组织工程化生物活性骨膜冻存后生物学特性的研究
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摘要
目的:观察冻存复苏后组织工程化生物活性骨膜的生物学特性和成骨活性。
方法:从新西兰大白兔骨髓中分离骨髓间充质干细胞(BMSCs),经体外成骨诱导培养后与猪小肠粘膜下层(SIS)构建组织工程化生物活性骨膜,另外将未诱导的骨髓间充质干细胞和猪小肠粘膜下层也构建组织工程化骨膜,而后用含10%二甲基亚砜、50%胎牛血清、40%DMEM冻存液,采用程序性降温法将两种工程化骨膜保存在-196℃液氮中。30天后复苏培养,用MTT法检测种子细胞在支架材料上的生长情况,并用扫描电镜观察种子细胞的生长状态,通过免疫印迹的方法来检测复苏后组织工程骨膜分泌Ⅰ型胶原和骨钙素量的变化,酶组织化学法检测碱性磷酸酶(ALP)分泌的量。
结果:冻存复苏后BMSCs仍能在SIS上保持良好的生长状况,构建的两种组织工程化生物活性骨膜复苏后在含有成骨诱导剂的培养基中培养可分泌一定量的Ⅰ型胶原、骨钙素和碱性磷酸酶。而且,骨髓间充质干细胞诱导为成骨细胞与小肠粘膜下层构成的工程化骨膜(M1)所含的Ⅰ型胶原的量,与未诱导的骨髓间充质干细胞和小肠粘膜下层构建的工程化骨膜(M2)所含的量差异无统计学意义(P＞0.05)。但是M2所分泌的骨钙素和碱性磷酸酶的量高于M1所分泌的量,差异有统计学意义(P＜0.05)。
结论:组织工程化生物活性骨膜冻存复苏后培养仍保持较稳定的生物活性,种子细胞在支架材料上附着生长,并稳定地分泌一定量的Ⅰ型胶原、碱性磷酸酶和骨钙素,具有较强的成骨活性。M2表达成骨活性高于M1。
Objective:To observe the biological characteristics of cryopreserved tissue-engineered periosteum and to examine the osteogenic potential.
Methods:The bone marrow mesenchymal stem cells(BMSCs)of New Zealand rabbit were first cultivated and osteogenically induced as seeding cells in vitro,then co-cultivated with porcine small intestinal submucosa(SIS).Other BMSCs were not osteogenically induced and co-cultivated with SIS as well.They were both cryopreserved at -196℃in liquid nitrogen for 30 days banking in cryoprotectant, which contains 10%DMSO,50%fetal calf serum by programmed cooling procedure. Subsequently,the biological characteristics of cryopreserved tissue-engineered periosteum were analyzed by scan electronic microscopy(SEM),MTT and ALP detection after resuscitation.In the meantime,the quantity of typeⅠcollagen and osteocalcin secreted by the tissue-engineered periosteum(TEP)was detected by western blotting.
Rusults:The growth status of BMSCs remains good after resuscitation.The quantity of typeⅠcollagen,osteocalcin and ALP secreted by the two kinds of TEP were higher than SIS.The quantity of typeⅠcollagen in the TEP which was constructed with BMSCs and SIS(M1 group)was not significantly different from the TEP which was constructed with osteoblasts and SIS(M2 group).(P＞0.05)Other wise, the quantity of osteocalcin and ALP in the M2 group was all significantly higher than the M1 group.(P＜0.05)
Conclusion:The biology activity of tissue engineered periosteum remained stable and the quantity of typeⅠcollagen,ALP and osteocalcin was secreted at a certain level.The tissue-engineered periosteum showed a highly osteogenetic activity. The expression level of osteogenetic activity in M2 group was higher than M1 group.
方法:从新西兰大白兔骨髓中分离骨髓间充质干细胞(BMSCs),经体外成骨诱导培养后与猪小肠粘膜下层(SIS)构建组织工程化生物活性骨膜,另外将未诱导的骨髓间充质干细胞和猪小肠粘膜下层也构建组织工程化骨膜,而后用含10%二甲基亚砜、50%胎牛血清、40%DMEM冻存液,采用程序性降温法将两种工程化骨膜保存在-196℃液氮中。30天后复苏培养,用MTT法检测种子细胞在支架材料上的生长情况,并用扫描电镜观察种子细胞的生长状态,通过免疫印迹的方法来检测复苏后组织工程骨膜分泌Ⅰ型胶原和骨钙素量的变化,酶组织化学法检测碱性磷酸酶(ALP)分泌的量。
结果:冻存复苏后BMSCs仍能在SIS上保持良好的生长状况,构建的两种组织工程化生物活性骨膜复苏后在含有成骨诱导剂的培养基中培养可分泌一定量的Ⅰ型胶原、骨钙素和碱性磷酸酶。而且,骨髓间充质干细胞诱导为成骨细胞与小肠粘膜下层构成的工程化骨膜(M1)所含的Ⅰ型胶原的量,与未诱导的骨髓间充质干细胞和小肠粘膜下层构建的工程化骨膜(M2)所含的量差异无统计学意义(P＞0.05)。但是M2所分泌的骨钙素和碱性磷酸酶的量高于M1所分泌的量,差异有统计学意义(P＜0.05)。
结论:组织工程化生物活性骨膜冻存复苏后培养仍保持较稳定的生物活性,种子细胞在支架材料上附着生长,并稳定地分泌一定量的Ⅰ型胶原、碱性磷酸酶和骨钙素,具有较强的成骨活性。M2表达成骨活性高于M1。
Objective:To observe the biological characteristics of cryopreserved tissue-engineered periosteum and to examine the osteogenic potential.
Methods:The bone marrow mesenchymal stem cells(BMSCs)of New Zealand rabbit were first cultivated and osteogenically induced as seeding cells in vitro,then co-cultivated with porcine small intestinal submucosa(SIS).Other BMSCs were not osteogenically induced and co-cultivated with SIS as well.They were both cryopreserved at -196℃in liquid nitrogen for 30 days banking in cryoprotectant, which contains 10%DMSO,50%fetal calf serum by programmed cooling procedure. Subsequently,the biological characteristics of cryopreserved tissue-engineered periosteum were analyzed by scan electronic microscopy(SEM),MTT and ALP detection after resuscitation.In the meantime,the quantity of typeⅠcollagen and osteocalcin secreted by the tissue-engineered periosteum(TEP)was detected by western blotting.
Rusults:The growth status of BMSCs remains good after resuscitation.The quantity of typeⅠcollagen,osteocalcin and ALP secreted by the two kinds of TEP were higher than SIS.The quantity of typeⅠcollagen in the TEP which was constructed with BMSCs and SIS(M1 group)was not significantly different from the TEP which was constructed with osteoblasts and SIS(M2 group).(P＞0.05)Other wise, the quantity of osteocalcin and ALP in the M2 group was all significantly higher than the M1 group.(P＜0.05)
Conclusion:The biology activity of tissue engineered periosteum remained stable and the quantity of typeⅠcollagen,ALP and osteocalcin was secreted at a certain level.The tissue-engineered periosteum showed a highly osteogenetic activity. The expression level of osteogenetic activity in M2 group was higher than M1 group.
引文
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