EVALUATION OF AN OPTIMIZING PROTOCOL FOR FABRICATING A SCAFFOLD DERIVED FROM PORCINE SKELETAL MUSCLE EXTRACELLULAR MATRIX_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

ZHANGDi ^1,2 , TANQiuwen ^1,2 , TANGShenli ^1,2 , LUOJingcong ² , ZHANGYi ² ,  LÜQing ¹

1. Center of Breast Surgery, West China Hospital, Sichuan University, Chengdu Sichuan, 610041, P. R. China;
2. State Key Laboratory of Biotherapy, Division of Stem Cell and Tissue Engineering, West China Hospital, Sichuan University, Chengdu Sichuan, 610041, P. R. China;

Corresponding author：

LÜQing, Email: huaxilvqing@163.com

Keywords：

Tissue engineering; Scaffold; Skeletal muscle; Decellularized matrix; Biocompatibility; Neovascularization

DOI：

10.7507/1002-1892.20160261

Video：

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Objective To explore an optimized protocol of decellularization to fabricate an ideal scaffold derived from porcine skeletal muscle acellular matrix. Methods Serial-step protocol of homogenating-milling-detergent method was used to fabricate decellularized porcine muscle tissue (DPMT) derived from native porcine skeletal muscle tissue from adult pig waist. Histological method was used to assess the effects of decellularization and degreasing. Sirius red staining was used to analyze collagen components. Scanning electron microscopy, BCA assay, and PicoGreen assay were used to evaluate the ultrastructure, total protein content, and DNA content in DPMT. The adipose derived stem cells (ADSCs), NIH3T3 cells, and human umbilical vein endothelial cells (HUVECs) were cultured in extraction liquor of DPMT in different concentrations for 1, 3, and 5 days, then the relative growth rate was calculated with cell counting kit 8 to assess the toxicity in vitro. Live/dead cell staining was used to evaluate the cytocompatibility by seeding HUVECs on the surface of DPMT and co-cultured in vitro for 3 days. For in vivo test, DPMT was subcutaneously implanted at dorsal site of male specific-pathogen free Sprague Dawley rats and harvested after 3, 7, 14, and 28 days. Gross obersvation was done and transverse diameter of remained DPMT in vivo was determined. HE staining and immunohistochemical staining of CD31 were used to assess inflammatory response and new capillary rings formation. Results Decellularization of the porcine skeletal muscle tissue by homogenating-milling-detergent serial steps protocol was effective, time-saving, and simple, which could be finished within only 1 day. The decellularizarion and degreasing effect of DPMT was complete. The main component of DPMT was collagen type I and type IV. The DNA content in DPMT was (15.902±1.392) ng/mg dry weight, the total protein content was 68.94% of DPMT dry weight, which was significantly less than those of fresh skeletal muscle tissue[(140.727±10.422) ng/mg and 93.14%] (P<0.05). The microstructure of DPMT was homogeneous and porous. The result of cytocompatibility revealed that the cytotoxicity of DPMT was 0-1 grade, and HUVECs could stably grow on DPMT. In vivo study revealed DPMT could almost maintain its structural integrity at 14 days and it degraded completely at 28 days after implantation. The inflammatory response peaked at 3 days after implantation, and reduced obviously at 7 days. Difference was significant in the number of inflammatory cells between 2 time points (P<0.05). Neovascularization was observed at 7 days after implantation and the number of new vessels increased at 14 days, showing significant difference between at 7 and 14 days (P<0.05). Conclusion The homogenating-milling-detergent serial-steps protocol is effective, time-saving, and reproducible. The DPMT reveals to be cell and lipid free, with highly preserved protein component. DPMT has good biocompatibility both in vitro and in vivo and may also have potential in promoting neovascularization.

Citation： ZHANG Di, TAN Qiuwen, TANG Shenli, LUO Jingcong, ZHANG Yi, LÜ Qing. EVALUATION OF AN OPTIMIZING PROTOCOL FOR FABRICATING A SCAFFOLD DERIVED FROM PORCINE SKELETAL MUSCLE EXTRACELLULAR MATRIX. Chinese Journal of Reparative and Reconstructive Surgery, 2016, 30(10): 1282-1289. doi: 10.7507/1002-1892.20160261 Copy

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16.	Keane TJ, Badylak SF. The host response to allogeneic and xenogeneic biological scaffold materials. J Tissue Eng Regen Med, 2015, 9(5):504-511.
17.	Keane TJ, Swinehart IT, Badylak SF. Methods of tissue decellularization used for preparation of biologic scaffolds and in vivo relevance. Methods, 2015, 84:25-34.
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20.	Vindigni V, Mazzoleni F, Rossini K, et al. Reconstruction of ablated rat rectus abdominis by muscle regeneration. Plast Reconstr Surg, 2004, 114(6):1509-1515.
21.	Zhang XY, Xue H, Liu JM, et al. Chemically extracted acellular muscle:a new potential scaffold for spinal cord injury repair. J Biomed Mate Res A, 2012, 100(3):578-587.
22.	Lin CH, Yang JR, Chiang NJ, et al. Evaluation of decellularized extracellular matrix of skeletal muscle for tissue engineering. Int J Artif Organs, 2014, 37(7):546-555.
23.	Mligiliche N, Kitada, M, Ide C. Grafting of detergent-denatured skeletal muscles provides effective conduits for extension of regenerating axons in the rat sciatic nerve. Arch Histol Cytol, 2001, 64(1):29-36.
24.	卿泉, 秦廷武. 大鼠骨骼肌脱细胞处理方法的优化研究. 中国修复重建外科杂志, 2009, 23(7):836-839.

1. 陈万青, 郑荣寿, 张思维, 等. 2012年中国恶性肿瘤发病和死亡分析. 中国肿瘤, 2016, 25(1):1-8.
2. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2015. CA Cancer J Clin, 2015, (1):5-29.
3. ASPS. 2015 Plastic Surgery Statistics Report. http://www.plasticsurgery.org/news/plastic-surgery-statistics.
4. 熊炳钧, 谭秋雯, 吕青. 自体脂肪移植在乳房修复重建中的应用与研究进展. 中国修复重建外科杂志, 2016, 30(1):123-128.
5. Maxwell GP, Gabriel A. Bioengineered breast:concept, technique, and preliminary results. Plast Reconstr Surg, 2016, 137(2):415-421.
6. Fu Y, Fan X, Tian C, et al. Decellularization of porcine skeletal muscle extracellular matrix for the formulation of a matrix hydrogel:a preliminary study. Journal of Cellular and Molecular Medicine, 2016, 20(4):740-749.
7. 田春祥, 范雪娇, 陈晓禾, 等. 骨骼肌无细胞基质的制备及其生物相容性研究. 中国修复重建外科杂志, 2012, 26(6):749-754.
8. 中华人民共和国国家质量监督检验检疫总局中国国家标准化管理委员会. GB/T 16886.12-2005/ISO 10993-12:2002 中华人民共和国国家标准:医疗器械生物学评价第12部分:样品制备与参照样品. 北京:中国标准出版社, 2005.
9. Reing JE, Brown BN, Daly KA, et al. The effects of processing methods upon mechanical and biologic properties of porcine dermal extracellular matrix scaffolds. Biomaterials, 2010, 31(33):8626-8633.
10. Lu P, Takai K, Weaver VM, et al. Extracellular matrix degradation and remodeling in development and disease. Cold Spring Harb Perspect Biol, 2011, 3(12):pii:a005058.
11. Pradhan S, Farach-Carson MC. Mining the extracellular matrix for tissue engineering applications. Regen Med, 2010, 5(6):961-970.
12. Brown BN, Badylak SF. Extracellular matrix as an inductive scaffold for functional tissue reconstruction. Transl Res, 2014, 163(4):268-285.
13. Burk J, Plenge A, Brehm W, et al. Induction of tenogenic differentiation mediated by extracellular tendon matrix and short-term cyclic stretching. Stem Cells Int, 2016, 2016:7342379.
14. Wang W, Zhang X, Chao NN, et al. Preparation and characterization of pro-angiogenic gel derived from small intestinal submucosa. Acta Biomater, 2016, 29:135-148.
15. Greco KV, Francis L, Somasundaram M, et al. Characterisation of porcine dermis scaffolds decellularised using a novel non-enzymatic method for biomedical applications. J Biomater Appl, 2015, 30(2):239-253.
16. Keane TJ, Badylak SF. The host response to allogeneic and xenogeneic biological scaffold materials. J Tissue Eng Regen Med, 2015, 9(5):504-511.
17. Keane TJ, Swinehart IT, Badylak SF. Methods of tissue decellularization used for preparation of biologic scaffolds and in vivo relevance. Methods, 2015, 84:25-34.
18. Crapo PM, Gilbert TW, Badylak SF. An overview of tissue and whole organ decellularization processes. Biomaterials, 2011, 32(12):3233-3243.
19. Wolf MT, Daly KA, Reing JE, et al. Biologic scaffold composed of skeletal muscle extracellular matrix. Biomaterials, 2012, 33(10):2916-2925.
20. Vindigni V, Mazzoleni F, Rossini K, et al. Reconstruction of ablated rat rectus abdominis by muscle regeneration. Plast Reconstr Surg, 2004, 114(6):1509-1515.
21. Zhang XY, Xue H, Liu JM, et al. Chemically extracted acellular muscle:a new potential scaffold for spinal cord injury repair. J Biomed Mate Res A, 2012, 100(3):578-587.
22. Lin CH, Yang JR, Chiang NJ, et al. Evaluation of decellularized extracellular matrix of skeletal muscle for tissue engineering. Int J Artif Organs, 2014, 37(7):546-555.
23. Mligiliche N, Kitada, M, Ide C. Grafting of detergent-denatured skeletal muscles provides effective conduits for extension of regenerating axons in the rat sciatic nerve. Arch Histol Cytol, 2001, 64(1):29-36.
24. 卿泉, 秦廷武. 大鼠骨骼肌脱细胞处理方法的优化研究. 中国修复重建外科杂志, 2009, 23(7):836-839.

Chinese Journal of Reparative and Reconstructive Surgery

EVALUATION OF AN OPTIMIZING PROTOCOL FOR FABRICATING A SCAFFOLD DERIVED FROM PORCINE SKELETAL MUSCLE EXTRACELLULAR MATRIX

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