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Preparation and identification of the periosteal decellularized bioscaffold
CHEN Kai NI Jin-hu LI Jian-min JIN Ke-ke MA Yu-jie ZHANG Qi YE Yi-heng WANG Yang CHEN Lei*
Acta Anatomica Sinica ›› 2015, Vol. 46 ›› Issue (2) : 275-281.
Preparation and identification of the periosteal decellularized bioscaffold
Objective To prepare rabbit periosteal decellularized bioscaffold andprovide a natural bioscaffold for the treatment for bone defect or bone nonunion in bone tissue engineering. Methods Bilateral medial proximal tibia periosteum of healthy New Zealand rabbits was obtained. To prepare the scaffold, we used physical freeze thawing (-80℃, 24h), eluted detergent (triton-X 100, SDS) and enzyme digestion (DNA enzymes, RNA enzymes ). After decellularization process, normal periosteum and decellularized periosteum were examined by HE staining， 4’,6-diamidino-phenylindole(DAPI) staining, agarose gel electrophoresis and quantitative analysis of genomic DNA (n=5) to evaluate the residual cell components. The retention of main components of the extracellular matrix (collagen) was examined by Masson staining and the hydroxyproline measurement (n=6); Scanning electron microscopy (SEM) was used to observe the microstructure of the scaffold; The toxicity of leaching liquor from scaffolds was tested by CCK8 assay; The immunological rejection of the scaffold was evaluated by subcutaneous implantation (n=4). Results HE and DAPI staining showed that no cells remained in the scaffold. After separation, the visible DNA bands were not found in the agarose electrophoresis gel for the decellularized periosteum. The DNA quantitative analysis showed that more than 95% of periosteal cells were removed; Masson staining and hydroxyproline measurement revealed that the collagen of the extracellular matrix was preserved; SEM showed the loose three-dimensional network of the extracellular matrix. The CCK8 assays demonstrated that there was no significant difference of periosteal cells proliferation among different volume fractions of leaching liquor from scaffolds and the control group (normal medium ) (P＞0.05). The subcutaneous implantation showed no obvious immune response to the decellularized periosteum. Conclusion The decellularized periosteum obtained by the use of physical freeze thawing, eluted detergent and enzyme digestion methods was found to remove periosteal cells completely, while the extracellular matrix structure and the main components were well-preserved and the biocompatibility was excellent.
Decellularization / Periosteum / Collagen / Biocompatiblilty / Subcutaneous implantation / Masson staining / Scanning electron microscopy / Rabbit
［1］Zhang X, Awad HA, O’Keefe RJ, et al. A perspective: engineering periosteum for structural bone graft healing［J］. Clin Orthop Relat Res, 2008, 466(8):1777-1787.
［2］Cuthbert RJ, Churchman SM, Tan HB, et al. Induced periosteum a complex cellular scaffold for the treatment of large bone defects［J］. Bone, 2013, 57(2):484-492.
［3］Zhang ZhX, Liu LX. Therapeutic effect of the self-periosteum transplantation in healing of fracture［J］. China Journal of Orthopaedics and Traumatology, 2005, 18(2):117. (in Chinese)
张忠信, 刘兰秀. 自体骨膜移植促进骨折愈合的疗效观察［J］. 中国骨伤, 2005, 18(2):117.
［4］Ryu YM, Hah YS, Park BW, et al. Osteogenic differentiation of human periosteal-derived cells in a three-dimensional collagen scaffold［J］. Mol Biol Rep, 2011, 38(5):2887-2894.
［5］Zhao L, Zhao J, Wang S, et al. Evaluation of immunocompatibility of tissue-engineered periosteum［J］. Biomed Mater, 2011, 6(1):015005.
［6］Gilbert TW, Sellaro TL, Badylak SF. Decellularization of tissues and organs［J］. Biomaterials, 2006, 27(19):3675-3683.
［7］Gilbert TW. Strategies for tissue and organ decellularization［J］. J Cell Biochem, 2012, 113(7):2217-2222.
［8］Benders KE, van Weeren PR, Badylak SF, et al. Extracellular matrix scaffolds for cartilage and bone regeneration［J］. Trends Biotechnol, 2013, 31(3):169-176.
［9］Wainwright DJ. Use of an acellular allograft dermal matrix (AlloDerm) in the management of full-thickness burns［J］. Burns, 1995, 21(4):243-248.
［10］Lin XF, Shao YK, Wang H, et al. Preparation and identification of the pancreatic decellularized bio-derived scaffold in isolated rats［J］. Acta Anatomica Sinica, 2012, 43(5):717-722. (in Chinese)
林贤丰, 邵营宽, 王辉，等. 离体大鼠胰腺去细胞生物支架的制备与鉴定［J］. 解剖学报, 2012, 43(5):717-722.
［11］Zhang JS, Wang H, Shao PG, et al. Preparation and identification of a whole kidney decellularized bio-derived scaffold［J］. Acta Anatomica Sinica, 2012, 43(2):253-257. (in Chinese)
张建色, 王辉, 邵培刚, 等. 去细胞全肾生物支架的制备与鉴定［J］. 解剖学报, 2012, 43(2):253-257.
［12］Bilkay U, Tokat C, Helvaci E, et al. Osteogenic capacities of tibial and cranial periosteum: a biochemical and histologic study［J］. J Craniofac Surg, 2008, 19(2):453-458.
［13］Lin Z, Fateh A, Salem DM, et al. Periosteum: biology and applications in craniofacial bone regeneration［J］. J Dent Res, 2014, 93(2):109-116.
［14］Colnot C, Zhang X, Knothe Tate ML. Current insights on the regenerative potential of the periosteum: molecular, cellular, and endogenous engineering approaches［J］. J Orthop Res, 2012, 30(12):1869-1878.
［15］Keating JF, Simpson AH, Robinson CM. The management of fractures with bone loss［J］. J Bone Joint Surg Br, 2005, 87(2):142-150.
［16］Allen MR, Hock JM, Burr DB. Periosteum: biology, regulation, and response to osteoporosis therapies［J］. Bone, 2004, 35(5):1003-1012.
［17］Squier CA, Ghoneim S, Kremenak CR. Ultrastructure of the periosteum from membrane bone［J］. J Anat, 1990, 171:233-239.
［18］Kanou M, Ueno T, Kagawa T, et al. Osteogenic potential of primed periosteum graft in the rat calvarial model［J］. Ann Plast Surg, 2005, 54(1):71-78.
［19］Chen AC, Lin SS, Chan YS, et al. Osteogenesis of prefabricated vascularized periosteal graft in rabbits［J］. J Trauma, 2009, 67(1):165-167.
［20］Langer R, Vacanti JP. Tissue engineering［J］. Science, 1993, 260(5110):920-926.
［21］Dawson E, Mapili G, Erickson K, et al. Biomaterials for stem cell differentiation［J］. Adv Drug Deliv Rev, 2008, 60(2):215-228.
［22］Zhang J, Li L. Stem cell niche: microenvironment and beyond［J］. J Biol Chem, 2008, 283(15):9499-9503.
［23］Crapo PM, Gilbert TW, Badylak SF. An overview of tissue and whole organ decellularization processes［J］. Biomaterials, 2011, 32(12):3233-3243.
［24］Song JJ, Ott HC. Organ engineering based on decellularized matrix scaffolds［J］. Trends Mol Med, 2011, 17(8):424-432.
［25］Shi S, Kirk M, Kahn AJ. The role of type I collagen in the regulation of the osteoblast phenotype［J］. J Bone Miner Res, 1996, 11(8):1139-1145.
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