多孔磷酸钙陶瓷在动态SBF中类骨磷灰石形成的研究

本实验在模拟体液（SBF）以静止、等于或大于骨骼肌组织内体液正常生理流率（2ml/100ml.min)的流动条件下，研究多孔磷酸钙陶瓷上类骨磷灰石的形成，结果表明，SBF在生理流率（2ml/100ml.min)情况下，材料仅在多孔磷酸钙陶瓷孔隙内部形成类骨磷灰石，静态（0ml/100ml.min)情况下，多孔磷酸钙陶瓷材料表面有类骨磷灰石形成；高于生理流率（10ml/100ml/min)时，表面和断面均无类骨磷灰石形成，但如果增加SBF中的Ca^2 ,HPO4^2-的浓度，则在孔隙内部有类骨磷灰石形成，生理流条件下的结果与多数肌肉内植入磷酸钙陶瓷试验的结果一致。仅在多孔材料内部成骨，这个结果表明，比起通常使用的静态浸泡试验，SBF以生理流率流动的动态体外试验能够更好地模拟类骨 磷灰石生长的体内环境，动态SBF对了解类骨磷灰石形成，进而了解磷酸钙陶瓷在体内诱导成骨机理是十分有用的。     

在动态模拟体液中致密CaP陶瓷表面形貌对类骨磷灰石层形成的影响研究

磷酸钙陶瓷植入体内后其表面类骨磷灰石层的形成是诱导成骨的先决条件。本实验在模拟体液 (Simu-lated body fluid,SBF)以人体骨骼肌组织的正常生理流率 (2 ml/ 10 0 m l· min)下 ,研究在动态 SBF中致密磷酸钙陶瓷表面形貌对类骨磷灰石层形成的影响。结果表明 :在生理流速条件下 ,材料的粗糙表面有利于类骨磷灰石层的形成 ,加大 SBF中 Ca2 +、HPO4 2 -离子浓度 ,类骨磷灰石层的形成速度加快。本研究进一步证实了材料的几何形貌对类骨磷灰石形成的影响 ,加深了对磷酸钙陶瓷在体内诱导成骨机理的理解     

多孔羟基磷灰石陶瓷诱导成骨的作用

多孔羟基磷灰石陶瓷种植体埋植于狗背部肌肉内,分别于植入后1、2、4和8月处死动物,取出种植体,将其固定,脱钙,切片、HE染色,在光镜下作组织形态学观察发现:一月的种植体孔隙内充满了血管丰富的结缔组织;二、四和八月的种植体孔隙内有较多量的新骨组织形成,种植体固有完整的纤维结缔组织包膜。本研究从组织形态学说明了多孔羟基磷灰石陶瓷具有诱导骨形成的能力。     

免股骨多孔磷灰石陶瓷植入材料的矿化和钙固定

虽然磷酸钙陶瓷材料表面组织相容性已被证实,但其性质至今还不清楚,特别是在生理学方面,合成材料与骨组织之间的降解动力学一直不了解。而作者在本文中采用了多孔磷灰石陶瓷材料作为动物股骨缺陷的植入,研究磷酸钙有关的物化行为和骨与植入物界面的矿化过程。其方法是羟基磷灰石和β-磷酸钙按10:1比例制成陶瓷混合园柱体(长0.8cm,直径1.0cm),植入20只兔的股骨,并且用髓内针固定,植入后每个月用X光照相检查植入部位情况,然后分别按植入后3个月(3只动物)、6个月(17只动物)处死。取出植入物及其周围组织,用甲基丙烯酸甲酯包埋,制作厚切片,用扫描电镜观察分析,而在植入前后用孔率计及X光衍射分析。通过能量散射X光微探分析确定骨与     

骨诱导性磷酸钙陶瓷为支架的组织工程骨修复狗下颌骨箱状缺损*☆◆

背景：应用不外加生长因子或细胞而具有骨诱导性的生物材料,在非骨部位构建骨移植物,即体内组织工程骨,其在修复箱状及节段性骨缺损方面,具有更可行的前景。 目的：采用骨诱导性钙磷陶瓷材料构建体内组织工程化类骨移植物,探索其应用于修复实验动物下颌骨箱状骨缺损的可行性。 方法：以骨诱导性磷酸钙陶瓷材料为支架植入狗肌肉内构建体内组织工程骨,同期在狗自体下颌骨左右两侧各拔除牙弓中段牙2颗,形成约20 mm无牙区。8周后在无牙区形成箱状缺损,同期取出支架即刻移植入一侧自体下颌骨缺损区,对侧骨缺损区直接移植入未经体内构建的磷酸钙陶瓷作为对照。 结果与结论：经肌肉内构建的体内组织工程骨移植物的力学性能较单纯磷酸钙陶瓷有明显提高。颌骨缺损区的核吸收强度明显强于对照区,其移植物内长入的骨组织较多,两者的成骨面积差异有非常显著性意义(P < 0.01)。说明在修复颌骨大范围缺损中,体内组织工程骨移植物较单纯骨磷酸钙陶瓷替代材料表现出明显的力学和生物学优势,修复效果显著,有良好的应用前景。关键词：骨诱导性磷酸钙陶瓷;体内组织工程骨;骨缺损;支架;组织工程 缩略语注释：BCP：biphasic calcium phosphate,双相磷酸钙 doi:10.3969/j.issn.1673-8225.2012.16.001     

锶磷灰石多孔陶瓷复合成骨细胞的体内异位成骨研究

目的比较不同孔隙率的锶磷灰石（含5%锶的羟磷灰石,Sr-HA）陶瓷复合成骨细胞后体内异位成骨能力。方法实验试样分复合成骨细胞的不同孔隙率组和未复合成骨细胞的不同孔隙率组。先抽取健康兔的骨髓组织,体外培养,诱导分化为成骨细胞,分别与孔径约为450～700μm、孔隙率分别为50%、60%、70%的Sr-HA及孔隙率为70%的纯羟磷灰石陶瓷体复合,自体回植到兔左侧背脊肌内;未复合成骨细胞的不同孔隙率组试样则种植在兔右侧背脊肌内作为阴性对照。植入后4、12、24周取材,行大体观察、组织学检查。结果复合成骨细胞的各组陶瓷在植入兔背脊肌内4周后均有类骨质形成,随着植入时间的延长,骨组织的数量不断增加;孔隙率为70%的Sr-HA陶瓷和HA陶瓷的成骨性能明显优于50%、60%孔隙率的Sr-HA陶瓷。未复合细胞的陶瓷材料在组织学检查中未见有类骨质形成。结论Sr-HA多孔陶瓷是较理想的骨组织工程支架材料,成骨细胞复合Sr-HA陶瓷用于骨缺损的修复,具有广阔的临床应用前景。     

钙磷多孔陶瓷表面明胶处理及体外细胞相容性

背景:烧结的钙磷多孔陶瓷由于其表面结构致密,在诱导细胞黏附和生长方面仍存在不足。目的:观察表面明胶处理对钙磷多孔陶瓷细胞相容性的影响。方法:以羟基磷灰石和磷酸钙为主要原料,采用有机泡沫浸渍工艺制备钙磷多孔陶瓷,然后将多孔陶瓷浸于5%的明胶溶液中进行表面处理。扫描电镜观察处理前后样品的孔隙形貌和表面结构,阿基米德法测试样品的孔隙率,WD-10A型电子万能材料试验机测定压缩强度;将材料与兔骨髓基质干细胞进行体外复合培养,通过MTT实验和扫描电镜观察细胞在材料上的生长情况。结果与结论:表面明胶处理后,多孔陶瓷的孔壁表面形成了较均匀的明胶涂层,样品的孔隙特征并没有受到显著影响。然而它们的平均压缩强度却从(1.04±0.15)MPa提高到了(5.17±0.17)MPa。扫描电镜观察和MTT实验结果表明,表面涂覆处理前后的多孔材料都具有良好的细胞相容性。与烧结的多孔陶瓷相比,表面明胶处理的样品更能增进细胞在材料表面的早期黏附和增殖。结果表明表面明胶处理在不破坏多孔陶瓷孔隙特征的情况下,不仅提高了钙磷多孔陶瓷的力学性能,而且改善了多孔陶瓷的细胞相容性。     

多孔碳酸钙陶瓷与干细胞源性成骨细胞的相容性

背景：国内外的研究证实普通碳酸钙陶瓷作为骨替代材料时具有细胞支架作用。 目的：观察多孔碳酸钙陶瓷与成骨细胞的相容性,及作为骨组织工程支架的可能性。 方法：SD大鼠骨髓基质干细胞经矿化诱导培养、扩增并检测证实其已具成骨细胞表型后,分别与多孔碳酸钙陶瓷支架、普通羟基磷灰石陶瓷支架体外复合培养。 结果与结论：骨髓基质干细胞经体外诱导形成成骨细胞,钙结节、Ⅰ型胶原和碱性磷酸酶免疫染色结果阳性。多孔碳酸钙陶瓷支架材料与羟基磷灰石陶瓷材料皆有细胞附着生长,但多孔碳酸钙陶瓷支架材料细胞的黏附能力、增殖活力及成骨活性均强于羟基磷灰石陶瓷材料。提示多孔碳酸钙陶瓷支架材料与SD大鼠骨髓基质干细胞源性成骨细胞有良好相容性。     

复合抗肿瘤珊瑚羟基磷灰石人工骨的体内成骨

背景：复合抗肿瘤珊瑚羟基磷灰石人工骨在体内外有良好的缓释效果及抗肿瘤作用,但由于其所复合药物量较大,植入体内骨缺损处较高的局部药物浓度是否影响骨的正常诱导、传导及生长？ 目的：建立复合抗肿瘤珊瑚羟基磷灰石人工骨成骨模型,进一步分析复合抗肿瘤珊瑚羟基磷灰石人工骨的体内成骨效应及规律。 方法：分别将珊瑚羟基磷灰石人工骨及复合抗肿瘤珊瑚羟基磷灰石人工骨植入兔股骨两干骺端骨缺损模型,定期观察股骨X射线影像,并取材行组织病理切片,观察材料降解和被新骨替代的速度、骨与材料界面的结合情况,材料内部新骨生长情况。 结果与结论：珊瑚羟基磷灰石人工骨植入后与周围骨形成组织及骨桥连接较复合抗肿瘤珊瑚羟基磷灰石人工骨快,植入4周后X射线片影像及组织切片示珊瑚羟基磷灰石人工骨边缘开始逐渐不清,并逐步与动物骨形成骨愈合。复合抗肿瘤珊瑚羟基磷灰石人工骨植入后早期8周内局部以抑制组织细胞生长为主,6~12周逐渐有组织结构向材料孔隙内生长且逐渐出现成骨细胞、骨基质及骨细胞,新生骨逐渐生长替代融合,26周左右与周围骨形成骨愈合。说明复合抗肿瘤珊瑚羟基磷灰石人工骨植入早期虽对骨愈合有一定的抑制作用,但最终仍可自行与周围骨缺损达到骨愈合。     

亚微米拓扑结构磷酸三钙陶瓷的研制及骨诱导性能

背景：人工合成的磷酸钙陶瓷材料与天然骨组织无机成分相似,通过表面形貌和化学组成进行功能化设计可赋予其优异的骨传导和骨诱导性能,研发具有骨诱导性能的磷酸钙陶瓷材料是目前的研究热点。目的：通过材料形貌调控和功能化设计赋予亚微米拓扑结构磷酸三钙陶瓷骨诱导性能,检测其理化性能及骨诱导性能。方法：采用高温烧结法制备亚微米拓扑结构的磷酸三钙陶瓷,以市场可供商品化的骨修复材料Bio-Oss骨粉为对照组,表征两种材料的表面形貌、蛋白吸附能力及体外矿化性能。将第3代人牙周膜干细胞与两种材料浸提液共培养,采用CCK-8法检测细胞增殖,茜素红染色检测细胞矿化性能;将第3代人牙周膜干细胞分别接种至两种材料表面,采用碱性磷酸酶染色检测早期成骨,qR T-PCR检测成骨相关因子的表达。结果与结论：(1)扫描电镜下可见两种材料均具有颗粒状纹理的微孔表面,Bio-Oss颗粒明显小于磷酸三钙陶瓷,两种材料的总孔隙度、大孔隙度和微孔隙度相似,磷酸三钙陶瓷主要为亚微米级孔隙,晶粒粒径100 nm-1.0μm,Bio-Oss骨粉主要为纳米级孔隙;体外矿化实验显示,磷酸三钙陶瓷表面诱导骨磷灰石沉积的能力强于Bio-Oss骨粉;...     

Ectopic osteogenesis with biphasic ceramics of hydroxyapatite and tricalcium phosphate in rabbits.

Porous calcium phosphate ceramics consisting of hydroxyapatite (HA) and tricalcium phosphate (TCP) with different HA to TCP ratio were implanted intramuscularly in rabbits for six months in order to carry out a comparative study on osteogenic activity of the ceramics. Bone formation was detected only in HT73 (HA to TCP ratio, 7-3) specimens. Other implants, HT28 (2-8) and HT010 (0-10), could not induce bone. After a six-month period of implantation, HT28 and HT010 implants showed obvious degradation of the implants changing their shape and size macro and microscopically. Microscopically, they showed aggregates of fine particles and appearance of multinucleated cells. However, HT73 implants was less degraded and could maintain their original structure macro and microscopically. This study showed that HT73 ceramics can induce bone in rabbit muscle tissue and it is considered that maintenance of porous structure, that is, degradation rate of the materials may be one of the affecting factors in ceramic-induced osteogenesis.     

Osteoinduction by biomaterials--physicochemical and structural influences

Osteoinduction by biomaterials has been shown to be a real phenomenon by many investigators in the last decade. The exact mechanism of this phenomenon is, however, still largely unknown. This in vivo study in goats was performed to get insight into processes governing the phenomenon of osteoinduction by biomaterials and had four main goals: (i) to further investigate the influence of physicochemical properties and structure on biomaterial osteoinductive potential, (ii) to investigate the influence of implant size on the amount of induced bone, (iii) to investigate implantation site dependence, and (iv) to investigate changes occurring on the surface of the material after implantation. Intramuscular implantations of four different biphasic calcium phosphate ceramics, consisting of hydroxyapatite and beta-tricalcium phosphate and a carbonated apatite ceramic, indicated that, for a maximal osteoinductive potential, there is an optimal specific surface area for each material type. It was further shown that a decrease of the implant size with a half significantly decreased the relative amount of induced bone. In addition, subcutaneous implantation did not give rise to ectopic bone formation in any of the animals, while bone was induced in most animals intramuscularly. Analysis of the surfaces of the materials after subcutaneous implantation inside diffusion chambers indicated that the increased specific surface area leads to more surface reactivity, which is hypothesized to be essential for osteoinductivity by biomaterials.     

Transformation of biphasic calcium phosphate ceramics in vivo: ultrastructural and physicochemical characterization

Specially prepared biphasic calcium phosphate (BCP) macroporous ceramics consisting of an intimate association of beta tricalcium phosphate (beta-TCP) and hydroxyapatite (HA) with beta-TCP/HA weight ratios of 15/85, 35/65, and 85/15 were implanted in surgically created periodontal osseous defects in dogs and recovered after 6 months. A decrease in average size of crystals in BCP ceramics and an increase in the size of microporosities in the surface and at the core of the ceramic after implantation were observed, indicating that in vivo dissolution has taken place. The resorbability (reflecting in vivo dissolution) of BCP ceramics depended on their beta-TCP/HA ratios, the higher the ratio, the greater the resorbability. The formation of microcrystals with crystallographic properties and Ca/P ratio similar to those of bone apatite crystals were also observed. The abundance of these crystals were directly related to the beta-TCP/HA ratio of the BCP ceramic before implantation. The formation of the bone apatite-like crystals may be due to the precipitation of calcium and phosphate ions released from the dissolving ceramic crystals (the beta-TCP component dissolving preferentially to the HA component). Results from this study suggested that one of the means of controlling resorbability (in vivo dissolution) of BCP ceramic is by varying its beta-TCP/HA ratio.     

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.     

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。     

3D microenvironment as essential element for osteoinduction by biomaterials

In order to unravel the mechanism of osteoinduction by biomaterials, in this study we investigated the influence of the specific surface area on osteoinductive properties of two types of calcium phosphate ceramics. Different surface areas of the ceramics were obtained by varying their sintering temperatures. Hydroxyapatite (HA) ceramic was sintered at 1150 and 1250 degrees C. Biphasic calcium phosphate (BCP) ceramic, consisting of HA and beta-tricalcium phosphate (beta-TCP), was sintered at 1100, 1150 and 1200 degrees C. Changes in sintering temperature did not influence the chemistry of the ceramics; HA remained pure after sintering at different temperatures and the weight ratio of HA and beta-TCP in the BCP was independent of the temperature as well. Similarly, macroporosity of the ceramics was unaffected by the changes of the sintering temperature. However, microporosity (pore diameter <10 microm) significantly decreased with increasing sintering temperature. In addition to the decrease of the microporosity, the crystal size increased with increasing sintering temperature. These two effects resulted in a significant decrease of the specific surface area of the ceramics with increasing sintering temperatures. Samples of HA1150, HA1250, BCP1100, BCP1150 and BCP1200 were implanted in the back muscles of Dutch milk goats and harvested at 6 and 12 weeks post implantation. After explantation, histomorphometrical analysis was performed on all implants. All implanted materials except HA1250 induced bone. However, large variations in the amounts of induced bone were observed between different materials and between individual animals. Histomorphometrical results showed that the presence of micropores within macropore walls is necessary to make a material osteoinductive. We postulate that introduction of microporosity within macropores, and consequent increase of the specific surface area, affects the interface dynamics of the ceramic in such a way that relevant cells are triggered to differentiate into the osteogenic lineage.     

Osteoinductivity and biomechanics of a porous ceramic with autogenic periosteum

Twenty-one dogs were used to study the osteoinductivity and biomechanical properties of a biphasic porous ceramic with autogenic periosteum implanted in muscle. The ceramic implants were swathed in fresh periosteum derived from the same animals and implanted in the femur muscles. The other two groups of animals served as controls using the same material implanted in the femur bones and muscles without periosteum. Biomechanical measurements showed that, in the muscles, the experimental group had a higher bending strength than the unswathed group by the time the samples were harvested. Six months postoperatively, the strength of the samples in the experimental group had almost reached that of normal bones. The results of X-ray diffraction and infrared spectrometric analysis suggested that the degradation rate and speed of tricalcium phosphate (TCP) of the ceramic in the experimental group were faster than in the unswathed samples, but slower than in samples implanted in bones. The bone replacement and bone-inducing activity were excellent in the periosteum-swathed samples. Histologically, satisfactory bone repair was seen in the experimental samples. All results indicate that autogenic periosteum could increase bioactivity of ceramics in heterosites and improve bone formation in the surroundings of porous calcium phosphate ceramics. The data also infer that the complicated procedure of culturing bone growth factors with biomaterials in vitro to obtain bioactive grafts could be replaced by this relatively simple method.     

Comparison of bone graft matrices for human mesenchymal stem cell-directed osteogenesis

Scaffolds to support cell-based tissue engineering are critical determinants of clinical efforts to regenerate and repair the body. Bone tissue engineering requires materials that are biocompatible, well vascularized, mechanically suited for bone function, integrated with the host skeleton, and support osteoinduction of the implanted cells that form new bone. The aim of this study was to compare the osteogenic potential of bone marrow-derived, culture expanded human mesenchymal stem cells (hMSCs) adherent to different scaffolds composed of various calcium phosphates. Cells were loaded onto 2 x 2 mm cubes of coral calcium carbonate-derived apatite, bovine bone-derived apatite, synthetic hydroxyapatite (HA)/tricalcium phosphate (TCP) (60:40%), or synthetic HA/TCP (20:80%) and placed into the dorsum of SCID mice for 5 weeks. Subsequent histomorphometric analysis of bone formation within the cubes revealed the absence of bone formation within the coral-derived apatite and the bovine bone-derived apatite. Bone formation within synthetic HA/TCP scaffolds was measured to be 8.8% (+/-2.7%) and 13.8% (+/-3.6%) of the total tissue present for the 60:40% and 20:80% materials, respectively. Minimal resorption was observed at this early time point. Scanning electron microscopy evaluation of loaded scaffolds indicates that cell loading was not a variable affecting the different bone formation outcomes in these four scaffolds. In this ectopic model, different apatite-containing scaffolds of similar morphology and porosity demonstrated marked differences in their ability to support osteoinduction by implanted hMSCs. The necessary induction of hMSCs along the osteoblastic lineage may be dependent, in part, on the local microenvironment established by the scaffold chemistry and interactions with the host.