骨髓间充质干细胞复合强化磷酸三钙/富血小板血浆凝胶支架材料体内成骨与血管化初步实验研究
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摘要
目的探讨骨髓间充质干细胞复合强化磷酸三钙/富血小板血浆凝胶支架材料体外定向成骨诱导后植入比格犬胫骨上段骨缺损巾构建组织工程骨并修复体内骨缺损能力及血管化的情况。
方法(1)比格犬骨髓间充质干细胞及强化磷酸三钙/富血小板血浆凝胶复合支架材料的制备,包括BMSCs的分离、纯化和扩增,富血小板血浆的制备。(2)比格犬骨髓间充质干细胞复合单纯强化磷酸三钙支架材料及强化磷酸三钙/富血小板血浆凝胶支架体外培养和诱导1周。(3)细胞复合单纯强化磷酸三钙支架材料复合物、自体髂骨及细胞复合强化磷酸三钙/富血小板血浆凝胶支架材料复合物随机植入比格犬双后肢胫骨平台下方骨缺损处。观察植入物的表面变化、骨痂形成、骨缺损修复情况及周围组织反应。(4)术后4、8、12周取材行HE染色计数微血管密度(MVD),同时行骨钙素蛋白免疫组织化学染色观察其表达情况。(5)RT-PCR检测体内各组在4、8、12周时OCN、VEGF的mRNA的表达情况,并对两者的相关性进行分析。
结果(1)各组材料植入比格犬胫骨骨缺损后,伤口生长良好,没有全身和局部的炎症、毒性反应及排斥反应,伤口一期愈合。细胞复合强化磷酸三钙/富血小板血浆凝胶支架材料组材料降解较快。(2)术后4、8、12周取材行MVD测定细胞复合强化磷酸三钙/富血小板血浆凝胶支架材料组MVD值与髂骨组接近,高于细胞复合单纯材料组。(3)术后4、8、12周取材行骨钙素蛋白免疫组织化学染色可见各组均可观察到新生软骨成骨情况,强化磷酸三钙/自体富血小板血浆凝胶复合材料组表达较细胞复合单纯强化磷酸三钙材料组更强。(4)各组在4、8、12周各组均可见OCN、VEGF的mRNA表达条带出现。比较4、8、12周各组目的条带和内参灰度值,8、12周时Ⅱ组和Ⅲ组各项表达强于Ⅰ组,Ⅱ组和Ⅲ组之间无明显差异。OCN、VEGF的mRNA表达呈正相关。
结论(1)犬骨髓间充质干细胞复合强化磷酸三钙/自体富血小板血浆凝胶复合支架材料经体外诱导后在犬体内能获得良好的血管化。(2)犬骨髓间充质干细胞复合强化磷酸三钙/自体富血小板血浆凝胶支架材料犬体内成骨量与血管化的程度密切相关。
Objective To investigate the ability of constructing tissue engineering bone and repairing bone defection as well as vascularizing with dog bone marrow mesenchymal stem cells in intensified beta-tricalcium phosphate/platelet-rich plasma gel multiple-scaffold implanted dog's tibia upper segment after directed differentiation of stem cells into osteoblasts in vitro.
Methods(1) Preparing dog bone marrow mesenchymal stem cells in intensified beta-tricalcium phosphate/platelet-rich plasma gel multiple-scaffold including the isolation、purification and proliferation of BMSCs,the preparation of PRP and so on.(2) Dog BMSCs was seeded in and then cultured or osteo-induced with intensified beta-tricalcium phosphate/platelet-rich plasma gel multiple-scaffold in vitro for 1 weeks.(3) The cells compound with alone beta-tricalcium phosphate(groupⅠ) or intensified beta-tricalcium phosphate/platelet-rich plasma gel multiple-scaffold(groupⅢ) were randomly implanted in dog's left or right circular tibial upper segment and were compared with autogenetic Ilium(groupⅡ).The changes of implants surface、the form of osteotylus、the reparation of bone defection and tissue reaction around the implant were observed.(4) All of implant were performed examination of HE stain and immunocytochemical stain of osteocalcin protein to observed the expression after operation 4、8、12 weeks,and to calculate microvessel density(MVD).(5) The expression of OCN、VEGF mRNA were detected by RT-PCR and the correlation of them was analyzed after operation 4、8、12 weeks.
Results(1) The wound growth all right,inflammation and toxicity reaction in body or local part were not observed in all groups. The material in the group of the cells compound with intensified beta-tricalcium phosphate/platelet-rich plasma gel multiple-scaffold were degraded faster than groupⅠ.(2)The numerical of microvessel density(MVD) in the group of the cells compound with intensified beta-tricalcium phosphate/platelet-rich plasma gel multiple-scaffold nearly reached the level of the autogenetic ilium,and was higher than groupⅠ.(3) The ossifications of new cartilages were observed in all groups by HE stain and immunocytochemical stain of osteocalcin protein.The expression of the cell/intensified beta-tricalcium phosphate/ platelet-rich plasma gel multiple-scaffold compounds were better positively than the cells/ intensified beta-tricalcium phosphate compounds.(4) The expression of osteocalcin,VEGF protein mRNA were showed positive at all groups aider operation for 4、8 and 12 weeks. The groupⅡ、Ⅲexpressed all protein mRNA more positive than groupⅠat 8 and 12 weeks.And there was no different between the expression of groupⅡ、Ⅲ,and there was positive correlation between the mRNA expression of OCN and VEGF.
Conclusion(1)Good vascularization could be acquired after canine BMSCs seeded in the intensified beta-tricalcium phosphate/platelet-rich plasma gel multiple-scaffold were transplanted in vivo.(2) There is close relationship between vascularization and ossification could after canine BMSCs seeded in the intensified beta-tricalcium phosphate/platelet-rich plasma gel multiple-scaffold were transplanted in vivo.
方法(1)比格犬骨髓间充质干细胞及强化磷酸三钙/富血小板血浆凝胶复合支架材料的制备,包括BMSCs的分离、纯化和扩增,富血小板血浆的制备。(2)比格犬骨髓间充质干细胞复合单纯强化磷酸三钙支架材料及强化磷酸三钙/富血小板血浆凝胶支架体外培养和诱导1周。(3)细胞复合单纯强化磷酸三钙支架材料复合物、自体髂骨及细胞复合强化磷酸三钙/富血小板血浆凝胶支架材料复合物随机植入比格犬双后肢胫骨平台下方骨缺损处。观察植入物的表面变化、骨痂形成、骨缺损修复情况及周围组织反应。(4)术后4、8、12周取材行HE染色计数微血管密度(MVD),同时行骨钙素蛋白免疫组织化学染色观察其表达情况。(5)RT-PCR检测体内各组在4、8、12周时OCN、VEGF的mRNA的表达情况,并对两者的相关性进行分析。
结果(1)各组材料植入比格犬胫骨骨缺损后,伤口生长良好,没有全身和局部的炎症、毒性反应及排斥反应,伤口一期愈合。细胞复合强化磷酸三钙/富血小板血浆凝胶支架材料组材料降解较快。(2)术后4、8、12周取材行MVD测定细胞复合强化磷酸三钙/富血小板血浆凝胶支架材料组MVD值与髂骨组接近,高于细胞复合单纯材料组。(3)术后4、8、12周取材行骨钙素蛋白免疫组织化学染色可见各组均可观察到新生软骨成骨情况,强化磷酸三钙/自体富血小板血浆凝胶复合材料组表达较细胞复合单纯强化磷酸三钙材料组更强。(4)各组在4、8、12周各组均可见OCN、VEGF的mRNA表达条带出现。比较4、8、12周各组目的条带和内参灰度值,8、12周时Ⅱ组和Ⅲ组各项表达强于Ⅰ组,Ⅱ组和Ⅲ组之间无明显差异。OCN、VEGF的mRNA表达呈正相关。
结论(1)犬骨髓间充质干细胞复合强化磷酸三钙/自体富血小板血浆凝胶复合支架材料经体外诱导后在犬体内能获得良好的血管化。(2)犬骨髓间充质干细胞复合强化磷酸三钙/自体富血小板血浆凝胶支架材料犬体内成骨量与血管化的程度密切相关。
Objective To investigate the ability of constructing tissue engineering bone and repairing bone defection as well as vascularizing with dog bone marrow mesenchymal stem cells in intensified beta-tricalcium phosphate/platelet-rich plasma gel multiple-scaffold implanted dog's tibia upper segment after directed differentiation of stem cells into osteoblasts in vitro.
Methods(1) Preparing dog bone marrow mesenchymal stem cells in intensified beta-tricalcium phosphate/platelet-rich plasma gel multiple-scaffold including the isolation、purification and proliferation of BMSCs,the preparation of PRP and so on.(2) Dog BMSCs was seeded in and then cultured or osteo-induced with intensified beta-tricalcium phosphate/platelet-rich plasma gel multiple-scaffold in vitro for 1 weeks.(3) The cells compound with alone beta-tricalcium phosphate(groupⅠ) or intensified beta-tricalcium phosphate/platelet-rich plasma gel multiple-scaffold(groupⅢ) were randomly implanted in dog's left or right circular tibial upper segment and were compared with autogenetic Ilium(groupⅡ).The changes of implants surface、the form of osteotylus、the reparation of bone defection and tissue reaction around the implant were observed.(4) All of implant were performed examination of HE stain and immunocytochemical stain of osteocalcin protein to observed the expression after operation 4、8、12 weeks,and to calculate microvessel density(MVD).(5) The expression of OCN、VEGF mRNA were detected by RT-PCR and the correlation of them was analyzed after operation 4、8、12 weeks.
Results(1) The wound growth all right,inflammation and toxicity reaction in body or local part were not observed in all groups. The material in the group of the cells compound with intensified beta-tricalcium phosphate/platelet-rich plasma gel multiple-scaffold were degraded faster than groupⅠ.(2)The numerical of microvessel density(MVD) in the group of the cells compound with intensified beta-tricalcium phosphate/platelet-rich plasma gel multiple-scaffold nearly reached the level of the autogenetic ilium,and was higher than groupⅠ.(3) The ossifications of new cartilages were observed in all groups by HE stain and immunocytochemical stain of osteocalcin protein.The expression of the cell/intensified beta-tricalcium phosphate/ platelet-rich plasma gel multiple-scaffold compounds were better positively than the cells/ intensified beta-tricalcium phosphate compounds.(4) The expression of osteocalcin,VEGF protein mRNA were showed positive at all groups aider operation for 4、8 and 12 weeks. The groupⅡ、Ⅲexpressed all protein mRNA more positive than groupⅠat 8 and 12 weeks.And there was no different between the expression of groupⅡ、Ⅲ,and there was positive correlation between the mRNA expression of OCN and VEGF.
Conclusion(1)Good vascularization could be acquired after canine BMSCs seeded in the intensified beta-tricalcium phosphate/platelet-rich plasma gel multiple-scaffold were transplanted in vivo.(2) There is close relationship between vascularization and ossification could after canine BMSCs seeded in the intensified beta-tricalcium phosphate/platelet-rich plasma gel multiple-scaffold were transplanted in vivo.
引文
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