BMP活性多肽复合羟基磷灰石聚乳酸在大鼠体内异位成骨的实验研究

A study on the ectopic osteogenesis experiment of bone morphogenetic protein active polypeptide combined with hydroxyapatite and poly lactic acid in rats

目的通过检测不同浓度骨形态发生蛋白(bone morphogenetic proteins,BMP)BMPIII与BMPIV活性多肽在大鼠体内异位成骨情况,来评价两种多肽在动物体内骨诱导能力;并与已检测BMPI0.4 g/L浓度进行对比,以选出最优多肽组及浓度。方法应用随机抽签法,将48只SD大鼠随机分为8组(A、B、C、D、E、F、G、H组),每组6只。将2种多肽的三种不同浓度(0.2 g/L、0.4 g/L、0.8 g/L的BMPIII和0.2 g/L、0.4 g/L、0.8 g/L的BMPIV)分别与羟基磷灰石聚乳酸(hydroxyapatite and poly lactic acid,HA/PLA)组成复合材料植入大鼠臀部肌肉浅层,A、B、C、D、E、F组;将0.4 g/L浓度BMPI多肽与HA/PLA组成的复合材料植入的实验对照为G组,仅植入HA/PLA框架材料空白对照为H组。术后3周摄背部正位X线片;术后5周时进行X线、CT照射。并于术后3周、5周分别取出标本,采用HE、Masson染色在光镜下观察,应用Masson染色评分并进行非参数统计分析大鼠术后5周的成骨特性。结果所有动物均顺利完成手术,术后观察期间,动物饮食、活动良好,伤口愈合良好,全部存活。植入材料各组硬度均增加。(1)X线检查:术后3周,手术植入多肽材料部位均有轻度模糊显影区,其中C、F组显影明显;术后5周,A、D组植入区内有较浅显影,B、E、G组有较浅较大显影,C、F组显影较明显;H组未见明显显影。(2)CT显示:除A、D组可见较模糊低密度显影外,B、C、E、F、G组均显影明显,H组未见显影。(3)HE染色:植入多肽材料的各组术后3周可见少量成骨细胞长入多孔材料,贴附于孔壁;术后5周材料部分降解,其中B、C、E、F、G组有较多软骨基质和软骨细胞形成,H组框架材料分解极少。(4)Masson染色:术后3周,A、D组在降解材料周围仅有少量软骨胶原形成,且B、C组和E、F组软骨胶原量分别明显多于A组和D组,G组蓝色面积较小,H组降解材料周围软骨胶原极少;术后5周,植入多肽材料的各组蓝色面积明显多于术后3周,H组降解材料周围软骨胶原较少,仅见少量蓝色软骨胶原形成。(5)Masson评分:B、C、E、F组分别为(2.22±0.45)分、(2.44±0.3)分、(2.28±0.25)分、(2.44±0.35)分均明显大于A组(1.06±0.39)分、D组(0.72±0.25)分,差异有统计学意义(P<0.05);B、C、E、F组各组间评分两两比较,差异均无统计学意义(P>0.05);G组评分(2.67±0.30)分较B、E组大,差异均有统计学意义(P<0.05);H组评分最少,为(0.222±0.27)分。结论 BMPIII与BMPIV两种活性多肽人工复合材料均具有骨诱导能力;0.4 g/L和0.8 g/L浓度骨诱导能力较0.2 g/L浓度大;0.4 g/L BMPI骨诱导能力较BMPIII与BMPIV大,更适于成为骨诱导材料。

Objective To evaluate osteoinductive potential of bone morphogenetic protein （ BMP ） III and BMPIV active polypeptides in animals by detecting ectopic bone formation ability of the 2 active polypeptides in different concentrations in rats, and to elect the best polypeptide in the best concentration by comparing with the osteoinductive activity of 0.4 g / L BMPI.Methods A total of 48 Sprague-Dawley （ SD ） rats were randomly divided into 8 groups （ groups A, B, C, D, E, F, G, H ）, with 6 rats in each group. The composite materials of 0.2 g / L, 0.4 g / L and 0.8 g / L BMPIII and 0.2 g / L, 0.4 g / L and 0.8 g / L BMPIV with hydroxyapatite and poly lactic acid （ HA / PLA ） were implanted into superifcial muscles of the hips of the rats in group A, B, C, D, E and F respectively. The rats in the experimental control group （ group G ） received the composite materials of 0.4 g / L BMPI with HA / PLA and the rats in the blank control group （ group H ） only received HA / PLA. At 3 weeks after the operation, the anteroposterior X-ray films of the back were taken. The X-ray and CT images were observed at 5 weeks after the operation. The samples were taken out, stained by HE and Masson and observed under the light microscope at 3 and 5 weeks after the operation respectively. Using the method of nonparametric statistics, the osteogenic properties of the rats were analyzed at 5 weeks after the operation.Results All the rats underwent operation successfully, and their diets, activities and wound healing were ifne. All the rats were alive. The hardness of implantation materials was increased in each group. （ 1 ） According to the X-ray iflms, the following results were found. At 3 weeks after the operation, there were slightly fuzzy shadows in the implantation area, which were more obvious in group C and F. At 5 weeks after the operation, there were slight shadows in group A and D, which were more obvious in group C and F. There were no shadows in group H. （ 2 ） The CT images showed there were fuzzy low-density shadows in group A and D, which were more obvious in group B, C, E, F and G, and no shadows in group H. （ 3 ） A few osteoblasts were noticed in the porous materials and attached to the hole wall in group A, B, C, D, E, F and G at 3 weeks after the operation based on the HE staining results. Part of them were degraded at 5 weeks after the operation. There were more cartilage matrix and cells in group B, C, D, E, F and G. Little framework material was degraded in group H. （ 4 ） According to Masson staining, there was a little chondrogen around degradable materials in group A and D. The chondrogen in group B and C and group E and F was more than that in group A and group D respectively. The blue area was relatively small in group G. There was little chondrogen around degradable materials in group H. At 5 weeks after the operation, the blue area in group A, B, C, D, E, F and G was obviously larger than that at 3 weeks after the operation. There was little chondrogen around degradable materials in group H, only a little blue chondrogen was found. （ 5 ） The Masson scores in group B, C, E and F were obviously larger than that in group A and D, and the differences were statistically signiifcant （P〈0.05 ）. There were no statistically signiifcant differences when the Masson scores were compared between each 2 of group B, C, E and F （P〉0.05 ）. The Masson score in group G was larger than that in group B and F, and the differences were statistically significant （ P〈0.05 ）.Conclusions The artificial composite materials of BMPIII and BMPIV active polypeptides possess osteoinductive potential. The osteoinductive potential of 0.4 g / L and 0.8 g / L BMPIII and BMPIV is stronger than that of 0.2 g / L BMPIII and BMPIV. The osteoinductive potential of 0.4 g / L BMPI is stronger than that of BMPIII and BMPIV, which is the best osteoinductive material.