应用3D适形打印制备网状复合体对兔颅骨缺损的修复作用及安全性研究

目的:探讨采用3D适形打印技术制备的羟基磷灰石/聚乳酸网状复合体在兔颅骨缺损中的修复作用及安全性。方法:以24只新西兰兔为研究对象,以羟基磷灰石/聚乳酸为材料,采用3D适形打印技术制备网状复合体,于兔颅骨顶部制成两个颅骨全层缺损,分别为孔A(左)和孔B(右),孔A(阳性对照组)以自体颅骨为修复材料,孔B(实验组)以复合体为修复材料,观察缺损修复区域的形态学、影像学(X线及CT扫描)及组织学检查结果。结果:植入后24周时,形态学显示:阳性对照组可见致密的骨组织修复,与缺损边缘界限不清,实验组中支架孔隙内纤维组织由新生骨质取代,且新生骨成熟度较提高,材料表面有部分吸收。CT扫描观察显示:冠状面上,阳性对照组缺损修复区域与周围正常骨组织融合为一体,实验组修复材料与缺损边缘融合紧密,与周围正常骨组织结合良好,部分边缘结合不连贯。组织学观察显示:实验组材料部分降解,材料间隔可见新生骨小梁。研究中无实验动物死亡,皮肤切口处缝合良好,无皮下积液,无移植物脱出、红肿感染等情况出现。结论:以3D适形打印技术制备的羟基磷灰石/聚乳酸复合体对兔颅骨缺损有较好的修复作用,能促进缺损区域新骨的形成和生长,且安全性较高。     

硫酸钙作为微小颗粒骨载体的实验研究

目的：研究硫酸钙作为微小颗粒骨载体,解决微小颗粒骨的自身缺点的实际效果,为其临床应用提供依据。方法：将49只日本大耳白兔随机分成4组并通过手术造成双侧桡骨中段1．5cm骨缺损,以植入硫酸钙为载体的自体微小颗粒骨为实验组,同时设立单纯植入自体微小颗粒骨,单纯植入硫酸钙和不植入任何物质的空白对照组。术后4周和8周分别行大体观察。X线摄片,组织学观察,骨生物力学测定。结果：以硫酸钙为栽体的自体微小颗粒骨组比单纯自体微小颗粒骨组及单纯硫酸钙组能更有效地修复骨缺损,单纯颗粒骨组成骨效果优于单纯硫酸钙组。空白组无骨愈合迹象;组织学观察示以硫酸钙为载体的自体微小颗粒骨实验组的成骨效果最好,单纯自体微小颗粒骨组次之;生物力学测定证明以硫酸钙为载体的自体微小颗粒骨实验组的力学强度优于单纯自体微小颗粒骨组及单纯硫酸钙组。结论：硫酸钙是微小颗粒骨的优良载体,以硫酸钙为载体的自体微小颗粒骨成骨速度快,成骨量多,质量高,骨的机械强度高,修复骨缺损的能力较单纯应用微小颗粒骨和硫酸钙强;二者结合可充分发辉各自的优势。     

两种壳聚糖仿生复合材料对家兔桡骨骨缺损修复的实验研究

目的:评价壳聚糖/碳酸钙三维复合材料(CS/CaCO3)和壳聚糖/羟基磷灰石复合材料(CS/HA)用于骨缺损修复的可行性.方法:家兔24只,随机分为对照、CS/CaCO3、CS/HA三组.左前肢去毛后,2%巴比妥钠(30mg/kg,iv)麻醉,距桡骨远端3cm处截骨1cm,形成骨缺损,分别植入相应材料.术后4w、8w、12w分别处死动物,X线摄片后,取骨缺损标本,进行大体与组织学观察.结果:术后4周植入块颜色变红,周围有较多量的新生骨样组织包裹,骨痂增多,向植入块内移行;术后8周,植入块周围有明显新骨生成,将材料分隔包围,新骨中央区可见材料呈蜂窝状残留.术后12周缺损区大部分编织骨被成熟的板层骨组织替代,并形成髓腔.结论:CS/CaCO3和CS/HA两种仿生复合材料能明显促进兔桡骨骨缺损修复,诱导骨痂生成.     

胶原蛋白/BMP复合材料的制备和成骨性能研究

以胶原膜(含87.5 mg I型胶原蛋白)为载体, 复合3.5 mg rhBMP-2(人基因重组骨形成蛋白-2), 制备胶原蛋白/BMP复合材料。复合材料首先在兔背阔肌中埋置, 预构新生骨组织, 并采用ALP染色、Von Kossa染色和HE染色等观察复合材料的成骨过程和组织形态。然后将形成的新骨组织游离移植修复自体下颌骨体部洞穿性缺损; 并设以胶原为载体的rhBMP-2复合骨修复材料直接修复为对照组, 骨缺损不修复组为空白组。采用X线、抗压强度、硬组织切片、四环素荧光染色、骨形态计量检查, 观察复合材料修复骨缺损的质量和效果。结果表明, 胶原蛋白/BMP复合材料在兔背阔肌中4～6周成骨, 胶原材料于3～5周降解; 成骨过程为是以软骨成骨为主的方式, 新骨形态为编织骨, 可见明显的微血管分布; 游离移植修复自体下颌骨缺损, 6周缺损区为骨性愈合, 与对照组在抗压强度(P = 0.041)、新骨量(P = 0.034)均有显著性差异。胶原蛋白/BMP复合材料在骨骼肌中形成的新生骨组织可作为供骨修复一定范围的骨缺损。     

胶原蛋白/BMP复合材料的制备和成骨性能研究

以胶原膜(含87.5 mg I型胶原蛋白)为载体, 复合3.5 mg rhBMP-2(人基因重组骨形成蛋白-2), 制备胶原蛋白/BMP复合材料。复合材料首先在兔背阔肌中埋置, 预构新生骨组织, 并采用ALP染色、Von Kossa染色和HE染色等观察复合材料的成骨过程和组织形态。然后将形成的新骨组织游离移植修复自体下颌骨体部洞穿性缺损; 并设以胶原为载体的rhBMP-2复合骨修复材料直接修复为对照组, 骨缺损不修复组为空白组。采用X线、抗压强度、硬组织切片、四环素荧光染色、骨形态计量检查, 观察复合材料修复骨缺损的质量和效果。结果表明, 胶原蛋白/BMP复合材料在兔背阔肌中4～6周成骨, 胶原材料于3～5周降解; 成骨过程为是以软骨成骨为主的方式, 新骨形态为编织骨, 可见明显的微血管分布; 游离移植修复自体下颌骨缺损, 6周缺损区为骨性愈合, 与对照组在抗压强度(P = 0.041)、新骨量(P = 0.034)均有显著性差异。胶原蛋白/BMP复合材料在骨骼肌中形成的新生骨组织可作为供骨修复一定范围的骨缺损。     

TGF-β3与TIMP-2联合转染兔骨髓间充质干细胞复合丝素蛋白/壳聚糖生物支架修复兔软骨缺损(英文)

目的:TGF-β3广泛存在于骨组织、软骨组织中,能诱导体外培养的间充质干细胞向软骨分化、生长。TIMP-2能抑制MMP对软骨基质的降解,保护新生软骨组织。本实验探讨单纯TGF-β3和TGF-β3,TIMP-2联合转染兔骨髓间充质干细胞复合丝素蛋白壳聚糖/(silk fibrin/chitosan,SF/CS)生物支架植入动物体内修复兔膝关节软骨缺损的可行性及效果差异。方法:将新西兰大白兔20只分为4组,每组5只(支架组、未转染组、reAAV-TGF-β3转染组、reAAV-TGF-β3,reAAV-TIMP-2联合转染组)。在无菌条件下取兔第三代对数生长期骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs),用携带目的基因的重组腺相关病毒进行转染,将转染成功的BMSCs与SF-CS生物支架复合,分别植入兔膝关节软骨缺损处:未转染组植入SF-CS生物支架,未转染组植入未转染的BMSCs复合SF-CS生物支架,reAAV-TGF-β3转染组植入reAAV-TGF-β3转染的BMSCs复合SF-CS生物支架,reAAV-TGF-β3,TIMP-2联合转染组植入reAAV-TGF-β3,TIMP-2联合转染的BMSCs复合SF-CS生物支架。两月后处死家兔,肉眼观察以及HE染色评定缺损软骨修复情况。并进行软骨细胞特征性染色即甲苯胺蓝染色及II型胶原免疫组化染色鉴定。结果:两个月后除支架组外各实验组兔膝关节软骨缺损处均有软骨样物质形成,且联合转染组诱导的新生成分更接近缺损处周围正常软骨。联合转染组与reAAV-TGF-β3转染组;联合转染组与未转染组;reAAV-TGF-β3转染组与未转染组的评分差异均具有统计学意义(P0.05)。HE染色结果提示联合转染组软骨修复效果较单纯TGF-β3转染组更好。结论:单纯TGF-β3转染兔骨髓间充质干细胞对兔膝关节软骨缺损有修复作用,TGF-β3与TIMP-2联合转染组修复缺损效果更明显,提示TIMP-2与TGF-β3具有协同效应。     

羟磷灰石—氧化铝复合陶瓷修复下颌骨缺损的实验研究

本文选择以氧化铝为基质,表面烧结羟磷灰石涂层的新型生物复合陶瓷与单纯氧化铝陶瓷为对照,修复犬下颌骨缺损。选用杂种犬8只,同体两侧下颌分别植入上述两种不同种植材料,术后4、8、12及24周处死动物取材对界面进行组织学及扫描电镜观察和元素探针分析。发现复合陶瓷材料植入后,具有(1)可引导界面新生骨组织生长,最终与涂层材料呈骨性结合。(2)伴随有钙、磷等活性离子的释放、迁移和钙—磷富集层的形成和变化。认为此过程在骨性结合中起着重要作用。结果表明:复合陶瓷克服了单纯羟磷灰石生物相容性虽好,但机械性能差,难以塑形的缺点,是理想的骨代用品。     

石墨化炭/炭复合材料＋羟基磷灰石涂层（C/C＋HA）复合骨植入材料研究

目的：通过建立兔股骨缺损的动物实验模型,对采用等温化学气相沉积法和等离子喷涂技术所制备的石墨化炭/炭复合材料＋羟基磷灰石涂层（C/C＋HA）复合骨植入材料进行骨植入实验的的生物相容性进行评价,探索该复合材料作为植入机体骨组织的可行性依据。方法：采用骨科钻在实验动物股骨髁上钻孔的方法建立骨缺损的动物实验模型,将待研究比较的实验材料分别植入实验动物的股骨髁内,持续观察8周,在术后第2、4、8周时应用X线照片、组织学染色和扫描电镜技术,分别观察所研究材料在机体内对骨缺损愈合及其对机体的影响,进行组间比较和相关性分析。结果：石墨化炭/炭复合材料＋羟基磷灰石涂层（C/C＋HA）复合骨植入材料的骨植入实验生物相容性良好,材料与骨组织结合牢固,界面中成骨细胞生长明显,且炭颗粒脱落现象少,未见炎症细胞浸润。植入动物体内的材料在植入期未引起机体局部的炎症浸润反应且表面脱落的碳颗粒在机体组织中也未引起局部严重的炎症反应。在实验动物植入材料后的连续8周观察期中,组织学观察显示：表面涂有HA的炭/炭复合材料对骨组织形态改建上表现良好,其与骨组织接界处所形成的纤维结缔组织膜层厚度明显比未涂HA的材料要小,与骨组织结合更为紧密和牢固;碳颗粒出现脱落游离的现象明显减少。结论：在炭/炭复合材料表面涂以HA生物涂层对骨的形态改建和促进骨小梁生长等方面具有良好的作用,是一种具有发展潜力的骨修复材料。     

转基因细胞片复合PLGA支架修复兔软骨缺损的研究

细胞片技术是应用组织工程方法使培养细胞从培养表面分离而形成含有细胞外基质的一层完整片状结构,弥补了传统组织工程技术的不足,是获取种子细胞以及对种子细胞进行转移的一项新技术。为探讨体外生长分化因子-5(GDF5)基因转染修饰的BMSCs细胞片与GDF5转基因BMSCs负载的PLGA支架形成的共聚物修复兔甲状软骨缺损的效果,实验通过腺病毒转染GDF5基因至四代兔BMSCs,温度敏感性培养皿制备GDF5转基因细胞片并与负载有转染GDF5基因BMSCs的PLGA支架复合,移植至同种兔甲状软骨缺损处,分别于术后4、8周行大体观察和组织学检测其修复效果。实验分3组:(A)转基因细胞片包裹负载有转基因BMSCs的PLGA支架组;(B)负载有转基因BMSCs的PLGA支架组;(C)负载BMSCs的PLGA支架组。结果显示,体外成功收获了完整的GDF5转基因细胞片,Real time PCR检测到GDF5 mRNA的表达,行大体组织的II型胶原免疫组化和阿利新蓝染色显示:A组和B组均表达II型胶原和糖胺聚糖(GAG),但A组表达高于B组,有统计学意义(P0.05)。由此可得,转基因细胞片包裹负载转基因BMSCs PLGA支架较传统转基因BMSCs负载PLGA支架方法具有更加优越的成软骨能力,能更有效地促进软骨缺损的修复。     

探讨生物活性接骨板研制的动物实验模型

目的探索生物活性接骨板修复骨折和骨缺损的动物实验模型,为研制不同强度的生物活性接骨板、观察骨折和骨缺损修复的机制提供实验对象。方法通过观察2.0cm兔股骨骨折和去骨膜缺损动物模型、2.0cm兔胫骨骨折和去骨膜缺损动物模型以及2.0cm兔桡骨骨折和去骨膜缺损动物模型,比较三种不同动物实验模型在兔节段性骨折和缺损实验修复中的大体标本及其术后4周的X线图片,了解三种动物实验模型在生物活性接骨板研制中的优缺点。结果2.0cm兔股骨骨折和去骨膜缺损组及2.0cm兔胫骨骨折和去骨膜缺损组术后均出现固定石膏脱落,生物活性接骨板断裂,骨折和缺损部位移位等现象;而2.0cm兔桡骨骨折和去骨膜缺损组术后4周X片发现生物活性接骨板的固定和复位良好,并且有骨痂形成。结论2.0cm兔桡骨骨折和去骨膜缺损动物模型,较好地模拟人体骨折和骨缺损的愈合过程,可能是研制生物活性接骨板的最佳动物实验模型之一。     

Placenta- versus bone-marrow-derived mesenchymal cells for the repair of segmental bone defects in a rabbit model

Tissue-engineered bones (TEBs) constructed with bone-marrow-derived mesenchymal stem cells (BMSCs) seeded on biomaterial scaffolds have achieved good results for bone defect repair in both animal experiments and clinical trials. This has been limited, however, by the source and quantity of BMSCs. We here explored TEBs constructed by placenta-derived mesenchymal stem cells (PMSCs) and compared their effect for the repair of critical-sized segmental osteoperiosteal defects with TEBs constructed with BMSCs. PMSCs were isolated from rabbit placenta by gradient centrifugation and in vitro monolayer culturing, and BMSCs were isolated from the hindlimb bone marrow of newborn rabbit. Primary cultured PMSCs and BMSCs were uniformly in a spindle shape. Immunocytochemistry indicated that both types of cells are positive for CD44 and CD105, and negative for CD34 and CD40L, confirming that they are mesenchymal stem cells. BrdU-labeled PMSCs and BMSCs were respectively co-cultured with bio-derived bone materials to construct TEBs in vitro. Critical-sized segmental osteoperiosteal defects of radii were created in 24 rabbits by surgery. The defects were repaired with TEBs constructed with PMSCs and BMSCs. The results showed that TEBs constructed by both PMSCs and BMSCs could repair the osteoperiosteal defects in a 'multipoint' manner. Measurement of radiography, histology, immunohistochemistry, alkaline phosphatase activity, osteocalcin assaying and biomechanical properties have found no significant difference between the two groups at 2, 4, 8 and 12 weeks after the transplantation (P > 0.05). Taken together, our results indicate that PMSCs have similar biological characteristics and osteogenic capacity to BMSCs and can be used as a new source of seeding cells for TEBs.     

Ectopic Osteoid and Bone Formation by Three Calcium-Phosphate Ceramics in Rats,Rabbits and Dogs

Calcium phosphate ceramics with specific physicochemical properties have been shown to induce de novo bone formation upon ectopic implantation in a number of animal models. In this study we explored the influence of physicochemical properties as well as the animal species on material-induced ectopic bone formation. Three bioceramics were used for the study: phase-pure hydroxyapatite (HA) sintered at 1200°C and two biphasic calcium phosphate (BCP) ceramics, consisting of 60 wt.% HA and 40 wt.% TCP (β-Tricalcium phosphate), sintered at either 1100°C or 1200°C. 108 samples of each ceramic were intramuscularly implanted in dogs, rabbits, and rats for 6, 12, and 24 weeks respectively. Histological and histomorphometrical analyses illustrated that ectopic bone and/or osteoid tissue formation was most pronounced in BCP sintered at 1100°C and most limited in HA, independent of the animal model. Concerning the effect of animal species, ectopic bone formation reproducibly occurred in dogs, while in rabbits and rats, new tissue formation was mainly limited to osteoid. The results of this study confirmed that the incidence and the extent of material-induced bone formation are related to both the physicochemical properties of calcium phosphate ceramics and the animal model.     

Utility of tricalcium phosphate and osteogenic matrix cell sheet constructs for bone defect reconstruction

AIM: To determine the effects of transplanting osteogenic matrix cell sheets and beta-tricalcium phosphate (TCP) constructs on bone formation in bone defects.METHODS: Osteogenic matrix cell sheets were prepared from bone marrow stromal cells (BMSCs), and a porous TCP ceramic was used as a scaffold. Three experimental groups were prepared, comprised of TCP scaffolds (1) seeded with BMSCs; (2) wrapped with osteogenic matrix cell sheets; or (3) both. Constructs were implanted into a femoral defect model in rats and bone growth was evaluated by radiography, histology, biochemistry, and mechanical testing after 8 wk.RESULTS: In bone defects, constructs implanted with cell sheets showed callus formation with segmental or continuous bone formation at 8 wk, in contrast to TCP seeded with BMSCs, which resulted in bone non-union. Wrapping TCP constructs with osteogenic matrix cell sheets increased their osteogenic potential and resulting bone formation, compared with conventional bone tissue engineering TCP scaffolds seeded with BMSCs. The compressive stiffness (mean ± SD) values were 225.0 ± 95.7, 30.0 ± 11.5, and 26.3 ± 10.6 MPa for BMSC/TCP/Sheet constructs with continuous bone formation, BMSC/TCP/Sheet constructs with segmental bone formation, and BMSC/TCP constructs, respectively. The compressive stiffness of BMSC/TCP/Sheet constructs with continuous bone formation was significantly higher than those with segmental bone formation and BMSC/TCP constructs.CONCLUSION: This technique is an improvement over current methods, such as TCP substitution, and is useful for hard tissue reconstruction and inducing earlier bone union in defects.     

Combination of bone tissue engineering and BMP-2 gene transfection promotes bone healing in osteoporotic rats

OBJECTIVE: The aim of this study was to develop a feasible approach to promote bone healing in osteoporotic rats using autogenous bone tissue-engineering and gene transfection of human bone morphogenetic protein 2 (hBMP-2). METHODS: Bone marrow stromal cells (BMSCs) from the left tibia of osteoporotic rats were transfected with the hBMP-2 gene in vitro which was confirmed by immunohistochemistry, in situ hybridization and Western blotting. Autogenous transfected or untransfected BMSCs were seeded on macroporous coral hydroxyapatite (CHA) scaffolds. Each cell-scaffold construct was implanted into a defect site which was created in the ramus of the mandible of osteoporotic rats. Four or eight weeks after implantation in situ hybridization was performed in BMSCs transfected with hBMP-2, X-ray examinations, histological and histomorphological analyses were used to evaluate the effect of tissue-engineered bone on osseous defect repair. RESULTS: Newly formed bone was observed at the margin of the defect 4 weeks after implantation with BMSCs transfected with BMP-2. Mature bone was observed 8 weeks after treatment. In the control group there was considerably less new bone and some adipose tissue was observed at the defect margins 8 weeks after implantation. CONCLUSIONS: Autogenous cells transfected with hBMP-2 promote bone formation in osteoporotic rats. BMSC-mediated BMP-2 gene therapy used in conjunction with bone tissue engineering may be used to successfully treat bone defects in osteoporotic rats. This method provides a powerful tool for bone regeneration and other tissue engineering.     

Effects of Vascular Endothelial Growth Factor 165 on Bone Tissue Engineering

To study the relationship between vascular endothelial growth factor (VEGF) and formation and repair of engineering bone, second-generation bone marrow stromal cells (BMSCs) of New Zealand white rabbits that were separated in vitro were transfected with VEGF 165 gene vectors by adenovirus to detect gene expressions. Transfected BMSCs and β-tricalcium phosphate material were complexed and implanted at the femoral injury sites of the study group (n = 12), and the control group (n = 12) were implanted with engineering bones that were not transfected with VEGF. Femoral recoveries of the two groups were observed on the 15th, 30th, 45th and 60th days, and their vascularization and ossification statuses were observed by immunohistochemical methods. The BMSCs transfected with VEGF highly expressed VEGF genes and excreted VEGF. The two groups both experienced increased vascularization and bone volume after implantation (t = 7.92, P<0.05), and the increases of the study group were significantly higher than those of the control group (t = 6.92, P<0.05). VEGF is clinically applicable because it can accelerate the formation and repair of engineering bone by promoting vascularization and ossification.     

Posterolateral spinal fusion with ostegenesis induced BMSC seeded TCP/HA in a sheep model

Autogenous bone graft is the gold standard for fusion procedure. However, pain at donor site and inconsistent outcome have left a surgeon to venture into some other technique for spinal fusion. The objective of this study was to determine whether osteogenesis induced bone marrow stem cells with the combination of ceramics granules (HA or TCP/HA), and fibrin could serve as an alternative to generate spinal fusion. The sheep's bone marrow mesenchymal stem cells (BMSCs) were aspirated form iliac crest and cultured for several passages until confluence. BMSCs were trypsinized and seeded on hydroxyapatite scaffold (HA) and tricalcium phosphate/hydroxyapatite (TCP/HA) for further osteogenic differentiation in the osteogenic medium one week before implantation. Six adult sheep underwent three-level, bilateral, posterolateral intertransverse process fusions at L1–L6. Three fusion sites in each animal were assigned to three treatments: (a) HA constructs group/L1–L2, (b) TCP/HA constructs group/L2–L3, and (c) autogenous bone graft group/L5–L6. The spinal fusion segments were evaluated using radiography, manual palpation, histological analysis and scanning electron microscopy (SEM) 12 weeks post implantation. The TCP/HA constructs achieved superior lumbar intertransverse fusion compared to HA construct but autogenous bone graft still produced the best fusion among all.     

骨诱导无机材料BAM 修复牙种植体周围骨缺损的实验研究

目的:评价骨诱导磷酸钙生物陶瓷(BAMOICPC)与可吸收胶原膜(BME-10X医用胶原膜)在牙种植体周围骨缺损中的修复能力。方法:在兔股骨上植入羟基磷灰石涂层BLB种植体,然后在其侧壁制造高4 mm、宽3 mm、深2 mm的骨缺损。对照组为单纯侧壁骨缺损,实验A组骨缺损区仅覆盖BME-10X膜,B组骨缺损区植入BAMOICPC,C组骨缺损区植入BAMOICPC并加盖BME-10X膜。于术后6个月取带种植体的骨段,通过HE染色和扫描电镜(SEM)分析。结果:对照组骨缺损区种植体表面见纤维包裹,实验A组骨缺损边界区少许骨质移行覆盖,实验B组下半部分缺损区新生骨覆盖。C组新生骨完全覆盖骨缺损区,且较B组硬度高,扫描电镜见与种植体结合更紧密。组织学观察B、C两实验组新生骨均可见比较成熟的哈弗氏管系统。结论:骨诱导磷酸钙生物陶瓷BAMOICPC是一种较理想的骨替代材料,联合运用胶原膜修复种植体周骨缺损效果佳。     

Enhanced bone regeneration with a synthetic cell-binding peptide--in vivo results

This in vivo study compared the regenerative processes within defined defects of the porcine skull after delivery of a porous algae-derived hydroxyapatite (adHA), a similar, experimental adHA carrying the cell binding peptide P-15, used solely and in combination with 25% autogenous bone (AB). Particulated AB served as a control group. During an observation period of 26 weeks, microradiography and histology were performed at four specific times. Significantly higher mineralization rates (p=0.008) were found 4 weeks after application of the bioactive material in combination with AB. At 12 weeks there was a significantly higher mineralization (p=0.036) following the application of the bioactive form alone. This study showed significantly higher mineralization after use of a P-15 bioactivated material at early stages. Thus, it can be concluded that the application of the P-15 sequence to an hydroxyapatite accelerates the process of early bone formation, whereas no long-term effect was traced.