Three-dimensional printing beta-tricalcium phosphate scaffold loaded with icariin particles for repairing osteonecrosis of the femoral head in rabbits

doi:10.3969/j.issn.2095-4344.1673

Abstract

Abstract:

BACKGROUND: Preliminary study has prepared three-dimensional printing β-tricalcium phosphate scaffold loaded with icariin.

OBJECTIVE: To investigate the role of three-dimensional printing β-tricalcium phosphate scaffold loaded with icariin in the repair of rabbit models of osteonecrosis of the femoral head.

METHODS: New Zealand white rabbits (provided by Qinglongshan Laboratory Animal Center of Nanjing) were selected to establish the steroid-induced osteonecrosis of the femoral head. The 27 model rabbits underwent core decompression and debridement, were randomly divided into three groups, and then implanted with autologous bone, β-tricalcium phosphate scaffold, three-dimensional printing β-tricalcium phosphate scaffold loaded with icariin (composite scaffold group), respectively. The micro-CT scanning and pathological observation were performed at 4, 8, and 12 weeks after implantation.

RESULTS AND CONCLUSION: (1) Micro-CT showed that at 4 weeks after implantation, trabecular bone was observed around and in implants in each group. In the autologous bone group, there were a large number of trabecular bones in the grafting area at 8 weeks, and the trabecular bone structure was dense at 12 weeks after implantation. In the tricalcium phosphate and composite scaffold groups, the scaffolds were well integrated with the bone interface. At 4 weeks after implantation, there was a certain amount of trabecular bone surrounding the scaffold, and trabecular grew into the scaffold until 8 weeks in the composite scaffold group. At 4 weeks after implantation, few thin trabecular bone was visible, and extensive trabecular bone formation was observed around the scaffold at 8 weeks in the tricalcium phosphate group. (2) Hematoxylin-eosin staining results showed that there were many mature osteoblasts, and few cartilage matrix, newly born bones integrated well to the implants at 12 weeks in the autologous bone and tricalcium phosphate groups. In the composite scaffold group, there were many cartilage matrixes, and newly born bones integrated poorly to the implants. (3) Masson staining showed that at 12 weeks after implantation, the osteogenic capacity in the composite scaffold group was lower than that in the autologous bone group (P < 0.05), but higher than that in the tricalcium phosphate group (P < 0.05). (4) TRAP staining results at 12 weeks after implantation revealed that the amount of osteoclast in composite scaffold group was less than that in the tricalcium phosphate group (P < 0.05), and was not significantly different from the autologous bone group (P > 0.05). (5) Immunohistochemical staining at 12 weeks after implantation revealed that the positive rate of vascular endothelial growth factor in the composite scaffold group was higher than that in the tricalcium phosphate group (P < 0.05), and lower than that in the autologous bone group (P < 0.05). (6) In summary, three-dimensional printing β-tricalcium phosphate scaffold loaded with icariin implanted into the rabbit model of osteonecrosis of the femoral head can promote the proliferation and differentiation of osteoblasts, inhibit the viability of osteoclasts, promote the angiogenesis, and contribute to the repair of osteonecrosis of the femoral head in rabbits.

Key words: Femur Head Necrosis;, Drugs, Chinese Herbal;, Calcium Phosphates, Tissue Engineering

CLC Number:

Peng Chenjian, Du Bin, Sun Guangquan, Liu Xin, Xue Peng, Cao Liangquan. Three-dimensional printing beta-tricalcium phosphate scaffold loaded with icariin particles for repairing osteonecrosis of the femoral head in rabbits[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(14): 2162-2168.

Figures/Tables 5

2.1 实验动物数量分析造模期间死亡2只兔，均为腹泻死亡。造模结束后，对兔子进行MRI影像学评价，头内或头颈交界区出现T1低信号，T2高信号，压脂像高信号作为判断标准来诊断兔子是否出现股骨头坏死。选用40只兔进行造模，共31只兔子造模成功，造模成功率为77.5%，选取1只行股骨头苏木精-伊红染色，显示骨小梁结构紊乱、断裂，伴有纤维结构增生。随机选取27只造模成功兔作为实验对象。 2.2 micro-CT影像学分析自术后第4周起，3组植入物周围及内部均有骨小梁生成。自体骨组4周后植骨区边缘即可见点状高密度影，为自体骨与周围骨组织的爬行替代产生的新生骨，术后8周植骨区有大量骨小梁，术后12周骨小梁结构致密，提示强度良好。磷酸三钙组、复合支架组支架与骨界面整合良好，且随术后时间延长，支架周围骨小梁逐渐增多，排列有序；复合支架组在第4周时即可见支架附近有一定量的骨小梁生成，至第8周时可见支架上有骨小梁长入；磷酸三钙组在术后第4周时仅可见微量薄弱骨小梁，第8周时才可见支架周围广泛骨小梁生成，12周时骨小梁仍较其余两组稀疏，见图3。除磷酸三钙组术后第8周有1只发生股骨头塌陷外，其他支架始终显示其形态学结构稳定，表明支架力学强度良好，同时具有较好的骨传导性和生物相容性。通过测定以下4种植入物-宿主骨区域骨小梁参数判断骨修复情况：①新生骨量：即植入物与骨交界区骨小梁体积占总体积的百分比。随着时间的推移，3组新生骨量有所增加。复合支架组术后不同时间点的新生骨总量多于磷酸三钙组(P=0.002，0.009，0.006)，但仍少于自体骨组(P=0.007，0.011，0.027)；②新生骨小梁厚度：随着术后时间延长，3组新生骨小梁厚度均有所增加。术后第4，8周时，自体骨组新生骨小梁厚度较复合支架组高(P < 0.05)，但第12周时两组间无差异。复合支架组术后不同时间点的新生骨小梁厚度均显著高于磷酸三钙组；③新生骨小梁数目：复合支架组术后不同时间点的新生骨小梁显著多于磷酸三钙组(P < 0.05)、少于自体骨组(P < 0.05)。④新生骨小梁间隙：术后第4周时，磷酸三钙组新生骨小梁间隙较自体骨组、复合支架组大(P < 0.05)，自体骨组术后第8，12周的新生骨小梁间隙小于复合支架组(P < 0.05)，磷酸三钙组术后第4，8，12周的新生骨小梁间隙均显著大于复合支架组(P < 0.05)，见图4。"

2.3 组织学观察结果 2.3.1 苏木精-伊红染色术后第4周时，自体骨组交界区骨陷窝内有较多细胞核较大的成熟成骨细胞，骨小梁排列较规则；磷酸三钙、复合支架组骨小梁排列不规则，细胞较为幼稚，复合支架组可见较多软骨基质及蓝染细胞，提示软骨化骨过程较为活跃，而磷酸三钙组显示较多的纤维组织增生。术后第8周时，自体骨组、复合支架组存在较多软骨细胞，空骨陷窝较少，有较多成熟骨小梁；磷酸三钙组新生骨小梁及软骨基质较少，空骨陷窝较4周时减少。术后第12周时，自体骨组、复合支架组细胞分化成熟，细胞核规则，软骨基质减少，新生骨组织与植入物整合良好，局部骨小梁致密有序；磷酸三钙组骨小梁排列较为有序，仍存一定软骨基质，新生骨与植入物尚未完成整合。 2.3.2 Masson染色术后4周时，自体骨组骨表面成骨细胞明显可见，软骨细胞及软骨基质较多；复合支架组骨组织表面可见少量成骨细胞，并见大量幼稚软骨细胞；磷酸三钙组成骨细胞更少，未见软骨样基质及软骨细胞。术后第8周时，自体骨组植骨区与自体骨界限模糊，大量骨小梁生成，染色变红；复合支架组骨表面成骨细胞稍增多，支架与骨组织间隙填充大量由蓝绿色向红色过渡的新生骨小梁；磷酸三钙组也可见骨小梁生成，但局部炎性反应较重，骨小梁间隙较大。术后第12周时，自体骨组基本已经完成爬行替代；复合支架组交界区界限稍模糊；磷酸三钙组新生骨及成骨细胞较前几周增生活跃，但交界区仍存在明显界限。在第4，8周时，复合支架组、自体骨组成骨细胞数比较无明显差异(P > 0.05)，但都显著高于磷酸三钙组(P=0.009，0.006)；术后第12周时，复合支架组成骨细胞增殖弱于自体骨组(P=0.023)，但仍高于磷酸三钙组(P=0.011)，见图5。 2.3.3 TRAP染色随着时间的推移，3组破骨细胞数目减少，复合支架组术后不同时间点的破骨细胞数均明显少于磷酸三钙组(P=0.001，0.006，0.035)，与自体骨组比较差异无显著性意义(P > 0.05)，见图6。 2.3.4 血管内皮生长因子免疫组织化学染色复合支架组术后第4，8，12周的血管内皮生长因子阳性率均显著大于磷酸三钙组(P=0.003，0.009，0.019)，但均低于自体骨组，见图7。"

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