摘要
目的研究低温保存对人骨髓基质干细胞(BMSCs)在脱钙骨(DBM)上生长特性及成骨能力的影响。方法取3例志愿者骨髓(3～5mL),密度梯度离心、差速贴壁法获得BMSCs。第3代BMSCs在-196℃下保存24h,37℃复苏,测定细胞成活率。低温保存前、后的BMSCs分别用成骨诱导液诱导培养,至90%融合时,收集细胞接种在DBM支架上,并测定细胞在DBM上的粘附率。DiI荧光染料标记BMSCs,例置相差显微镜、荧光显微镜和SEM观察低温保存前、后的BMSCs在DBM上的生长及基质分泌情况,MTT法测定细胞在DBM上的增殖活性。通过测定碱性磷酸酶(ALP)活性和骨钙素(OCN)含量观察细胞在DBM上的成骨能力。结果复苏细胞的存活率为(90．24±0．02)%。低温保存前、后的BMSCs在DBM上的粘附率分别为(97．25±1．17)%和(97．00±1．09)%。倒置相差显微镜及SEM观察显示低温保存前、后的BMSCs在DBM上粘附、生长良好,有大量细胞外基质分泌、沉积。低温保存前、后的BMSCs在DBM上MTT吸光度值和ALP活性的检测结果差异无显著性意义(P＞0．05)；体外培养12d时,MTT吸光度值及ALP活性同时达到峰值。低温保存前、后的BMSCs在DBM上分泌OCN的量差异无显著性意义(P＞0．05),OCN含量随着培养时间延长而不断上升,在观察期(16d)内未出现平台期。结论低温保存对人BMSCs在DBM上的体外增殖、粘附及成骨能力影响差异无显著性意义,低温保存的人BMSCs可作为组织工程骨的种子细胞。
Objective To investigate the effects of cryopreservation on the growth and osteogenesis capahility of human hone marrow stromal cells （BMSCs） on demineralized bone matrix （DBM）. Methods Bone marrow aspirates were ohtained from the iliac crests of three donors. The BMSCs were isolated from the bone marrow by density gradient centrifugation. Cells of passage 3 were cryopreserved in liquid nitrogen for 24 hours, and then recovered. The non-cryopreserved BMSCs were used as the control. The cryopreserved and control BMSCs were cultured in osteogenic media, collected and labeled with Dil to be seeded onto the DBM when cells were confluent. The percentage of BMSCs adhered to the DMB was detected. The cell morphology and matrices secreted by BMSCs on the DBM were observed by the inverted phase-contrasted microscope, fluorescence microscope and scanning electron microscope （SEM）. The growth and viability of BMSCs on the DBM were determined using the modified MTT assay. The osteogenesis ability of BMSCs on the DBM was determined by assessment of the alkaline phosphatase （ALP） activity and osteocalcin （OCN） content. Results The percentages of the cryopreserved and control cells adhered to DBM were （97.25 ± 1. 17） % and （97. 00 ± 1.09） % respectively. The cells adhered well to the DBM and grew rapidly. Large amounts of matrices on the DBM were observed by the light microscope and SEM. The cells embedded in the matrices could be observed by fluorescence microscope. There were no significant differences in the assay values of MTT, ALP and OCN hetween the cryopreserved and control BMSCs on the DBM. Conclusion Since cryopreservation does not affect the growth and osteogenesis capability of BMSCs on DBM, the cryopreserved BMSCs can be used as a cell source in bone tissue engineering.
出处
《中华创伤骨科杂志》
CAS
CSCD
2006年第10期933-937,共5页
Chinese Journal of Orthopaedic Trauma
基金
国家高科技研究发展计划(863)资助项目(2002AA205011)
关键词
低温保存
骨髓基质干细胞
脱钙骨
组织工程
Cryopreservation
Bone marrow stromal cells （BMSCs）
Demineralized bone matrix （DBM）
Tissue engineering
