Introduction of Ectopic Bone Formation by BMP-2 Incorporated Biomimetically into Calcium Phosphate Coatings of Titanium-Alloy Implants
Introduction of Ectopic Bone Formation by BMP-2 Incorporated Biomimetically into Calcium Phosphate Coatings of Titanium-Alloy Implants
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钛合金植入物磷酸钙涂层中仿生 BMP-2 引入异位骨形成
DOI：
10.4028/www.scientific.net/kem.240-242.667
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发表时间：
2002
期刊：
影响因子：
--
通讯作者：
P. Layrolle
中科院分区：
文献类型：
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作者：
Y. Liu;E. Hunziker;K. de Groot;P. Layrolle
我们最近发现蛋白质可以与磷酸钙晶体在钛合金植入物表面生物共沉淀。重组人骨形态发生蛋白2（rhBMP-2）与无机基质复合后，仍保持其体外骨诱导活性。此外，在含BMP-2的磷酸钙涂层上生长的大鼠骨髓基质细胞的成骨性（碱性磷酸酶活性）高于在塑料基质上培养并暴露于自由悬浮药物的细胞。在本研究中，将含有BMP-2的磷酸钙涂层的钛合金圆盘异位（皮下）植入大鼠体内，并在随后的1-5周内监测骨形成过程。骨组织在植入后2周出现，此后质量稳定增加。因此，将BMP-2掺入钛合金植入物的磷酸钙涂层中保持其作为体内骨诱导剂的生物活性。该系统的原理可用于矫形外科和牙种植外科。骨形态发生蛋白（BMPs）不仅能够在形成骨系统的一部分的组织内诱导骨形成，例如在人类骨不愈合的部位[1]，而且能够异位诱导骨形成，例如在实验动物的背克莱斯内。骨形成蛋白也被证明在体外刺激骨祖细胞的碱性磷酸酶活性。然而，在体内，骨形成的诱导不能简单地通过局部注射其游离形式的BMP来引起。需要合适的载体，例如磷酸钙。最近已经可以使用仿生方法将蛋白质掺入沉积在钛合金植入物上的磷酸钙层中[2]。这样的涂层可以提供用于缓慢但以持续且足够高的速率递送BMP的载体，以支持和维持它们在体内局部的成骨活性。在本研究中，钛合金种植体表面涂有含BMP-2的磷酸钙层，并将其植入大鼠皮下，在5周的过程中对骨形成过程进行组织学监测。关键工程材料在线：2003-05-15 ISSN：1662-9795，Vols. 240-242，pp 667-670 doi：10.4028/www.scientific.net/KEM.240-242.667 © 2003 Trans Tech Publications Ltd，Switzerland版权所有。未经Trans Tech Publications Ltd（www.scientific.net）的书面许可，不得以任何形式或任何方式复制或传播本文的任何内容。（Semanticscholar.org-13/03/20，21：20：54）2方法含rhBMP-2的磷酸钙涂层的制备首先将钛合金（Ti6 A14 V）盘（直径1cm）在高度成核条件下在37 ℃下浸入浓缩的刺激体液中24小时，然后在无菌条件下在37 ℃下浸入含rhBMP-2（10μg/ml）的磷酸钙过饱和溶液（pH 7.4）中48小时。用扫描电镜和傅里叶变换红外光谱对涂层进行了表征。磷酸钙涂层的rhBMP-2含量通过ELISA测定掺入每个涂层中的rhBMP-2的量。将每个涂层从其下面的钛合金盘上剥离，称重，然后溶解在1 ml的20%EDTA中。将浸提液样品稀释10倍和100倍，并将100 μl等份的每种溶液转移至96孔微孔板中。向每个孔中加入100 μl抗BMP-2 [（Sigma，B-1520）在PBS中1：10.000稀释]和等体积的抗rabbi t IgG [（Sigma，A 0418）在PBS中1：5000稀释]，并使用酶标仪在405 nm的吸收波长下测量有色反应产物的强度。使用校准曲线将颜色强度转换为rhBMP-2的纳克数。使用上述设置制备，用等体积的含标准量rhBMP-2的溶液代替100 μl等份的共提取物。将6个带有含rhBMP-2磷酸钙涂层的钛合金圆盘皮下植入相同数量的大鼠中。在5周内每隔7天取出样品，固定在10%福尔马林中，在乙醇中浸泡并包埋在甲基丙烯酸甲酯中。切取300 μ m厚的切片，抛光，用碱性品红、麦克尼尔四色和甲苯胺蓝O进行表面染色，准备在光学显微镜下进行组织学分析。结果磷酸钙与rhBMP-2仿生共沉淀法制备的涂层的特性：每1 mg磷酸钙中含有0.5 μg rhBMP-2，每片钛合金表面均匀地涂有25 μ m厚的涂层。在不存在和存在rhBMP-2的情况下制备的涂层在扫描电子显微镜中具有相似的形态学表现，每个涂层完全由板状晶体组成（图1）。傅立叶变换红外光谱显示磷酸钙晶体具有典型的碳酸磷灰石结构。rhBMP-2的存在未引起结构变化。668生物陶瓷15
We have recently shown that proteins can be biomimetically co-precipitate d with calcium phosphate crystals upon the surfaces of titanium-alloy impl ants. When recombinant human bone morphogenetic protein 2 (rhBMP-2) was thus incorporate d into the inorganic matrix, it retained its bioactivity as an osteoinducti ve agent in vitro. Furthermore, the osteogenicity (alkaline phosphatase activity) of rat bone-marrow stromal cells grown on BMP-2-containing calcium phosphate coatings was highe r than that of cells cultured on a plastic substratum and exposed to the freely-suspended dr ug. In the present study, titanium-alloy discs bearing BMP-2-containing calcium phospha te coatings were implanted ectopically (subcutaneously) in rats and the bone-formation pr ocess monitored during the ensuing 1-5 weeks. Osseous tissue appeared 2 weeks after implantation and thereafter increased steadily in mass. Hence, BMP-2 incorporate d in o the calcium phosphate coatings of titanium-alloy implants retains its bioact ivity as an osteoinductive agent in vivo. The principle of this system may be put to good use i n orthopaedic and dental implant surgery. Introduction Bone morphogenetic proteins (BMPs) are capable of inducing bone forma ti n not only within tissues that form part of the osseous system, such as at s ites of bone disunion in humans [1], but also ectopically, as for instance within the dorsal mus cles of experimental animals. BMPs have also been demonstrated to stimulate the alkaline phosphatase activity of osteoprogenitor cells in vitro. In vivo, however, the induction of bone forma tion cannot be elicited simply by the local injection of BMPs in their fre e form. A suitable carrier, such as calcium phosphate, is required. It has recently become possible to incorporate proteins into the calcium phosphate layers deposited on titanium-alloy implants using a biomimetic approach [2]. Such coatings may afford a vehicle for delivering BMP s slowly but at a sustained and sufficiently high rate to support and sustain their osteog enic activities locally in vivo. In the present study, titanium-alloy implants coated with BMP-2containing calcium phosphate layers were implanted subcutaneously in a rat model and the bone-formation process monitored histologically during the course of 5 weeks. Key Engineering Materials Online: 2003-05-15 ISSN: 1662-9795, Vols. 240-242, pp 667-670 doi:10.4028/www.scientific.net/KEM.240-242.667 © 2003 Trans Tech Publications Ltd, Switzerland All rights reserved. No part of contents of this paper may be reproduced or transmitted in any form or by any means without the written permission of Trans Tech Publications Ltd, www.scientific.net. (Semanticscholar.org-13/03/20,21:20:54) 2 Methods Preparation of calcium phosphate coatings containing rhBMP-2 Titanium-alloy (Ti6A14V) discs (1cm in diameter) were immer sed first in concentrated stimulated body fluid under high-nucleation conditions for 24 hours at 37°C, and then in a supersaturated solution of calcium phosphate (pH 7.4) containing rhBMP-2 (10μg/ml) for 48 hours at 37°C under sterile conditions. The coatings were characteri zed by scanning electron microscopy and Fourier-transform infrared spectroscopy. RhBMP-2 content of calcium phosphate coatings The amount of rhBMP-2 incorporated into each coating was determined by ELISA. Each coating was peeled away from its underlying titanium-alloy disc, weighed, and then dissolved in 1ml of 20% EDTA. Samples of the extract were diluted 10and 100-fold, and 100-μl aliquots of each solution transferred to 96-well microplates. 100 μl of anti-BMP-2 [(Sigma, B-1520) diluted 1:10.000 in PBS] and an equivalent volume of anti-rabbi t IgG [(Sigma, A 0418) diluted 1:5000 in PBS] were added to each well and the i ntensity of the coloured reaction product measured using a microplate reader at an a bsorption wavelength of 405 nm. Colour intensity was converted to nanograms of rhBMP-2 using a c alibration curve. This was prepared using the set-up described above, the 100-μl aliquots of coatextract being replaced by an equivalent volume of solution containing s ta dard amounts of rhBMP-2. Subcutaneous implantation of coated discs in rats Six titanium-alloy discs bearing rhBMP-2-containing calcium phos ate coatings were implanted subcutaneously in the same number of rats. Samples were retrieved at 7-day intervals over a period of 5 weeks, fixed in 10% formalin, dehydrate in ethanol and embedded in methylmethacrylate. 300-μm-thick sections were cut, pol ished and surface stained with basic Fuchsine, McNeil’s Tetrachrome and Toluidine Bl u O in preparation for histological analysis in the light microscope. Results Characteristics of coatings prepared by the biomimetic co-precipitation of calcium phosphate and rhBMP-2 Each titanium-alloy disc was uniformly coated with a 25-μm-thic k layer containing 1.6 μg of rhBMP-2 at a concentration of 0.5 μg per mg of calcium phosphate. Coat ings prepared in the absence and presence of rhBMP-2 had a similar morphological a ppear nce in the scanning electron microscope, each being comprised entirely of plate -like crystals (Fig. 1). Fourier-transform infrared spectroscopy revealed the calcium phospha te crystals to have a typical carbonated apatite structure. No structural change was elicited by the presence of rhBMP-2. 668 Bioceramics 15