软骨细胞外基质源性微粒修复山羊负重区大面积软骨缺损
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摘要
背景:随着组织工程技术的发展,使用天然的生物材料作为支架可以加快新生软骨组织的生成,有利于软骨缺损的修复。目的:探究软骨细胞外基质源性微粒修复负重区软骨大面积缺损的能力。方法:刮取山羊膝关节软骨,通过脱细胞技术制备细胞外基质源性微粒。取12只中国山羊(解放军总医院动物实验中心提供),制作右股骨内外髁负重区直径8 mm、深2 mm的全层骨软骨缺损模型,随机分为2组,实验组(n=6)于缺损处植入同种异体软骨细胞外基质源性微粒,并用纤维蛋白胶固定;对照组(n=6)仅填入纤维蛋白胶。植入3,6个月取右膝关节股骨远端样本,进行组织学及生物力学评价。结果与结论:(1)苏木精-伊红染色:对照组植入3个月时缺损基本无修复,有少量纤维组织填充,缺损区凹陷,基质无异染,两侧交界面整合差;植入后6个月缺损区仍主要为纤维组织,少量纤维软骨填充,新生组织与周围正常组织仍有明显界限。实验组植入3个月时缺损处面积较对照组小,新生组织为纤维软骨与透明软骨复合体,内部可见典型软骨陷窝,细胞排列较为有序;植入6个月时缺损区组织透明软骨比例增大,表面较为平滑,与周围正常软骨组织类似,并整合良好,放大可见典型软骨陷窝,细胞排列有序;(2)番红O-固绿染色:植入3个月时,对照组缺损区基本无蛋白多糖红染,实验组缺损区显示明显蛋白多糖着色;植入6个月时,实验组新生组织中蛋白多糖着色明显多于对照组,且实验组新生组织透明软骨比例大,与周围正常软骨类似;(3)生物力学:植入后6个月,实验组平均杨氏模量明显高于对照组(P <0.05);(4)结果表明:使用软骨细胞外基质源性微粒可促进软骨缺损的修复。
BACKGROUND: With the development of tissue engineering technology, the use of natural biological materials as scaffolds can accelerate the formation of new cartilage tissue and facilitate the repair of cartilage defects. OBJECTIVE: To explore the ability of cartilage extracellular matrix-derived particles to repair cartilage defects in the weight-bearing area. METHODS: Extracellular matrix-derived particles were prepared by decellularization of goat knee cartilage. Twelve Chinese goats(provided by the Experimental Animal Center of PLA General Hospital in China) were selected to make a full-thickness osteochondral defect model with a diameter of 8 mm and a depth of 2 mm in the weight-bearing area of the inner and outer condyles of the right femur. These goat models were randomly divided into two groups. In the experimental group(n=6), the cartilage extracellular matrix-derived particles were implanted to the defect and fixed with fibrin glue. The control group(n=6) was only filled with fibrin glue. General, histological and biomechanical evaluations were conducted with distal right femur samples taken at 3 and 6 months after implantation. RESULTS AND CONCLUSION:(1) Hematoxylin-eosin staining: The defect was basically not repaired at 3 months after implantation in the control group, with a small amount of fibrous tissue filling, sag in the defect area, matrix staining and poor integration of the bilateral interface; at 6 months after implantation, the defect area in the control group was still mainly filled with fibrous tissues and contained a small amount of fibrous cartilage, and there was still a clear boundary between the new tissue and surrounding normal tissue. At 3 months after implantation, the defect area of the experimental group was smaller than that of the control group, and the new tissue was a complex of fibrocartilage and hyaline cartilage. At 6 months after implantation, the proportion of tissue hyaline cartilage in the defect area of the experimental group increased, and the surface was relatively smooth, similar to the surrounding normal cartilage tissue, and well integrated. Enlarged cartilage lacunae were visible and the cells are arranged in order.(2) Safranine O-Fast Green staining: At 3 months after implantation, the defect area of the control group was basically free of proteoglycan red stain, while the defect area of the experimental group showed obvious proteoglycan coloring. At 6 months after implantation, the amount of proteoglycan-stained new tissue in the experimental group was significantly higher than that in the control group, and the proportion of hyaline cartilage in the new tissue was higher in the experimental group, similar to that of surrounding normal cartilage.(3) Biomechanical analysis: Six months after implantation, the average Young's modulus of the experimental group was significantly higher than that of the control group(P < 0.05). All the findings reveal that the use of cartilage extracellular matrix-derived microparticles can promote the repair of cartilage defects.
BACKGROUND: With the development of tissue engineering technology, the use of natural biological materials as scaffolds can accelerate the formation of new cartilage tissue and facilitate the repair of cartilage defects. OBJECTIVE: To explore the ability of cartilage extracellular matrix-derived particles to repair cartilage defects in the weight-bearing area. METHODS: Extracellular matrix-derived particles were prepared by decellularization of goat knee cartilage. Twelve Chinese goats(provided by the Experimental Animal Center of PLA General Hospital in China) were selected to make a full-thickness osteochondral defect model with a diameter of 8 mm and a depth of 2 mm in the weight-bearing area of the inner and outer condyles of the right femur. These goat models were randomly divided into two groups. In the experimental group(n=6), the cartilage extracellular matrix-derived particles were implanted to the defect and fixed with fibrin glue. The control group(n=6) was only filled with fibrin glue. General, histological and biomechanical evaluations were conducted with distal right femur samples taken at 3 and 6 months after implantation. RESULTS AND CONCLUSION:(1) Hematoxylin-eosin staining: The defect was basically not repaired at 3 months after implantation in the control group, with a small amount of fibrous tissue filling, sag in the defect area, matrix staining and poor integration of the bilateral interface; at 6 months after implantation, the defect area in the control group was still mainly filled with fibrous tissues and contained a small amount of fibrous cartilage, and there was still a clear boundary between the new tissue and surrounding normal tissue. At 3 months after implantation, the defect area of the experimental group was smaller than that of the control group, and the new tissue was a complex of fibrocartilage and hyaline cartilage. At 6 months after implantation, the proportion of tissue hyaline cartilage in the defect area of the experimental group increased, and the surface was relatively smooth, similar to the surrounding normal cartilage tissue, and well integrated. Enlarged cartilage lacunae were visible and the cells are arranged in order.(2) Safranine O-Fast Green staining: At 3 months after implantation, the defect area of the control group was basically free of proteoglycan red stain, while the defect area of the experimental group showed obvious proteoglycan coloring. At 6 months after implantation, the amount of proteoglycan-stained new tissue in the experimental group was significantly higher than that in the control group, and the proportion of hyaline cartilage in the new tissue was higher in the experimental group, similar to that of surrounding normal cartilage.(3) Biomechanical analysis: Six months after implantation, the average Young's modulus of the experimental group was significantly higher than that of the control group(P < 0.05). All the findings reveal that the use of cartilage extracellular matrix-derived microparticles can promote the repair of cartilage defects.
引文
[1]Lourenco S,Lucas R,Araujo F,et al.Osteoarthritis medical labelling and health-related quality of life in the general population.Health Qual Life Outcomes.2014;12:146.
[2]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.
[3]Makris EA,Gomoll AH,Malizos KN,et al.Repair and tissue engineering techniques for articular cartilage.Nat Rev Rheumatol.2015;11:21-34.
[4]Huang H,Zhang X,Hu X,et al.A functional biphasic biomaterial homing mesenchymal stem cells for in vivo cartilage regeneration.Biomaterials.2014;35:9608-9619.
[5]Ko JY,Im GI.Chondrogenic and Osteogenic Induction from iPSCells.Methods Mol Biol.2016;1357:441-450
[6]Johnson K,Zhu S,Tremblay MS,et al.A stem cell-based approach to cartilage repair.Science.2012;336:717-721.
[7]Mei L,Shen B,Ling P,et al.Culture-expanded allogenic adipose tissue-derived stem cells attenuate cartilage degeneration in an experimental rat osteoarthritis model.PloSOne.2017;12:e0176107.
[8]Dubey NK,Mishra VK,Dubey R,et al.Combating Osteoarthritis through Stem Cell Therapies by Rejuvenating Cartilage:AReview.Stem Cells Int.2018;2018:5421019.
[9]Jones EA,English A,Henshaw K,et al.Enumeration and phenotypic characterization of synovial fluid multipotential mesenchymal progenitor cells in inflammatory and degenerative arthritis.Arthritis Rheum.2004;50(3):817-827.
[10]van Lent PL,van den Berg WB.Mesenchymal stem cell therapy in osteoarthritis:advanced tissue repair or intervention with smouldering synovial activation?Arthritis Res Ther.2013;15(2):112.
[11]卢强,张莉,彭江,等.可注射性组织工程软骨源性微载体的微观结构及其生物相容性观察[J].中国矫形外科杂志,2009,17(9):688-691.
[12]Sutherland AJ,Converse GL,Hopkins RA,et al.The bioactivity of cartilage extracellular matrix in articular cartilage regeneration.Adv Healthc Mater.2015;4(1):29-39.
[13]Olvera D,Daly A,Kelly DJ.Mechanical Testing of Cartilage Constructs.Methods Mol Biol.2015;1340:279-287
[14]DiSilvestro MR,Suh JK.Biphasic poroviscoelastic characteristics of proteoglycan-depleted articular cartilage:simulation of degeneration.Ann of Biomed Eng.2002,30:792-800
[15]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.
[16]Hsieh YH,Shen BY,Wang YH,et al.Healing of Osteochondral Defects Implanted with Biomimetic Scaffolds of Poly(epsilon-Caprolactone)/Hydroxyapatite and Glycidyl-Methacrylate-Modified Hyaluronic Acid in a Minipig.Int J Mol Sci.2018;19(4).pii:E1125.doi:10.3390/ijms19041125.
[17]Fisher MB,Belkin NS,Milby AH,et al.Cartilage repair and subchondral bone remodeling in response to focal lesions in a mini-pig model:implications for tissue engineering.Tissue Eng Part A.2015;21(3-4):850-860.
[18]Doyran B,Tong W,Li Q,et al.Nanoindentation modulus of murine cartilage:a sensitive indicator of the initiation and progression of post-traumatic osteoarthritis.Osteoarthritis Cartilage.2017;25(1):108-117.
[19]Chang NJ,Lin CC,Shie MY,et al.Positive effects of cell-free porous PLGA implants and early loading exercise on hyaline cartilage regeneration in rabbits.Acta Biomater.2015;28:128-137.
[20]Mainil-Varlet P,Aigner T,Brittberg M,et al.Histological assessment of cartilage repair:a report by the Histology Endpoint Committee of the International Cartilage Repair Society(ICRS).J Bone Joint Surg Am.2003;85-A Suppl2:45-57.
[21]Mattei G,Gruca G,Rijnveld N,et al.The nano-epsilon dot method for strain rate viscoelastic characterisation of soft biomaterials by spherical nano-indentation.J Mech Behav Biomed Mater.2015;50:150-159.
[22]Jeznach O,Kolbuk D,Sajkiewicz P.Injectable hydrogels and nanocomposite hydrogels for cartilage regeneration.J Biomed Mater Res A.2018.doi:10.1002/jbm.a.36449.[Epub ahead of print]Review.
[23]方洪松,周建林,彭昊,等.组织工程支架材料修复关节软骨缺损[J].中国组织工程研究,2016,20(52):7891-7898
[24]Polat G,Ersen A,Erdil ME,et al.Long-term results of microfracture in the treatment of talus osteochondral lesions.Knee Surg Sports Traumatol Arthrosc.2016;24(4):1299-1303.
[25]Nooeaid P,Salih V,Beier JP,et al.Osteochondral tissue engineering:scaffolds,stem cells and applications.J Cell Mol Med.2012;16(10):2247-2270.
[26]Vinatier C,Guicheux J.Cartilage tissue engineering:From biomaterials and stem cells to osteoarthritis treatments.Ann Phys Rehabil Med.2016;59(3):139-144.
[27]Agarwal T,Narayan R,Maji S,et al.Gelatin/Carboxymethyl chitosan based scaffolds for dermal tissue engineering applications.Int J Biol Macromol.2016;93(Pt B):1499-1506.
[28]Cheng NC,Estes BT,Awad HA,et al.Chondrogenic differentiation of adipose-derived adult stem cells by a porous scaffold derived from native articular cartilage extracellular matrix.Tissue Eng Part A.2009;15(2):231-241.
[29]侯立刚,杨建义.骨修复中应用的生物降解可吸收材料[J].中国组织工程研究,2016,20(3):441-445..
[30]Christensen BB,Foldager CB,Olesen ML,et al.Experimental articular cartilage repair in the Gottingen minipig:the influence of multiple defects per knee.J Exp Orthop.2015;2(1):13.
[31]Gotterbarm T,Breusch SJ,Schneider U,et al.The minipig model for experimental chondral and osteochondral defect repair in tissue engineering:retrospective analysis of 180 defects.Lab Anim.2008;42(1):71-82.
[32]Baboolal TG,Mastbergen SC,Jones E,et al.Synovial fluid hyaluronan mediates MSC attachment to cartilage,a potential novel mechanism contributing to cartilage repair in osteoarthritis using knee joint distraction.Ann Rheum Dis.2016;75(5):908-915.
[33]Saarakkala S,Korhonen RK,Laasanen MS,et al.Mechano-acoustic determination of Young’s modulus of articular cartilage.Biorheology.2004;41(3-4):167-179.
[34]Bhumiratana S,Eton RE,Oungoulian SR,et al.Large,stratified,and mechanically functional human cartilage grown in vitro by mesenchymal condensation.Proc Natl Acad Sci U SA.2014;111(19):6940-6945.
[2]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.
[3]Makris EA,Gomoll AH,Malizos KN,et al.Repair and tissue engineering techniques for articular cartilage.Nat Rev Rheumatol.2015;11:21-34.
[4]Huang H,Zhang X,Hu X,et al.A functional biphasic biomaterial homing mesenchymal stem cells for in vivo cartilage regeneration.Biomaterials.2014;35:9608-9619.
[5]Ko JY,Im GI.Chondrogenic and Osteogenic Induction from iPSCells.Methods Mol Biol.2016;1357:441-450
[6]Johnson K,Zhu S,Tremblay MS,et al.A stem cell-based approach to cartilage repair.Science.2012;336:717-721.
[7]Mei L,Shen B,Ling P,et al.Culture-expanded allogenic adipose tissue-derived stem cells attenuate cartilage degeneration in an experimental rat osteoarthritis model.PloSOne.2017;12:e0176107.
[8]Dubey NK,Mishra VK,Dubey R,et al.Combating Osteoarthritis through Stem Cell Therapies by Rejuvenating Cartilage:AReview.Stem Cells Int.2018;2018:5421019.
[9]Jones EA,English A,Henshaw K,et al.Enumeration and phenotypic characterization of synovial fluid multipotential mesenchymal progenitor cells in inflammatory and degenerative arthritis.Arthritis Rheum.2004;50(3):817-827.
[10]van Lent PL,van den Berg WB.Mesenchymal stem cell therapy in osteoarthritis:advanced tissue repair or intervention with smouldering synovial activation?Arthritis Res Ther.2013;15(2):112.
[11]卢强,张莉,彭江,等.可注射性组织工程软骨源性微载体的微观结构及其生物相容性观察[J].中国矫形外科杂志,2009,17(9):688-691.
[12]Sutherland AJ,Converse GL,Hopkins RA,et al.The bioactivity of cartilage extracellular matrix in articular cartilage regeneration.Adv Healthc Mater.2015;4(1):29-39.
[13]Olvera D,Daly A,Kelly DJ.Mechanical Testing of Cartilage Constructs.Methods Mol Biol.2015;1340:279-287
[14]DiSilvestro MR,Suh JK.Biphasic poroviscoelastic characteristics of proteoglycan-depleted articular cartilage:simulation of degeneration.Ann of Biomed Eng.2002,30:792-800
[15]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.
[16]Hsieh YH,Shen BY,Wang YH,et al.Healing of Osteochondral Defects Implanted with Biomimetic Scaffolds of Poly(epsilon-Caprolactone)/Hydroxyapatite and Glycidyl-Methacrylate-Modified Hyaluronic Acid in a Minipig.Int J Mol Sci.2018;19(4).pii:E1125.doi:10.3390/ijms19041125.
[17]Fisher MB,Belkin NS,Milby AH,et al.Cartilage repair and subchondral bone remodeling in response to focal lesions in a mini-pig model:implications for tissue engineering.Tissue Eng Part A.2015;21(3-4):850-860.
[18]Doyran B,Tong W,Li Q,et al.Nanoindentation modulus of murine cartilage:a sensitive indicator of the initiation and progression of post-traumatic osteoarthritis.Osteoarthritis Cartilage.2017;25(1):108-117.
[19]Chang NJ,Lin CC,Shie MY,et al.Positive effects of cell-free porous PLGA implants and early loading exercise on hyaline cartilage regeneration in rabbits.Acta Biomater.2015;28:128-137.
[20]Mainil-Varlet P,Aigner T,Brittberg M,et al.Histological assessment of cartilage repair:a report by the Histology Endpoint Committee of the International Cartilage Repair Society(ICRS).J Bone Joint Surg Am.2003;85-A Suppl2:45-57.
[21]Mattei G,Gruca G,Rijnveld N,et al.The nano-epsilon dot method for strain rate viscoelastic characterisation of soft biomaterials by spherical nano-indentation.J Mech Behav Biomed Mater.2015;50:150-159.
[22]Jeznach O,Kolbuk D,Sajkiewicz P.Injectable hydrogels and nanocomposite hydrogels for cartilage regeneration.J Biomed Mater Res A.2018.doi:10.1002/jbm.a.36449.[Epub ahead of print]Review.
[23]方洪松,周建林,彭昊,等.组织工程支架材料修复关节软骨缺损[J].中国组织工程研究,2016,20(52):7891-7898
[24]Polat G,Ersen A,Erdil ME,et al.Long-term results of microfracture in the treatment of talus osteochondral lesions.Knee Surg Sports Traumatol Arthrosc.2016;24(4):1299-1303.
[25]Nooeaid P,Salih V,Beier JP,et al.Osteochondral tissue engineering:scaffolds,stem cells and applications.J Cell Mol Med.2012;16(10):2247-2270.
[26]Vinatier C,Guicheux J.Cartilage tissue engineering:From biomaterials and stem cells to osteoarthritis treatments.Ann Phys Rehabil Med.2016;59(3):139-144.
[27]Agarwal T,Narayan R,Maji S,et al.Gelatin/Carboxymethyl chitosan based scaffolds for dermal tissue engineering applications.Int J Biol Macromol.2016;93(Pt B):1499-1506.
[28]Cheng NC,Estes BT,Awad HA,et al.Chondrogenic differentiation of adipose-derived adult stem cells by a porous scaffold derived from native articular cartilage extracellular matrix.Tissue Eng Part A.2009;15(2):231-241.
[29]侯立刚,杨建义.骨修复中应用的生物降解可吸收材料[J].中国组织工程研究,2016,20(3):441-445..
[30]Christensen BB,Foldager CB,Olesen ML,et al.Experimental articular cartilage repair in the Gottingen minipig:the influence of multiple defects per knee.J Exp Orthop.2015;2(1):13.
[31]Gotterbarm T,Breusch SJ,Schneider U,et al.The minipig model for experimental chondral and osteochondral defect repair in tissue engineering:retrospective analysis of 180 defects.Lab Anim.2008;42(1):71-82.
[32]Baboolal TG,Mastbergen SC,Jones E,et al.Synovial fluid hyaluronan mediates MSC attachment to cartilage,a potential novel mechanism contributing to cartilage repair in osteoarthritis using knee joint distraction.Ann Rheum Dis.2016;75(5):908-915.
[33]Saarakkala S,Korhonen RK,Laasanen MS,et al.Mechano-acoustic determination of Young’s modulus of articular cartilage.Biorheology.2004;41(3-4):167-179.
[34]Bhumiratana S,Eton RE,Oungoulian SR,et al.Large,stratified,and mechanically functional human cartilage grown in vitro by mesenchymal condensation.Proc Natl Acad Sci U SA.2014;111(19):6940-6945.