Biocompatibility of bone marrow stromal stem cells with vascular endothelial growth factor/bone morphogenetic protein 2-poly(lactic-co-glycolic acid) copolymer/gelatin nanofibrous scaffold

doi:10.3969/j.issn.2095-4344.2017.33.005

Abstract

Abstract:

BACKGROUND: Using bone tissue engineering methods for reconstruction of bone repair is an ideal treatment for bone defects, and it is crucial to combine biological scaffolds carrying growth factors with seed cells.
OBJECTIVE: To observe the biocompatibility of rat bone marrow stromal stem cells (BMSCs) seeded onto vascular endothelial growth factor (VEGF)/bone morphogenetic protein 2 (BMP-2)-poly(lactic-co-glycolic acid) (PLGA) copolymer/ gelatin nanofibrous scaffolds.
METHODS: Rat BMSCs were divided into five groups: blank scaffold group, BMP-2 group, VEGF group, VEGF/BMP-2 groups (1.2:1, 0.8:1, 0.4:1). In each group, the nanofibrous scaffold was placed at the bottom of the cell culture plate, and then rat BMSCs were seeded into the culture plate. Cell adhesion was observed under scanning electron microscope; cell proliferation was detected by cell counting kit-8; and ALP, RUNX-2 and OCN expression was determined by RT-PCR.
RESULTS AND CONCLUSION: Under the scanning electron microscope, the BMSCs adhered to the scaffold and further grew into the scaffold. The adherent cells grew best in the 0.4:1 group, in which the cells were tightly integrated with the scaffold to form a cell-scaffold complex. Compared with the blank scaffold group, the cell proliferation and expression of ALP, RUNX-2 and OCN were both higher in the other groups, which was highest in the 0.4:1 group. To conclude, the VEGF/BMP-2-loaded PLGA copolymer/gelatin nanofibrous scaffold can promote cell adhesion, proliferation, osteogenic differentiation of rat BMSCs because of a good cytocompatibility. Moreover, the concentration ratio of VEGF/BMP-2 is optimized at 0.4:1.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Bone Morphogenetic Proteins, Vascular Endothelial Growth Factors, Tissue Scaffolds, Bone Marrow, Mesenchymal Stem Cells, Tissue Engineering

CLC Number:

R394.2

An Gang, Lu Bin, Zhang Wen-bo, Jiang Yu-dong. Biocompatibility of bone marrow stromal stem cells with vascular endothelial growth factor/bone morphogenetic protein 2-poly(lactic-co-glycolic acid) copolymer/gelatin nanofibrous scaffold[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(33): 5274-5279.

Figures/Tables 1

2.1 大鼠骨髓基质干细胞的形态及表面抗原的表达原代培养细胞呈圆形漂浮，培养3 d时细胞呈现长梭形以及多角形改变。随着培养时间延长，可见贴壁细胞增多，形状呈梭形，细胞成簇融合，单层生长，细胞排列多数呈现漩涡状，见图1。流式细胞术检测骨髓基质干细胞CD44、CD29、CD90表达率分别为(93.55±0.27)%、(94.68±0.55)%、(18.79± 2.30)%；CD45、CD34表达率分别为(2.30±0.12)%、(8.70± 0.25)%。根据细胞表面的抗原表达情况，可以确认所分离培养的细胞为骨髓基质干细胞。 2.2 骨髓基质干细胞在纳米纤维支架上的黏附生长大鼠骨髓基质干细胞种植于纳米纤维支架上，通过扫描电镜观察(图2)，均可见细胞在纤维支架上黏附，形态呈梭形，支架内部也有大量的细胞黏附和生长，向周围伸出伪足，细胞周围可见白色颗粒状、丝状基质物质，说明纳米纤维支架的孔隙率较大，有利于细胞进入支架内部生长，而不仅仅是在支架的表面生长，使得细胞可以稳定的附着于支架上。由图3可见，空白支架组细胞散在分布于纤维支架上，主要为未黏附状态的球形细胞，细胞之间的相互联系少，细胞呈独立状态。VEGF组纤维支架上的扁平状细胞增多，可见一定程度的细胞极性表现，支架表面及内部均可见细胞长入，细胞间相互联系增多。BMP-2组细胞形态呈梭形改变，细胞表面可见颗粒物质并且表面的丝状物质与纤维支架的连接更多。VEGF/BMP-2(1.2︰1)组细胞的数量及细胞之间的连接均少于BMP-2组。VEGF/BMP-2 (0.8︰1)组和VEGF/BMP-2(0.4︰1)组细胞广泛分布在支架内部以及表面，分泌的细胞外基质更多，细胞向纤维支架上伸出更多的丝状伪足，紧密连接。VEGF/BMP-2(0.4︰1)组的细胞黏附生长状态最好，细胞与支架紧密结合成为支架细胞复合体。 2.3 不同纳米纤维支架对骨髓基质干细胞增殖生长的影响由图4可见，在培养第7，14，21天，相对于空白支架组，负载生长因子各组吸光度值均明显增高(P < 0.05)，表明支架有促进细胞增殖的作用；在负载生长因子各组中单纯负载VEGF组细胞计数最低，BMP-2组和VEGF/ BMP-2各实验组在第3天时细胞数量差异无显著性意义，但培养7 d之后各组之间细胞增殖数量差异有显著性意义，随着培养时间的延长，这种差异越明显。VEGF/ BMP-2(0.8︰1)组及VEGF/BMP-2(0.4︰1)组的吸光度值在7，14，21 d时均明显高于BMP-2组(P < 0.05)，VEGF/ BMP-2(0.4︰1)组的细胞增殖数量最高。 2.4 RUNX-2、ALP、OCN基因表达由图5可见，在第7天成骨分化的早期，RUNX-2及ALP成骨基因在BMP-2组、VEGF组以及VEGF/BMP-2各实验组的表达明显高于空白对照组，其中VEGF组的表达低于其他各实验组。OCN基因表达各组之间差异无显著性意义。在成骨诱导分化的第14天，VEGF/BMP-2(0.8︰1)组及VEGF/BMP-2(0.4︰1)组的RUNX-2及ALP基因的表达明显高于其他各组，但是VEGF/BMP-2(1.2︰1)组的上述2种基因的表达水平均低于BMP-2组。OCN基因在BMP-2组、VEGF/BMP-2(0.8︰1)组和VEGF/BMP-2(0.4︰1)组中表达开始明显增高，其中VEGF/BMP-2(0.4︰1)组的OCN基因表达最高。在成骨诱导分化的第21天，各组RUNX-2及ALP的基因表达水平均呈现出下降趋势。OCN基因的表达呈现增高趋势。"

References

[1] Gruskin E, Doll BA, Futrell FW, et al. Demineralized bone matrix in bone repair: history and use. Adv Drug Deliv Rev. 2012;64(12):1063-1077.

[2] Bohner M. Resorbable biomaterials as bone graft substitutes. Materials Today, 2010;13(1-2):24-30.

[3] Hubbell JA. Biomaterials in tissue engineering. Biotechnology (N Y). 1995;13(6):565-576.

[4] Geethaa M, Singh AK, Asokamani R, et al. Ti based biomaterials, the ultimate choice for orthopaedic implants – A review. Progress in Materials Science. 2009;54(3):397-425.

[5] Kanczler JM, Ginty PJ, White L, et al. The effect of the delivery of vascular endothelial growth factor and bone morphogenic protein-2 to osteoprogenitor cell populations on bone formation. Biomaterials. 2010;31(6):1242-1250.

[6] An G, Zhang WB, Ma DK, et al. Influence of VEGF/BMP-2 on the proliferation and osteogenetic differentiation of rat bone mesenchymal stem cells on PLGA/gelatin composite scaffold. Eur Rev Med Pharmacol Sci. 2017;21(10):2316-2328.

[7] Deckers MM, van Bezooijen RL, van der Horst G, et al. Bone morphogenetic proteins stimulate angiogenesis through osteoblast-derived vascular endothelial growth factor A. Endocrinology. 2002;143(4):1545-1453.

[8] Seong JM, Kim BC, Park JH, et al. Stem cells in bone tissue engineering. Biomed Mater. 2010;5(6):062001.

[9] Yoshimoto H, Shin YM, Terai H, et al. A biodegradable nanofiber scaffold by electrospinning and its potential for bone tissue engineering. Biomaterials. 2003;24(12):2077-2082.

[10] Webster TJ, Waid MC, Mckenzie JL,et al. Nano-biotechnology: carbon nanofibres as improved neural and orthopaedic implants. Nanotechnology. 2004; 15(1): 48-54.

[11] Woo KM, Chen VJ, Ma PX. Nano-fibrous scaffolding architecture selectively enhances protein adsorption contributing to cell attachment. J Biomed Mater Res A. 2003;67(2):531-537.

[12] Binulal NS, Deepthy M, Selvamurugan N, et al. Role of nanofibrous poly(caprolactone) scaffolds in human mesenchymal stem cell attachment and spreading for in vitro bone tissue engineering--response to osteogenic regulators. Tissue Eng Part A. 2010;16(2):393-404.

[13] Wang J, Ma H, Jin X, et al. The effect of scaffold architecture on odontogenic differentiation of human dental pulp stem cells. Biomaterials. 2011;32(31):7822-7830.

[14] Bianco P, Robey PG. Stem cells in tissue engineering. Nature. 2001;414(6859):118-121.

[15] Cui F, Wang X, Liu X, et al. VEGF and BMP-6 enhance bone formation mediated by cloned mouse osteoprogenitor cells. Growth Factors. 2010;28(5):306-317.

[16] Kanczler JM, Ginty PJ, White L, et al. The effect of the delivery of vascular endothelial growth factor and bone morphogenic protein-2 to osteoprogenitor cell populations on bone formation. Biomaterials. 2010;31(6):1242-1250.

[17] Bauters C, Asahara T, Zheng LP, et al. Site-specific therapeutic angiogenesis after systemic administration of vascular endothelial growth factor. J Vasc Surg. 1995;21(2): 314-324.

[18] Peng H, Wright V, Usas A, et al. Synergistic enhancement of bone formation and healing by stem cell-expressed VEGF and bone morphogenetic protein-4. J Clin Invest. 2002;110(6): 751-759.

[19] Li G, Corsi-Payne K, Zheng B, et al. The dose of growth factors influences the synergistic effect of vascular endothelial growth factor on bone morphogenetic protein 4-induced ectopic bone formation.Tissue Eng Part A. 2009;15(8): 2123-2133.

[20] Liu TM, Lee EH. Transcriptional regulatory cascades in Runx2-dependent bone development. Tissue Eng Part B Rev. 2013;19(3):254-263.

[21] Liu W, Toyosawa S, Furuichi T, et al. Overexpression of Cbfa1 in osteoblasts inhibits osteoblast maturation and causes osteopenia with multiple fractures. J Cell Biol. 2001;155(1): 157-166.

[22] Neve A, Corrado A, Cantatore FP. Osteocalcin: skeletal and extra-skeletal effects. J Cell Physiol. 2013;228(6):1149-1153.

[23] Peng H, Wright V, Usas A, et al. Synergistic enhancement of bone formation and healing by stem cell-expressed VEGF and bone morphogenetic protein-4. J Clin Invest. 2002;110(6): 751-759.

[24] Li G, Corsi-Payne K, Zheng B, et al. The dose of growth factors influences the synergistic effect of vascular endothelial growth factor on bone morphogenetic protein 4-induced ectopic bone formation. Tissue Eng Part A. 2009;15(8): 2123-2133.