脱钙松质骨复合同种异体软骨细胞修复兔关节骨软骨缺损的实验研究
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摘要
目的评价脱钙松质骨(DCB)复合同种异体软骨细胞构建组织工程软骨修复兔关节骨软骨缺损的效果。方法分离1月龄雄性新西兰兔关节软骨细胞,原代培养后复合制备的DCB体外培养2周构建组织工程软骨。4~5月龄新西兰兔30只双侧股骨内髁制作直径3 mm、深3 mm,穿透软骨下骨板的骨软骨缺损模型,20只右侧关节缺损处植入构建的组织工程软骨(A组),左侧缺损处植入DCB(B组),10只双侧骨软骨缺损未予处理作为空白对照(C组)。分别于术后1、3、6月取修复组织标本,进行大体形态、组织学及Ⅱ型胶原染色;并对6月修复组织进行组织学评分,比较各组修复效果差异。结果制备的DCB为三维多孔的海绵结构,孔隙大小约为100~500μm,相互交通。DCB植入体内后1月开始降解,3月完全吸收。术后6月A组缺损处修复组织主要为透明样软骨,与周围正常软骨厚度基本一致,修复交界区整合良好,不易辨认。修复组织深层细胞在软骨陷窝内,呈柱状排列,基质蛋白多糖和Ⅱ型胶原染色接近正常软骨,软骨下骨板完整。B组缺损处以纤维软骨样组织修复为主。C组以纤维组织填充。组织学评分显示术后6月A组除软骨下骨板重建与B组比较无统计学差异外,其它各项评分均优于B组和C组,差异有统计学意义(P<0.05)。结论 DCB是一种较好的软骨组织工程支架材料,复合同种异体软骨细胞能修复关节骨软骨缺损,修复组织为透明样软骨。
Objective To evaluate the effect of demineralized cancellous bone(DCB) seeded with allogeneic chondrocytes for repairing articular osteochondral defects in rabbits. Methods Articular chondrocytes were isolated from a 1-month-old male New Zealand rabbit for primary culture. The passage 1 chondrocytes were seeded onto prepared rabbit DCB scaffold to construct tissue-engineered cartilage and cultured in vitro for 2 weeks. Full-thickness articular osteochondral defects(3 mm both in diameter and depth) were created on both sides of the femoral medial condyles in 30 New Zealand white rabbits(age 4-5 months). In 20 of the rabbits, the defects were filled with the tissue-engineered cartilage on the right side(group A) and with DCB only on the left side(group B); the remaining 10 rabbits did not receive any implantation in the defects to serve as the control(group C). At 1, 3, and 6 months after the implantation, tissue samples were collected from the defects for macroscopic observation and histological examination with Toluidine blue(TB) and collagen type II staining. The effect of defect repair using the tissue-engineered cartilage was assessed at 6 months based on the histological scores. Results The prepared DCB had a spongy 3 D structure with open and interconnected micropores of various sizes and showed good plasticity and mechanical strength. DCB began to degrade within 1 month after implantation and was totally absorbed at 3 months. At 6 months after implantation, the defects filled with the chondrocyte-seeded DCB were repaired mainly by hyaline-like cartilage tissues, which were well integrated to the adjacent cartilage without clear boundaries and difficult to recognize. The chondrocytes were located in the lacunate and arranged in vertical columns in the deep repaired tissue, where matrix proteoglycans and collagen type II were distributed homogeneously close to the normal cartilage. The subchondral bone plate was reconstructed completely. The defects implanted with DCB only were filled with fibrocartilage tissue, as compared with fibrous tissue in the control defects. The histological scores in group A were significantly superior to those in group B and C(P<0.05), but the scores for subchondral bone plate reconstruction were comparable between groups A and B at 6 months. Conclusion DCB is a good scaffold material for preparing tissue-engineered cartilage, and chondrocyte-seeded DCB can repair articular osteochondral defects by inducing the generation of hayline-like cartilage.
Objective To evaluate the effect of demineralized cancellous bone(DCB) seeded with allogeneic chondrocytes for repairing articular osteochondral defects in rabbits. Methods Articular chondrocytes were isolated from a 1-month-old male New Zealand rabbit for primary culture. The passage 1 chondrocytes were seeded onto prepared rabbit DCB scaffold to construct tissue-engineered cartilage and cultured in vitro for 2 weeks. Full-thickness articular osteochondral defects(3 mm both in diameter and depth) were created on both sides of the femoral medial condyles in 30 New Zealand white rabbits(age 4-5 months). In 20 of the rabbits, the defects were filled with the tissue-engineered cartilage on the right side(group A) and with DCB only on the left side(group B); the remaining 10 rabbits did not receive any implantation in the defects to serve as the control(group C). At 1, 3, and 6 months after the implantation, tissue samples were collected from the defects for macroscopic observation and histological examination with Toluidine blue(TB) and collagen type II staining. The effect of defect repair using the tissue-engineered cartilage was assessed at 6 months based on the histological scores. Results The prepared DCB had a spongy 3 D structure with open and interconnected micropores of various sizes and showed good plasticity and mechanical strength. DCB began to degrade within 1 month after implantation and was totally absorbed at 3 months. At 6 months after implantation, the defects filled with the chondrocyte-seeded DCB were repaired mainly by hyaline-like cartilage tissues, which were well integrated to the adjacent cartilage without clear boundaries and difficult to recognize. The chondrocytes were located in the lacunate and arranged in vertical columns in the deep repaired tissue, where matrix proteoglycans and collagen type II were distributed homogeneously close to the normal cartilage. The subchondral bone plate was reconstructed completely. The defects implanted with DCB only were filled with fibrocartilage tissue, as compared with fibrous tissue in the control defects. The histological scores in group A were significantly superior to those in group B and C(P<0.05), but the scores for subchondral bone plate reconstruction were comparable between groups A and B at 6 months. Conclusion DCB is a good scaffold material for preparing tissue-engineered cartilage, and chondrocyte-seeded DCB can repair articular osteochondral defects by inducing the generation of hayline-like cartilage.
引文
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[23]杨渊,林春博,陈维平.脱钙松质骨的骨支架材料制备及其生物学检测[J].中国组织工程研究与临床康复,2010,14(47):8769-72.
[24]Haaparanta AM,J?rvinen E,Cengiz IF,et al.Preparation and characterization of collagen/PLA,chitosan/PLA,and collagen/chitosan/PLA hybrid scaffolds for cartilage tissue engineering[J].J Mater Sci Mater Med,2014,25(4):1129-36.
[25]黄永波,卫小春,翁卫生,等.海藻酸钠-成年软骨细胞培养移植修复成年兔关节软骨缺损的研究[J].中国实验外科杂志,2004,21(8):988-90.
[26]Wei X,Gao J,Messner K.Maturation-dependent repair of untreated osteochondral defects in the rabbit knee joint[J].J Biomed Mater Res,1997,34(1):63-72.
[27]Schlichting K,Schell H,Kleemann RU,et al.Influence of scaffold stiffness on subchondral bone and subsequent cartilage regeneration in an ovine model of osteochondral defect healing[J].Am J Sports Med,2008,36(12):2379-91.
[28]Madry HC.Mueller-Gerbl M.the basic science of the subchondral bone[J].Knee Surg Sports Traumatol Arthrosc,2010,18(4):419-33.
[29]尹战海,张璐,王金堂,等.双相"组织工程软骨修复兔关节骨软骨缺损[J].中国修复重建外科杂志,2005,19(8):652-7.
[2]Musumeci G,Castrogiovanni P,Leonardi R,et al.New perspectives for articular cartilage repair treatment through tissue engineering:A contemporary review[J].World J Orthop,2014,5(2):80-8.
[3]Ringe J,Sittinger M.Regenerative medicine:Selecting the right biological scaffold for tissue engineering[J].Nat Rev Rheumatol,2014,10(7):388-9.
[4]Gaut C,Sugaya K.Critical review on the physical and mechanical factors involved in tissue engineering of cartilage[J].Regen Med,2015,10(5):665-79.
[5]Liao J,Qu Y,Chu B,et al.Biodegradable CSMA/PECA/graphene porous hybrid scaffold for cartilage tissue engineering[J].Sci Rep,2015,5(5):9879.
[6]Vázquez-Portalat?N N,Kilmer CE,Panitch A,et al.Characterization of collagen type I and II blended hydrogels for articular cartilage tissue engineering[J].Biomacromolecules,2016,17(10):3145-52.
[7]Kapur SP,Reddi AH.Chondrogenic potential of mesenchymal cells elicited by bone matrix in vitro[J].Differentiation,1986,32(3):252-9.
[8]Wang SJ,Jiang D,Zhang ZZ,et al.Chondrogenic potential of peripheral blood derived mesenchymal stem cells seeded on demineralized cancellous bone scaffolds[J].Sci Rep,2016,6(6):36400.
[9]Ziran BH,Smith WR,Morgan SJ.Use of calcium-based demineralized bone matrix/allograft for nonunions and posttraumatic reconstruction of the appendicular skeleton:preliminary results and complications[J].J Trauma,2007,63(6):1324-8.
[10]Kenneth JH,Lee MD,Jonathan G,et al.Demineralized bone matrix and spinal arthrodesis[J].The Spine Journal,2005,15(5):217-23.
[11]Li Q,Tang JC,Sun ZY,et al.Repairing full-thickness articular cartilage defects with homograft of mesenchymal stem cells seeded onto cancellous demineralized bone matrix[J].中国组织工程研究与临床康复,2008,12(45):8943-7.
[12]Yang B,Yin Z,Cao J,et al.In vitro cartilage tissue engineering using cancellous bone matrix gelatin as a biodegradable scaffold[J].Biomed Mater,2010,5(4):045003.
[13]Buma P,Pieper JS,van Tienen T,et al.Cross-linked type I and type II collagenous matrices for the repair of full-thickness articular cartilage defects--a study in rabbits[J].Biomaterials,2003,24(19):3255-63.
[14]Ramallal M,Maneiro E,López E,et al.Xeno-implantation of pig chondrocytes into rabbit to treat localized articular cartilage defects:an animal model[J].Wound Repair Regen,2004,12(3):337-45.
[15]Huang BJ,Hu JC,Athanasiou KA.Cell-based tissue engineering strategies used in the clinical repair ofarticular cartilage[J].Biomaterials,2016,98(7):1-22.
[16]Makris EA,Gomoll AH,Malizos KN,et al.Repair and tissue engineering techniques for articular cartilage[J].Nat Rev Rheumatol,2015,11(1):21-34.
[17]Goyal D,Keyhani S,Lee EH,et al.Evidence-based status of microfracture technique:a systematic review of level I and II studies[J].Arthroscopy,2013,29(9):1579-88.
[18]Minas T,Von Keudell A,Bryant T,et al.The john insall award:a minimum 10-year outcome study of autologous chondrocyte implantation[J].Clin Orthop Relat Res,2014,472(1):41-51.
[19]Shekkeris A,Perera J,Bentley G,et al.Histological results of 406BIOPSIES following ACI/MACI procedures forosteochondral defects in the knee[J].Bone Joint J,2012,94-B:SUPP XXXVI12.
[20]Camarero-Espinosa S,Rothen-Rutishauser B,Foster EJ,et al.Articular cartilage:from formation to tissue engineering[J].Biomater Sci,2016,4(5):734-67.
[21]宋红星,曹峻岭,刘淼,等.同种异体软骨细胞移植修复兔膝关节软骨缺损的免疫反应[J].中国矫形外科杂志,2002,9(1):32-5.
[22]Gruskin E,Doll BA,Futrell FW,et al.Demineralized bone matrix in bone repair:history and use[J].Adv Drug Deliv Rev,2012,64(12):1063-77.
[23]杨渊,林春博,陈维平.脱钙松质骨的骨支架材料制备及其生物学检测[J].中国组织工程研究与临床康复,2010,14(47):8769-72.
[24]Haaparanta AM,J?rvinen E,Cengiz IF,et al.Preparation and characterization of collagen/PLA,chitosan/PLA,and collagen/chitosan/PLA hybrid scaffolds for cartilage tissue engineering[J].J Mater Sci Mater Med,2014,25(4):1129-36.
[25]黄永波,卫小春,翁卫生,等.海藻酸钠-成年软骨细胞培养移植修复成年兔关节软骨缺损的研究[J].中国实验外科杂志,2004,21(8):988-90.
[26]Wei X,Gao J,Messner K.Maturation-dependent repair of untreated osteochondral defects in the rabbit knee joint[J].J Biomed Mater Res,1997,34(1):63-72.
[27]Schlichting K,Schell H,Kleemann RU,et al.Influence of scaffold stiffness on subchondral bone and subsequent cartilage regeneration in an ovine model of osteochondral defect healing[J].Am J Sports Med,2008,36(12):2379-91.
[28]Madry HC.Mueller-Gerbl M.the basic science of the subchondral bone[J].Knee Surg Sports Traumatol Arthrosc,2010,18(4):419-33.
[29]尹战海,张璐,王金堂,等.双相"组织工程软骨修复兔关节骨软骨缺损[J].中国修复重建外科杂志,2005,19(8):652-7.