取总 RNA, 经逆转录成 cDNA 后,用实时相对定量PCR的方法检测 BMP4 mRNA 的表达.结果表明:在本实验周期,BMP4 mRNA 在磷酸钙陶瓷构建的体内组织工程骨中的表达量高于在自然再生骨中的表达量,但BMP4 mRNA 在体内组织工程骨和自然再生骨中有相同的基因表达时序性.作为类似自体骨的骨替代物,磷酸钙陶瓷构建的体内组织工程骨具有切实可行的应用前景.     

脂肪间充质干细胞复合骨诱导性磷酸钙陶瓷支架的异位成骨CSCD

背景:生物材料的骨诱导现象已经在多种动物实验中被证实。目的:考察磷酸钙陶瓷自身固有的诱导骨生成能力在其作为骨组织工程支架时的表现。方法:取健康家犬10只,在每只的背部肌肉内分别植入骨诱导性磷酸钙陶瓷与自体脂肪间充质干细胞复合物、非骨诱导性磷酸钙陶瓷与自体脂肪间充质干细胞复合物、骨诱导性磷酸钙陶瓷及非骨诱导性磷酸钙陶瓷,植入后8,12周,取出植入材料及其周围组织进行Micro-CT检测和组织形态学检测,评价成骨情况。结果与结论:组织学观察结果显示,骨诱导性磷酸钙陶瓷组及骨诱导性磷酸钙陶瓷与自体脂肪间充质干细胞复合物组均有有异位骨生成,并且骨诱导性磷酸钙陶瓷与自体脂肪间充质干细胞复合物组的成骨量显著大于骨诱导性磷酸钙陶瓷组(P<0.05);其余两组均无异位成骨。Micro-CT检测结果与组织形态学检测结果一致。结果表明骨诱导性磷酸钙陶瓷作为骨组织工程支架材料有明显的成骨优势,而脂肪间充质干细胞作为种子细胞对异位成骨有明显的促进作用。     

预分化脂肪干细胞与纤维蛋白胶和磷酸钙骨水泥自体异位构建血管化移植物

背景：预构骨皮瓣研究启发人们构建预构血管化骨进行游离移植来替代带血管蒂游离自体骨移植修复大段骨缺损的想法。 目的：设计一种基于预分化脂肪干细胞、纤维蛋白胶和多孔磷酸钙骨水泥支架复合体的血管化移植物。 方法：将体外分离培养的大鼠脂肪干细胞在条件培养基中进行血管内皮细胞定向分化,经鉴定活性后,复合至纤维蛋白胶和多孔磷酸钙骨水泥构建血管化组织工程支架。将血管化组织工程支架、纤维蛋白胶/多孔磷酸钙骨水泥支架及多孔磷酸钙骨水泥支架分别植入SD大鼠股四头肌肌袋内,植入后2,4周进行组织学检测、血管定量分析和Western blot检测。 结果与结论：向血管内皮细胞分化的脂肪干细胞与纤维蛋白胶和多孔磷酸钙骨水泥共培养7 d,可见细胞密度适中,与支架组织结合较好。植入实验中,各组支架孔隙中充填有纤维血管组织和脂肪组织,血管化组织工程组支架孔隙中均长入大量血管,并有小动脉长入,不同时间点的血管直径和数量及血管内皮生长因子C的表达量均优于纤维蛋白胶/多孔磷酸钙骨水泥组和多孔磷酸钙骨水泥组(P < 0.01)。表明构建的血管化组织工程支架能够实现可靠迅速血管化。中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程全文链接：     

颌面部及牙周组织工程骨的血管化和成骨策略

背景：颌面部骨缺损以及牙周组织缺损是影响人类功能和美观的重要疾病之一,组织工程骨成为了修复颌面部骨和牙周组织缺损的主要手段。目前已形成了种子细胞及细胞共培养、支架材料、微环境三者复合以构建组织工程骨的基本模式,将组织工程骨进行预血管化并促进其快速成骨可以减少植入物的坏死、吸收,提升骨缺损修复的成功概率。 目的：归纳总结近5年内组织工程骨在口腔颌面部及牙周组织上的研究新进展。 方法：对CNKI数据库和PubMed数据库2010年至2015年文献进行检索,中文检索关键词为：颌面;组织工程骨;骨再生;血管化;基因修饰;种子细胞;支架材料;微环境,英文检索词为：oral and maxillofacial,bone tissue engineering, bone regeneration,vascularization,Genetic modification,Seed cells,Support material, Micro environment。排除无关性研究、重复性研究,保留68篇中英文文献进行归纳总结。 结果与结论：组织工程骨在修复口腔颌面部及牙周缺损上的应用得到了巨大发展,在三维结构的支架材料上加载基因修饰的种子细胞可以有效的促进血管化进程,提高成骨效应,增加颌骨缺损修复术的成功概率。此外,在牙槽嵴缺损处或新鲜拔牙窝处植入组织工程骨材料,可以有效的恢复和保存牙槽嵴高度和宽度,为后续的修复治疗保证了良好的骨条件。在植入组织工程骨后,可在缺损处加载不同的外部环境刺激,可以通过调节细胞外基质成分或信号通路来改变血管化进程,而血管化是建立有效血循环,保证植入支架成功的前提条件。 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

咬合刺激调控胶原骨粉修复骨缺损过程中骨改建的形态学分析

背景：咬合刺激对于颌骨的功能及改建至关重要,但人们对其调节骨移植修复骨缺损过程中骨改建的作用尚缺少清楚的认识。 目的：应用形态学方法分析咬合刺激对胶原骨粉修复骨缺损中骨改建的可能调控作用。 方法：在成年SD大鼠左侧下颌骨及顶骨区分别建立标准骨缺损模型,随即植入胶原骨粉充填骨缺损区。术后12周,通过X射线、苏木精-伊红染色、Gomori染色、抗酒石酸酸性磷酸酶染色及骨形成蛋白2免疫组织化学染色观察两骨缺损区的差异。 结果与结论：下颌骨及顶骨骨缺损区均有新骨形成,顶骨骨缺损区的板层骨形成量明显多于下颌骨骨缺损区,新骨形成矿化度明显高于下颌骨骨缺损区,说明顶骨骨缺损区的骨改建优于下颌骨骨缺损区;顶骨骨缺损区抗酒石酸酸性磷酸酶及骨形成蛋白2积分吸光度值均低于下颌骨骨缺损区,说明顶骨骨缺损区的破骨细胞及成骨细胞活性低于下颌骨骨缺损区。表明咬合刺激可能通过影响骨的矿化及成熟而导致下颌骨骨缺损修复过程中骨改建的延迟。 中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程     

磷酸钙生物材料固有骨诱导性的研究现状与展望

磷酸钙陶瓷因与骨的无机组成相似,具有良好生物相容性和骨引导性,无抗原性,被广泛应用于骨缺损修复,但一直认为磷酸钙陶瓷是一类仅具有骨引导性,而无骨诱导性的生物活性材料。大量研究表明通过材料自身组成和结构优化,可赋予磷酸钙陶瓷骨诱导性。本文对磷酸钙骨诱导现象发现和确证、骨诱导过程和机制、影响骨诱导性的材料学因素和骨诱导与动物种属关系、骨诱导相关的间充质细胞来源、以及骨诱导性材料的应用研究现状进行综述,并对磷酸钙生物材料骨诱导性及相关的研究方向予以展望。     

瓦楞状组织工程骨支架对种子细胞接种和成骨的影响

背景：不同形状工程骨支架负载种子细胞修复骨缺损的研究效果评价不一,而负载细胞数量的多少是影响疗效的重要因素之一,目前该方面研究证据不多。 目的：自制瓦楞状组织工程骨支架和其他3种形状的支架,比较4种不同形状支架负载种子细胞的数量,以及瓦楞状组织工程骨支架体内成骨时凹槽的优势及特点。 方法：①体外实验：将体积和样本数相同的4组支架分为单纯瓦楞状支架组、无瓦楞支架组、圆柱状支架组和带中空管柱状支架组,分别以相同密度、相同容积的成骨诱导兔骨髓间充质干细胞悬液接种于支架表面,孵育、培养、消化、收集,进行细胞计数、吸光度值检测以及碱性磷酸酶和茜素红染色。②体内实验：将兔随机分为重组人骨形态发生蛋白2/瓦楞状自固化磷酸钙人工骨组,瓦楞状自固化磷酸钙人工骨组和松质骨组,将体积相同的3组支架植入兔L5-6两侧横突间,植入后4,8,12周行大体、组织学观察。 结果与结论：体外实验显示,瓦楞状工程骨支架上滴注的细胞液能充分停留在表面,由于表面瓦楞状凹槽和液体的表面张力以及支架本身的无孔隙性使得细胞液不向培养皿流失,每个样本平均消化下来的正常形态的细胞数量高于其他3组(P < 0.05),吸光度值差异无显著性意义(P > 0.05)。体内实验显示,各时间点重组人骨形态发生蛋白2/瓦楞状自固化磷酸钙人工骨组的成骨量比瓦楞状自固化磷酸钙人工骨组明显多(P < 0.05),与松质骨组之间比较差异无显著性意义(P > 0.05)。结果证实,实验自制的瓦楞状工程骨支架的形状特点有利于种子细胞负载,从数量上保证了接种种子细胞的有效性,可促进支架大量成骨和段状骨缺损的愈合。中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程全文链接：     

A comparison of bone formation in biphasic calcium phosphate (BCP) and hydroxyapatite (HA) implanted in muscle and bone of dogs at different time periods

Physicochemical modification could implement synthetic materials into osteoinductive materials, which induce bone formation in nonosseous tissues. We hereby studied the relevance between the osteogenic capacities of osteoinductive materials in nonosseous tissues and in osseous sites. Biphasic calcium phosphate ceramic (BCP) and hydroxyapatite ceramic (HA) were implanted in femoral muscles and femoral cortical bone of dogs for 7, 14, 21, 30, 45, 60, 90, 180, and 360 days, respectively. Two dogs were used in each time point. In each dog, four cylinders (phi5x6 mm) per material were implanted in femoral muscles and 2 cylinders (phi5x6 mm) per material in femoral cortical bone. The harvested samples were processed for both histological and histomorphometric analyses. Bone was observed in BCP implanted in femoral muscles since day 30, while in HA since day 45. Quantitatively, more bone was formed in BCP than in HA at each time point after day 30 (p<0.05). The earlier and more bone formed in BCP than in HA suggests BCP a higher osteoinductive potential than HA in muscle. In femoral cortical bone defects, a bridge of bone in the defect with BCP was observed at day 21, while with HA at day 30. At days 14, 21, and 30, significantly more bone was formed in BCP than in HA (p<0.05). The results herein show that osteogenic capacities of osteoinductive materials in nonosseous tissues and osseous sites are correlated: the higher the osteoinductive potential of the material, the faster the bone repair.     

Material-dependent bone induction by calcium phosphate ceramics: a 2.5-year study in dog

Bone induction by different calcium phosphate biomaterials has been reported previously. With regard to (1) whether the induced bone would disappear with time due to the absence of mechanical stresses and (2) whether this heterotopically formed bone would give rise to uncontrolled growth, a long-time investigation of porous hydroxyapatite ceramic (HA), porous biphasic calcium phosphate ceramic (TCP/HA, BCP), porous alpha-tricalcium phosphate ceramic (alpha-TCP) and porous beta-tricalcium phosphate ceramic (beta-TCP) was performed in dorsal muscles of dog, for 2.5 years. Histological observation, backscattered scanning electron microscopy observation and histomorphometric analysis were made on thin un-decalcified sections of retrieved samples. Normal compact bone with bone marrow was found in all HA implants (n = 4) and in all BCP implants (n = 4), 48 +/- 4% pore area was filled with bone in HA implants and 41 +/- 2% in BCP implants. Bone-like tissue, which was a mineralised bone matrix with osteocytes but lacked osteoblasts and bone marrow, was found in all beta-TCP implants (n = 4) and in one of the four alpha-TCP implants. Both normal bone and bone-like tissues were confined inside the pores of the implants. The results show that calcium phosphate ceramics are osteoinductive in muscles of dogs. Although the quality and quantity varied among different ceramics, the induced bone in both HA and BCP ceramics did neither disappear nor grow uncontrollably during the period as long as 2.5 years.     

骨诱导性磷酸钙陶瓷材料修复牙槽突裂

BACKGROUND: In our previous studies, we had prepared calcium phosphate ceramics with better ectopic osteogenesis. OBJECTIVE: To explore the effect of novel osteoinductive calcium phosphate ceramics in the repairing of alveolar cleft. METHODS: Bilaterl alveolar defects were created in nine immature beagles. Three months later, osteoinductive calcium phosphate ceramics with high modular surface (experimental group) and smooth surface (control group) were randomly implanted in each side of the defect. Meanwhile, the corresponding material was implanted into the thigh muscle. New bone formation in the implanted region, osteogenesis in the implanted region and muscle, and respair results were respectively observed by fluorescence microscope, light microscope and CT at 4, 8 and 12 weeks after implantation. RESULTS AND CONCLUSION: (1) Fluorescence microscope observation: A circular permutation of red, yellow and green fluorescent strip could be observed in both two groups. (2) Light microscope observation: At 12 weeks after implantation, in the experimental group, the bone reconstruction was obvious, the implant material was decomposed gradually, the gap was filled with a large number of mature bone that combined with the rest material closely, and numerous Haversian canals appeared; the control group was similar but slightly inferior to the experimental group in the quality of new bone. The experimental group material successfully induced heterotopic osteogenesis in muscle, while the control did not. (3) CT examination: The two group materials restored the appearance and continuity of the alveolar ridge, and made no effect on the eruption of permanent teeth in both sides of the defect. To conclude, our findings suggest that the novel osteoinductive calcium phosphate ceramic exhibits advantages in alveolar cleft repair with earlier osteogenesis activation, faster osteogenesis rate and more bone formation than those traditional materials.     

The influence of genetic factors on the osteoinductive potential of calcium phosphate ceramics in mice

The efficacy of calcium phosphate (CaP) ceramics in healing large bone defects is, in general, not as high as that of autologous bone grafting. Recently, we reported that CaP ceramics with osteoinductive properties were as efficient in healing an ilium defect of a sheep as autologous bone graft was, which makes this subclass of CaP ceramics a powerful alternative for bone regeneration. Although osteoinduction by CaP ceramics has been shown in several large animal models it is sporadically reported in mice. Because the lack of a robust mouse model has delayed understanding of the mechanism, we screened mice from 11 different inbred mouse strains for their responsiveness to subcutaneous implantation of osteoinductive tricalcium phosphate (TCP). In only two strains (FVB and 129S2) the ceramic induced bone formation, and in particularly, in FVB mice, bone was found in all the tested mice. We also demonstrated that other CaP ceramics induced bone formation at the same magnitude as that observed in other animal models. Furthermore, VEGF did not significantly increase TCP induced bone formation. The mouse model here described can accelerate research of osteoinductive mechanisms triggered by CaP ceramics and potentially the development of therapies for bone regeneration.     

A preliminary study on osteoinduction of two kinds of calcium phosphate ceramics.

With respect to the effect of material factors on calcium phosphate biomaterial-induced osteogenesis, the osteoinductive property of two kinds of porous hydroxyapatite ceramics, which were made by different producers, was investigated in dorsal muscles of dogs. One hydroxyapatite ceramic (S-HA), macroporous implants with rough pore walls containing abundant micropores, was made by Sichuan Union University (Chengdu, China); the other hydroxyapatite ceramic (J-HA), porous implants with smooth macropore walls composed of regularly aligned crystal grains, was provided by Mitsubishi Ceramic Int. (Japan). Different tissue response was detected histologically and microradiographically after the ceramic samples had been implanted in dorsal muscles of dogs for 3 and 6 months. Bone formation was found in S-HA at 3 months, which increased at 6 months. In contrast, no bone formation was detected in J-HA at both 3 and 6 months. These results indicate that with the special architecture, calcium phosphate ceramic can induce bone formation in soft tissue. As both materials were very similar in their chemical and crystallographic structures, but varied in their microstructures, the latter seem to be an important factor affecting the osteoinductive capacity of calcium phosphate ceramics. These data suggest that, by controlling the preparation of calcium phosphate ceramic, bone substitutes with intrinsic osteoinductive property can be developed from calcium phosphates.     

晶须化磷酸钙多孔生物活性陶瓷修复犬股骨髁缺损

BACKGROUND: Previous studies have demonstrated that the internal microstructure of porous calcium phosphate ceramics after the whiskering process has some changes, and obtain good mechanical properties. OBJECTIVE: To further investigate the effect of whisker-covered porous calcium phosphate ceramics in repair of canine femoral condyle defects. METHODS: Highly interconnected porous calcium phosphate ceramics was prepared by placeholder method. The whiskering of the materials was finished by hydrothermal process. Fifteen healthy adult beagle dogs were selected in this study. A 10 mm×10 mm cylindrical inclusive bone defect was made bilaterally on the lateral femoral condyle with a drill. The porous calcium phosphate ceramics after the whiskering process was implanted onto the right femoral condyle as experiment group. The porous calcium phosphate ceramics without the whiskering process was implanted onto the left femoral condyle as control group. At 2, 4, 8, and 12 weeks after implantation, X-ray and dual-energy X-ray test were conducted in the bone defect area respectively. RESULTS AND CONCLUSION: (1) X-ray: With the increase of time, the interface between bone defect and normal bone of two groups gradually blurred, disappeared, and completely fused at 12 weeks. The material in the control group was partially dissolved, while there was no obvious dissolution in the experiment group. No significant difference in the X-ray scores was found between two groups at different time points. (2) Dual-energy X-ray: With the increase of time, the bone mineral density of the two groups both increased gradually, but there was no significant difference in the bone mineral density at different time points between these two groups. These results demonstrate that the porous calcium phosphate ceramics after the whiskering process has good ability to repair the defects of femoral condyle.       

Bone inductive properties of rhBMP-2 loaded porous calcium phosphate cement implants inserted at an ectopic site in rabbits

Recombinant human bone morphogenetic protein-2 (rhBMP-2) is known for its osteoinductive potential in bone tissue engineering. Calcium phosphate (Ca-P) cements are injectable, osteoconductive ceramic materials in which a macroporous structure can be induced during the setting reaction. In this study, the osteoinductive capability of rhBMP-2 loaded porous Ca-P cement was evaluated. Porous Ca-P cement discs were made and loaded with rhBMP-2 in vitro and implanted subcutaneously in the back of New Zealand white rabbits. The implantation period was either 2 or 10 weeks. Histological analysis of retrieved specimens revealed evident bone formation in the rhBMP-2 loaded Ca-P cement discs (pore fill: 18+/-6%) after 10 weeks of implantation. Bone formation occurred only in rhBMP-2 loaded porous Ca-P cement discs. Degradation of the Ca-P cement could not be confirmed after 10 weeks of implantation. The scaffold maintained its shape and stability during this time period. We conclude that porous Ca-P cement is a suitable carrier material for ectopic bone engineering.