Repair of calvarial defect using a tissue-engineered bone with simvastatin-loaded β-tricalcium phosphate scaffold and adipose derived stem cells in rabbits

Abstract

Abstract: PURPOSE: The osteogenic-angiogenic differentiation effects of simvastatin (Sim) were explored on adipose tissue-derived stem cells (ASCs). A tissue-engineered bone with simvastatin loaded β-tricalcium phosphate (β-TCP) scaffold and ASCs was constructed to repair the calvarial defect in rabbits. METHODS：ASCs were obtained from the groin of rabbits. After 14 days of osteogenic inducing culture, sufficient cells were expanded for the following experiments. Cell counting was conducted to ASCs in osteogenic inducing medium containing 0, 0.01, 0.1 and 1 μmol/L simvastatin. Concentrations of 0.05 and 0.1 μmol/L simvastatin were administrated to ASCs for real-time PCR of angiogenesis-osteogenesis related genes like RUNX2, OPN, OCN, and VEGF on day 1, 7. ALP staining was performed on day 7, Alizarin red staining for calcium deposits was carried out on day 14. Bilateral critical-sized defects were created on 12 New Zealand rabbits. Four groups of tissue-engineered bone were randomly allocated to them. Group A: β-tricalcium phosphate (β-TCP) (n=6); group B: β-TCP/Cell (n=6); group C: β-TCP/Sim (n=6); group D: β-TCP/Cell/Sim (n=6). Specimens were decalcified and stained by HE 8 weeks after operation. The data was statistically analyzed using SPSS 17.0 software package. RESULTS: The use of simvastatin with the concentration of 0.05 μmol/L enhanced the expression of angiogenic-osteogenic related genes like RUNX2, OPN, OCN, and VEGF. ALP activity and von Kossa were significantly stronger in osteogenic inducing medium containing 0.05 μmol/L simvastatin. The new bone formation area of β-TCP/Cell/Sim group at 8-week after implantation was significantly larger than the other groups. CONCLUSIONS: 0.05 μmol/L simvastatin enhances the angiogenic-osteogenic differentiation of ASCs. Simvastatin loaded β-TCP scaffold and ASCs successfully repair the calvarial defect in rabbits. These results indicate a promising future in application of simvastatin for bone regeneration.

Key words: Adipose-derived stem cells, Simvastatin, β-tricalcium phosphate, Tissue-engineered bone, Rabbit

CLC Number:

R318.08

XU Lian-yi, SUN Xiao-juan, ZHANG Xiu-li, JIN Yu-qin, WU Yu-qiong, JIANG Xin-quan. Repair of calvarial defect using a tissue-engineered bone with simvastatin-loaded β-tricalcium phosphate scaffold and adipose derived stem cells in rabbits[J]. Shanghai Journal of Stomatology, 2013, 22(4): 361-367.

References

[1] Zhou M, Peng X, Mao C, et al. Primate mandibular reconstruction with prefabricated, vascularized tissue-engineered bone flaps and recombinant human bone morphogenetic protein-2 implanted in situ[J]. Biomaterials, 2010, 31(18): 4935-4943.
[2] Wikesj? UM, Polimeni G, Qahash M. Tissue engineering with recombinant human bone morphogenetic protein-2 for alveolar augmentation and oral implant osseointegration: experimental observations and clinical perspectives[J]. Clin Implant Dent Relat Res, 2005, 7(2): 112-119.
[3] Du Z, Chen J, Yan F, et al. Effects of Simvastatin on bone healing around titanium implants in osteoporotic rats[J]. Clin Oral Implants Res, 2009, 20(2): 145-150.
[4] Song C, Guo Z, Ma Q, et al. Simvastatin induces osteoblastic differentiation and inhibits adipocytic differentiation in mouse bone marrow stromal cells[J]. Biochem Biophys Res Commun, 2003, 308(3): 458-462.
[5] van Staa TP, Wegman S, de Vries F, et al. Use of statins and risk of fractures[J]. JAMA, 2001, 285(14): 1850-1855.
[6] 万光勇, 蔡景龙, 张明宾. 人脂肪干细胞体外向成骨细胞定向诱导分化的实验研究[J]. 上海口腔医学, 2008, 17(6): 652-658.
[7] Wang S, Zhang Z, Xia L, et al. Systematic evaluation of a tissue-engineered bone for maxillary sinus augmentation in large animal canine model[J]. Bone, 46(1): 91-100.
[8] 李沁华, 王迪. 冻干法制备的海藻酸钙-壳聚糖组织工程复合材料支架[J]. 中国组织工程研究, 2012, 16(29): 5441-5444.
[9] Nyan M, Sato D, Kihara H, et al. Effects of the combination with α-tricalcium phosphate and simvastatin on bone regeneration[J]. Clin Oral Implants Res, 2009, 20(3): 280-287.
[10] Liu C, Wu Z, Sun HC. The effect of simvastatin on mRNA expression of transforming growth factor-β1, bone morphogenetic protein-2 and vascular endothelial growth factor in tooth extraction socket[J]. Int J Oral Sci, 2009, 1(2): 90-98.
[11] Chen PY, Sun JS, Tsuang YH, et al. Simvastatin promotes osteoblast viability and differentiation via Ras/Smad/Erk/BMP-2 signaling pathway[J]. Nutr Res, 2010, 30(3): 191-199.
[12] Takenaka M, Hirade K, Tanabe K, et al. Simvastatin stimulates VEGF release via p44/p42 MAP kinase in vascular smooth muscle cells[J]. Biochem Biophys Res Commun, 2003, 301(1): 198-203.
[13] Siddiqui AJ, Gustafsson T, Fischer H, et al. Simvastatin enhances myocardial angiogenesis induced by vascular endothelial growth factor gene transfer[J]. J Mol Cell Cardiol, 2004, 37(6): 1235-1244.
[14] Zuk PA, Zhu M, Mizuno H, et al. Multilineage cells from human adipose tissue: implications for cell-based therapies[J]. Tissue Eng, 2001, 7(2): 211-228.
[15] Kim WS, Park BS, Sung JH, et al. Wound healing effect of adipose-derived stem cells: a critical role of secretory factors on human dermal fibroblasts[J]. J Dermatol Sci, 2007, 48(1): 15-24.
[16] Xu Y, Malladi P, Wagner DR, et al. Adipose-derived mesenchymal cells as a potential cell source for skeletal regeneration[J]. Curr Opin Mol Ther, 2005, 7(4): 300-305.
[17] Stock P, Staege MS, Müller LP, et al. Hepatocytes derived from adult stem cells[J]. Transplant Proc, 2008, 40(2): 620-623.
[18] Safford KM, Hicok KC, Safford SD, et al. Neurogenic differentiation of murine and human adipose-derived stromal cells[J]. Biochem Biophys Res Commun, 2002, 294(2): 371-379.
[19] Lee JH, Kemp DM. Human adipose-derived stem cells display myogenic potential and perturbed function in hypoxic conditions[J]. Biochem Biophys Res Commun, 2006, 341(3): 882-888.
[20] Bai X, Alt E. Myocardial regeneration potential of adipose tissue-derived stem cells[J]. Biochem Biophys Res Commun,2010,401(3): 321-326.