In vitro cultivation, identification and osteoinduction of adult bone marrow
mesenchymal stem cells

doi:10.3969/j.issn.2095-4344.2013.45.019

Abstract

Abstract:

BACKGROUND: Bone marrow mesenchymal stem cells have the potential of self-proliferation and multi-directional differentiation, while mesenchymal stem cells are few in adult bone marrow. In vitro purification, amplification and osteoinduction are very important for the research of bone tissue engineering.
OBJECTIVE: To establish a simple and reliable in vitro cultivation and identification system of adult bone marrow mesenchymal stem cells, and to induce the mesenchymal stem cells to differentiate into osteoblasts.
METHODS: Bone marrow were extracted from adult anterior superior iliac, the density gradient centrifugation and adhesion method were used to isolate, purify, culture and amplify the bone marrow mesenchymal stem cells. Osteogenic medium was prepared by mixing appropriate amount of dexamethasone, β-glycerophosphate and ascorbic acid C. The cells were divided into osteoinduction group and blank control group for observation.
RESULTS AND CONCLUSION: Adult bone marrow mesenchymal stem cells were in typical long spindle-shape. The cells grew into rapid proliferation phase at 8-11 days and the growth curve was S-shape. CD44 and CD90 were in positive expression, while CD34 and CD45 were negative. The alkaline phosphatase activity was increased with culturing time prolonging, and reached the summit at the 12th day. The alkaline phosphatase activities of osteoinduction group were higher than those in the blank control group at different time points. These results suggested that in vitro cultivation, identification and osteoinduction system could obtain mesenchymal stem cells with high purity and good osteogenic differentiation capacity.

Key words: stem cells, cell differentiation, osteoblasts, tissue engineering

CLC Number:

R394.2

Zhang Ming-jie, Zhang Qing-wen, He Wei, Chen Zhen-qiu, Ou Zhi-xue, Jia Xiao-jun. In vitro cultivation, identification and osteoinduction of adult bone marrow
mesenchymal stem cells[J]. Chinese Journal of Tissue Engineering Research, doi: 10.3969/j.issn.2095-4344.2013.45.019.

Figures/Tables 11

References

[1] Granero-Molto F, Weis JA, Longobardi L, et al. Role of mesenchymal stem cells in regenerative medicine: application to bone and cartilage repair. Expert Opin Biol Ther. 2008;8(3):255-268.
[2] Kang SW,Lee JS,Park MS,et al.Enhancement of in vivo bone regeneration efficacy of human mesenchymal stem cells.J Microbiol Biotechnol.2008;18(5):975-982.
[3] Ciavarella S,De Matteo M,Cafforio P,et al.Mesenchymal stem cells and bone regeneration.Recenti Prog Med.2008;99(2): 75-82.
[4] Waese EY,Kandel RA,Stanford WL.Application of stem cells in bone repair.Skeletal Radiol.2008;37(7):601-608.
[5] Hernigou P,Beaujean F.Treatment of osteonecrosis with autologous bone marrow grafting.Clin Orthop Relat Res.2002; 405(12):14-23.
[6] 孙伟,李子荣,王佰亮,等.髓芯减压加自体骨髓干细胞移植治疗股骨头坏死的早期随访结果[J].中国组织工程研究与临床康复, 2008,12(12):2231-2234.
[7] 杨晓凤,王红梅,许忆峰,等.干细胞移植改善股骨头坏死缺血状态122例临床观察[J].中华外科杂志.2007,45(20):1428-1431.
[8] Solchaga LA, Tognana E, Penick K, et al. A rapid seeding technique for the assembly of large cell/scaffold composite constructs. Tissue Eng. 2006;12(7):1851-1863.
[9] Reger RL, Tucker AH, Wolfe MR. Differentiation and characterization of human MSCs. Methods Mol Biol. 2008; 449:93-107.
[10] Chung CH,Golub EE,Forbe E,et al.Mechanism of action of beta-glycerophosphate on bone cell mineralization.Calaif Tissue Int.1992;51(4):305-311.
[11] Bruder SP,Jaiswal N,Haynesworth SE.Growth kinetics,self-renewal,and the osteogenic potential of purified human mesenchymal stem cells during extensive subcultivation and following cryopreservation.J Cell Biochem. 1997;64(2):278-294.
[12] Tabata Y.Tissue regeneration based on tissue engineering technology.Congenit Anom(Kyoto).2004;44(3):111-124.
[13] Herbertson A,Aubin JE.Cell sorting enriches osteogenic populations in rat bone marrow stromal cell culture.Bone. 1997;21(6):491-500.
[14] Li X,Jin L,Cui Q,et al.Steroid effects on osteogenesis through mesenchymal cell gene expression.Osteoporos Int.2005; 16(1):101-108.
[15] White A,Lamb PW,Barrett JC.Frequent downregulation of the KAI1(CD82) metastasis suppressor protein in human cancer cell lines.Oncogene.1998;16(24):3143-3149.
[16] Abdallah BM,Jensen CH,Gutierrez G,et al.Regulation of human skeletal stem cells differentiation by Dlk1/Pref-1.J Bone Mineral Res. 2004;19(5):841-852.
[17] 孙伟,李子荣,王佰亮,等.磷酸三钙多孔生物陶瓷复合自体骨髓间充质干细胞修复股骨头坏死模型的超微结构观察[J].中国组织工程研究与临床康复,2009,13(3):457-460.
[18] 彭吾训,王蕾,邓进,等.一种组织工程骨修复兔股骨头坏死模型的效果评价[J].第三军医大学学报,2010,32(7):661-664.