血管基质混合物复合软骨细胞外基质源性微载体修复大鼠膝关节软骨缺损的实验研究
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摘要
背景:关节软骨的修复和再生能力很弱,利用软骨组织工程能更好地实现关节软骨的修复和再生。种子细胞、细胞载体作为组织工程的重要组成部分正在进行新的探索。目的:探讨自体脂肪组织来源的血管基质混合物(ASVF)复合软骨细胞外基质源性微载体(CEDPs)修复SD大鼠关节软骨缺损的可行性。方法:从大鼠腹股沟脂肪垫中分离获取ASVF,以湿法粉碎和脱细胞处理得到猪来源CEDPs,将两者混合,修复大鼠膝关节滑车直径2 mm全层软骨缺损。实验按移植物的不同分为空白组、CEDPs组、CEDPs+ASVF组。术后6周、12周取材,对标本进行Micro-CT分析,评估缺损去软骨下骨的重塑情况;标本切片后进行HE染色、甲苯胺蓝染色,以Wakitani评分进行组织学评估。结果:Micro-CT结果显示各组均有软骨下骨重塑,CEDPs+ASVF组的骨体积分数、骨小梁厚度均高于其他两组(P <0.05)。组织学分析显示CEDPs+ASVF组新生组织结构主要为透明软骨,其余两组为纤维组织与透明软骨混合分布,且CEDPs+ASVF组组织学评分高于其他两组。结论:自体ASVF复合CEDPs可用于修复SD大鼠膝关节全层软骨缺损,为修复关节软骨缺损提供了新的思路。
Background: Articular cartilage has a weak ability of repair and regeneration. The cartilage tissue engineering can better repair and regenerate articular cartilage. Seed cells and cell vectors, important parts of tissue engineering, are constantly developing. Objective: To explore the feasibility of autologous adipose-derived stromal vascular fraction(ASVF) in combination with cartilage ECM-derived particles(CEDPs) to repair articular cartilage defects in SD rats. Methods: ASVF was isolated from adipose tissue on the groin of SD rats. CEDPs were obtained by wet pulverization and decellularization. The mixture of ASVF and CEDPs was used to repair 2-mm diameter full-thickness cartilage defects, which were made in the femoral trochlea of SD rats. Rats were divided into three groups: blank control group, CEDPs group and CEDPs+ASVF group. At 6 and 12 weeks after operation, Micro-CT scanning, gross observation and histological evaluation were conducted to evaluate the cartilage repairs. Results: Micro-CT results showed that subchondral bone remodeling was seen in each group, and bone volume fraction and trabecular thickness in CEDPs+ASVF group were significantly higher than those in the other two groups(P<0.05). Histological analysis showed that the repaired tissue of CEDPs+ASVF group was mainly hyaline cartilage and the other two groups were mixed with fibrous tissue and hyaline cartilage. Histological score of CEDPs+ASVF group was higher than that in the other two groups. Conclusions: Autologous ASVF with CEDPs can be used to repair the articular cartilage defect in rats. This method is a promise strategy on regeneration of cartilage in the future.
Background: Articular cartilage has a weak ability of repair and regeneration. The cartilage tissue engineering can better repair and regenerate articular cartilage. Seed cells and cell vectors, important parts of tissue engineering, are constantly developing. Objective: To explore the feasibility of autologous adipose-derived stromal vascular fraction(ASVF) in combination with cartilage ECM-derived particles(CEDPs) to repair articular cartilage defects in SD rats. Methods: ASVF was isolated from adipose tissue on the groin of SD rats. CEDPs were obtained by wet pulverization and decellularization. The mixture of ASVF and CEDPs was used to repair 2-mm diameter full-thickness cartilage defects, which were made in the femoral trochlea of SD rats. Rats were divided into three groups: blank control group, CEDPs group and CEDPs+ASVF group. At 6 and 12 weeks after operation, Micro-CT scanning, gross observation and histological evaluation were conducted to evaluate the cartilage repairs. Results: Micro-CT results showed that subchondral bone remodeling was seen in each group, and bone volume fraction and trabecular thickness in CEDPs+ASVF group were significantly higher than those in the other two groups(P<0.05). Histological analysis showed that the repaired tissue of CEDPs+ASVF group was mainly hyaline cartilage and the other two groups were mixed with fibrous tissue and hyaline cartilage. Histological score of CEDPs+ASVF group was higher than that in the other two groups. Conclusions: Autologous ASVF with CEDPs can be used to repair the articular cartilage defect in rats. This method is a promise strategy on regeneration of cartilage in the future.
引文
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[21] Georgi N, van Blitterswijk C, Karperien M. Mesenchymal stromal/stem cell-or chondrocyte-seeded microcarriers as building blocks for cartilage tissue engineering. Tissue Eng Part A, 2014, 20(17-18):2513-2523.
[22] Pei M, He F, Li J, et al. Repair of large animal partial-thickness cartilage defects through intraarticular injection of matrix-rejuvenated synovium-derived stem cells. Tissue Eng Part A, 2013, 19(9-10):1144-1154.
[23] Song L, Baksh D, Tuan RS. Mesenchymal stem cell-based cartilage tissue engineering:cells, scaffold and biology. Cytotherapy, 2004, 6(6):596-601.
[24] Hong Y, Gao C, Xie Y, et al. Collagen-coated polylactide microspheres as chondrocyte microcarriers. Biomaterials, 2005,26(32):6305-6313.
[25] Sutherland AJ, Detamore MS. Bioactive microsphere-based scaffolds containing decellularized cartilage. Macromol Biosci, 2015,15(7):979-989.
[26] Guo J, Nguyen A, Banyard DA, et al. Stromal vascular fraction:A regenerative reality? Part 2:Mechanisms of regenerative action. J Plast Reconstr Aesthet Surg, 2016, 69(2):180-188.
[27] Nguyen A, Guo J, Banyard DA, et al. Stromal vascular fraction:A regenerative reality? Part 1:Current concepts and review of the literature. J Plast Reconstr Aesthet Surg, 2016, 69(2):170-179.
[28] Oberbauer E, Steffenhagen C, Wurzer C, et al. Enzymatic and non-enzymatic isolation systems for adipose tissue-derived cells:current state of the art. Cell Regen(Lond), 2015,4:7.
[2] Johnstone B, Alini M, Cucchiarini M, et al. Tissue engineering for articular cartilage repair--the state of the art. Eur Cell Mater, 2013, 25:248-267.
[3] Dell'accio F, Vincent TL. Joint surface defects:clinical course and cellular response in spontaneous and experimental lesions. Eur Cell Mater, 2010, 20:210-217.
[4] Komdrek J, Valis P, Repko M, et al. Treatment of deep cartilage defects of the knee with autologous chondrocyte transplantation:long-term results. Acta Chir Orthop Traumatol Cech,2010,77(4):291-295.
[5] Hurst JM, Steadman JR, O'Brien L, et al. Rehabilitation following microfracture for chondral injury in the knee. Clin Sports Med, 2010,29(2):257-265, viii.
[6] Bartha L, Vajda A, Duska Z, et al. Autologous osteochondral mosaicplasty grafting. J Orthop Sports Phys Ther, 2006, 36(10):739-750.
[7] Harris JD, Cavo M, Brophy R, et al. Biological knee reconstruction:a systematic review of combined meniscal allograft transplantation and cartilage repair or restoration. Arthroscopy, 2011, 27(3):409-418.
[8] Robert H. Chondral repair of the knee joint using mosaicplasty. Orthop Traumatol Surg Res, 2011,97(4):418-429.
[9] Pietschmann MF, Niethammer TR, Horng A, et al. The incidence and clinical relevance of graft hypertrophy after matrix-based autologous chondrocyte implantation. Am J Sports Med, 2012,40(1):68-74.
[10] Gaut C, Sugaya K. Critical review on the physical and mechanical factors involved in tissue engineering of cartilage.Regen Med, 2015, 10(5):665-679.
[11] Dahlin RL, Kinard LA, Lam J, et al. Articular chondrocytes and mesenchymal stem cells seeded on biodegradable scaffolds for the repair of cartilage in a rat osteochondral defect model. Biomaterials, 2014, 35(26):7460-7469.
[12] Brittberg M. Cell carriers as the next generation of cell therapy for cartilage repair:a review of the matrix-induced autologous chondrocyte implantation procedure. Am J Sports Med,2010,38(6):1259-1271.
[13] Dertinger S, S(o|¨)eder S, B(o|¨)sch H, et al. Matrix composition of cartilaginous anlagen in achondrogenesis typeⅡ(LangerSaldino). Front Biosci, 2005,10:446-453.
[14] Leijten JC, Georgi N, Wu L, et al. Cell sources for articular cartilage repair strategies:shifting from monocultures to cocultures. Tissue Eng Part B Rev, 2013, 19(1):31-40.
[15] Vieira NM, Brandalise V, Zucconi E, et al. Isolation, characterization, and differentiation potential of canine adipose-derived stem cells. Cell Transplant, 2010,19(3):279-289.
[16] Lin Y, Luo E, Chen X, et al. Molecular and cellular character-ization during chondrogenic differentiation of adipose tissuederived stromal cells in vitro and cartilage formation in vivo.J Cell Mol Med, 2005,9(4):929-939.
[17] Jang Y, Koh YG, Choi YJ, et al. Characterization of adipose tissue-derived stromal vascular fraction for clinical application to cartilage regeneration. In Vitro Cell Dev Biol Anim,2015,51(2):142-150.
[18] Jurgens WJ, Kroeze RJ, Bank RA, et al. Rapid attachment of adipose stromal cells on resorbable polymeric scaffolds facilitates the one-step surgical procedure for cartilage and bone tissue engineering purposes. J Orthop Res, 2011, 29(6):853-860.
[19] Semon JA, Zhang X, Pandey AC, et al. Administration of murine stromal vascular fraction ameliorates chronic experimental autoimmune encephalomyelitis. Stem Cells Transl Med,2013,2(10):789-796.
[20] Yin H, Wang Y, Sun Z, et al.Induction of mesenchymal stem cell chondrogenic differentiation and functional cartilage microtissue formation for in vivo cartilage regeneration by cartilage extracellular matrix-derived particles. Acta Biomater,2016, 33:96-109.
[21] Georgi N, van Blitterswijk C, Karperien M. Mesenchymal stromal/stem cell-or chondrocyte-seeded microcarriers as building blocks for cartilage tissue engineering. Tissue Eng Part A, 2014, 20(17-18):2513-2523.
[22] Pei M, He F, Li J, et al. Repair of large animal partial-thickness cartilage defects through intraarticular injection of matrix-rejuvenated synovium-derived stem cells. Tissue Eng Part A, 2013, 19(9-10):1144-1154.
[23] Song L, Baksh D, Tuan RS. Mesenchymal stem cell-based cartilage tissue engineering:cells, scaffold and biology. Cytotherapy, 2004, 6(6):596-601.
[24] Hong Y, Gao C, Xie Y, et al. Collagen-coated polylactide microspheres as chondrocyte microcarriers. Biomaterials, 2005,26(32):6305-6313.
[25] Sutherland AJ, Detamore MS. Bioactive microsphere-based scaffolds containing decellularized cartilage. Macromol Biosci, 2015,15(7):979-989.
[26] Guo J, Nguyen A, Banyard DA, et al. Stromal vascular fraction:A regenerative reality? Part 2:Mechanisms of regenerative action. J Plast Reconstr Aesthet Surg, 2016, 69(2):180-188.
[27] Nguyen A, Guo J, Banyard DA, et al. Stromal vascular fraction:A regenerative reality? Part 1:Current concepts and review of the literature. J Plast Reconstr Aesthet Surg, 2016, 69(2):170-179.
[28] Oberbauer E, Steffenhagen C, Wurzer C, et al. Enzymatic and non-enzymatic isolation systems for adipose tissue-derived cells:current state of the art. Cell Regen(Lond), 2015,4:7.