藻酸钙凝胶复合骨形态发生蛋白复合大鼠红骨髓对异位诱导成骨活性的影响(英文)

Effect of calcium alginate gel-bone morphogenetic protein-red bone marrow compound on the osteoinductivity

背景:红骨髓中的骨髓基质细胞是骨的非特异性生长因子,是骨形态发生蛋白的靶细胞,具有骨诱导作用和自身成骨作用;可注射性藻酸钙凝胶属于凝胶态可降解生物材料,其组织相容性好,为成骨、成软骨细胞的增殖、附着提供支架,有利于新生毛细血管的增殖。目的:制备骨形态发生蛋白、自体红骨髓及藻酸钙凝胶复合物,观察其诱导成骨活性的影响。设计:随机对照动物实验。单位:深圳市人民医院骨科,华中科技大学同济医学院附属协和医院骨科。材料:实验于2002-02/2003-02在深圳市人民医院完成,选用27只纯种SD大鼠,雌雄不拘,体质量(200±20)g,购于解放军第一军医大学动物实验中心,骨形态发生蛋白购于解放军第四军医大学附属西京医院。方法:随机摸球法将大鼠分为3组:藻酸钙凝胶-骨形态发生蛋白-红骨髓组、藻酸钙凝胶-骨形态发生蛋白组及藻酸钙凝胶组,每组9只,藻酸钙凝胶-骨形态发生蛋白-红骨髓组大鼠在麻醉后,取已制备的藻酸钙凝胶-骨形态发生蛋白复合物0.4mL,于股骨大粗隆处抽取红骨髓0.1mL,加入藻酸钙凝胶-骨形态发生蛋白复合物,搅拌混合后用注射器注入股后肌肉中,藻酸钙凝胶-骨形态发生蛋白组及藻酸钙凝胶组双侧肢体股后肌群分别植入藻酸钙凝胶-骨形态发生蛋白复合物及单纯藻酸钙凝胶。①术后观察动物麻醉后苏醒、切口愈合、饮食、活动情况,同时观察植入物的大小、硬度及血管分布情况。②分别在造模后1,2,4周3个时间点进行指标检测,每个时间点3只大鼠,取出标本切片进行光镜下观察及碱性磷酸酶活性的测定。主要观察指标:①术后动物一般观察及植入物大体观察。②组织病理观察。③碱性磷酸酶活性测定结果。结果:纳入SD大鼠27只,麻醉4～6h后完全清醒,可正常进饮食;24h切口无血肿、可正常活动;72h切口无感染征象;2周后切口完全愈合、缝线脱落;全部27只大鼠切口均Ⅰ期愈合,均进入结果分析。①植入物标本大体观察:藻酸钙凝胶-骨形态发生蛋白-红骨髓、藻酸钙凝胶-骨形态发生蛋白组1周时植入物体积无减少、有血管长入;2周时质地较硬,切面呈灰白色,有骨样组织沉积;4周时有大量血管长入,有大量骨样组织沉积,质地较硬。②组织病理观察结果:藻酸钙凝胶-骨形态发生蛋白-红骨髓组1周可见大量间充质细胞聚集,骨母细胞及软骨母细胞增生活跃;2周软骨细胞趋于成熟,有软骨样基质及骨样基质;4周骨细胞多见,有编织骨形成,较其他两组成骨多。③碱性磷酸酶活性:植入后1,2,4周,藻酸钙凝胶组分别为(0.179±0.018),(0.058±0.017),(0.027±0.018)IU/g,低于藻酸钙凝胶-骨形态发生蛋白-红骨髓组[(0.922±0.226),(1.169±0.249),(0.431±0.081)IU/g,P<0.01],植入后1,4周,藻酸钙凝胶-骨形态发生蛋白组活性分别为(0.447±0.015),(0.276±0.081)IU/g,低于藻酸钙凝胶-骨形态发生蛋白-红骨骨髓组(P<0.05)。结论:藻酸钙凝胶-骨形态发生蛋白-红骨髓复合物具有稳定而持久的诱导成骨活性作用。

BACKGROUND：The bone marrow stromal cells in red bone marrow （RBM） are the non-specific bone growth factors, and the target cells of bone morphogenetic protein （BMP）, and they have the activities of osteoinduction and osteogenesis, Injectable calcium alginate gel （CaAG） is a degradable biomaterial with good histocompatibility, it provides scaffold for the proliferation and adhesion of osteoblasts and chondroblasts, and it is good for the proliferation of new capillaries, OBJECTIVE： To prepare CaAG-BMP-RBM gel compound, and observe its osteoinductivity DESIGN ： A randomized controlled animal trial SETTINGS： Department of Orthopaedics, Shenzhen People＇s Hospital; Department of Orthopaedics, Union Hospital affiliated to Tongji Medical College, Huazhong University of Science and Technology. MATERIALS： The experiments were carried out in Shenzhen People＇s Hospital from February 2002 to February 2003. Twenty-seven pure SD rats, either male or female, weighing （200±20） g, were purchased from the Animal Experimental Center of Southern Medical University （No. SCXK2002-99）. BMP was bought from Xijing Hospital affiliated to the Fourth Military Medical University of Chinese PLA. METHODS ： The rats were randomly divided into three groups： CaAG -BMP-RBM group （n =9）, CaAG -BMP group （n =9） and CaAG group （n =9）. In the CaAG -BMP-RBM group, the rats were anesthetized, then 0.1 mL RBM collected from trochanteric section of femur was added into the prepared CaAG-BMP compound （0.4 mL）, sufficiently mixed, and then injected into posterior femoral muscle. The rats in the CaAG-BMP group and CaAG group were implanted with CaAG-BMP and CaAG into bilateral posterior femoral muscles respectively. （1） The conditions of becoming conscious after anesthesia, incision healing, diet and activities were observed, the size and consistency of the implants and distribution of vessels were also observed. （2） The indexes were detected at 1, 2 and 4 weeks after model establishment respectively, and 3 rats were used for each time point. The sections were observed under light microscope, and the activity of alkaline phosphatase （ALP） was determined. MAIN OUTCOME MEASURES： （1） General conditions of animals and gross observation of the implants;（2） Histopathological observation; （3） ALP activities, RESULTS： All the 27 SD rats were involved in the analysis of results. The rats became conscious at 4-6 hours after anesthesia, and they could eat normally; Hematom at incision disappeared, and the rats could move normally at 24 hours; No sign of infection at incision was observed at 72 hours; The incisions healed completely and suture shed after 2 weeks. The incisions were stage I healing in all the 27 rats. （1） Results of gross observation of the implants： At 1 week after implantation, the size of implant did not decrease and vessels in-grew in both the CaAG-BMP-RBM group and CaAG-BMP group; At 2 weeks, the quality was hard, and the section appeared as gray and white with deposition of bone-like tissues; At 4 weeks, a great amount of vessels in-grew, and there were many depositions of hard bone-like tissues. （2） Results of the histopathological observation under light microscope： In the CaAG-BMP-RBM group, many mesenchymal cells aggregated, osteoblasts and chondroblasts proliferated actively at 1 week; The chondrocytes tended to mature with cartilage-like and bone-like matrixes at 2 weeks; Many osteocytes were observed and fibrous bone formed at 4 weeks, which were more then those in the other two groups. （3） Results of ALP activity： At 1, 2 and 4 weeks after implantation, the ALP activities in the CaAG group [（0.179±0.018）, （0.058±0.017）, （0.027±0.018） IU/g] were lower than those in the CaAG-BMP-RBM group [（0.922±0.226）, （1.169±0.249）, （0.431 ±0.081）IU/g, P 〈 0.01）; At 1 and 4 weeks, the ALP activities in the CaAG-BMP group [（0.447±0.015）, （0.276±0.081） IU/g] were lower than those in the CaAG-BMP-RBM group （P 〈 0.05）.CONCLUSION ： The compound of CaAG-BMP-RBM possesses stable and lasting osteoinductivity.