同种异体幼年软骨微粒移植修复关节软骨缺损的可行性
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摘要
背景:同种异体幼年软骨微粒制备简单,获取容易,该技术在美国已经进入临床研究阶段,但临床上还处于"黑箱"操作阶段,对于移植的幼年软骨微粒是如何通过生物化学机制和基因信号转导发挥生物学效应,目前还不清楚。目前,中国还没有相关技术的报道。目的:探索同种异体幼年软骨微粒移植修复关节软骨缺损的可行性。方法:从幼年贵州小香猪(陆军军医大学实验动物中心提供)膝关节获取同种异体幼年软骨微粒,体外培养1,3,7 d进行Brdu免疫荧光检测。将同种异体幼年软骨微粒/纤维蛋白凝胶复合物移植于SCID大鼠(陆军军医大学实验动物中心提供)皮下,1个月后取材,进行苏木精-伊红染色、番红染色、免疫组织化学检测。在10只成年贵州小香猪(陆军军医大学实验动物中心提供)膝关节髌骨面制作直径8 mm的软骨缺损,随机分2组干预,实验组软骨缺损处移植同种异体幼年软骨微粒/纤维蛋白凝胶复合物,空白组不移植任何材料,3个月后取材,对修复组织进行苏木精-伊红、番红-固绿、甲苯胺蓝、免疫组织化学检测。结果与结论:(1)在同种异体幼年软骨微粒的体外培养中,第1天见到极少的软骨细胞脱落与增殖;第3天可见少量的软骨细胞脱落与增殖;第7天见到有明显的细胞脱落与增殖,且增殖的细胞分布在切缘周围;(2)皮下移植1个月后,移植的同种异体幼年软骨微粒仍然存活且周围有少量的软骨细胞增殖;(3)软骨缺损修复3个月后,空白组未见明显修复组织;实验组可见明显的修复组织,新生的软骨组织颜色与正常软骨组织颜色相似,且与周围正常软骨组织界面整合良好,细胞分布较均匀;(4)结果表明,同种异体幼年软骨微粒修复关节软骨缺损可取得良好的效果。
BACKGROUND: Particulated juvenile cartilage allograft is simple and easy to obtain, and relevant clinical studies are underway in the USA. However, how the transplanted juvenile cartilage fragments exert biological effects through biochemical mechanisms and genetic signal transduction is still unclear. There is as yet no report on this technology in China. OBJECTIVE: To explore the feasibility of articular cartilage defects repaired with particulated juvenile cartilage allograft. METHODS: The cartilage fragments were obtained from juvenile Pitman-Moore strains(provided by the Laboratory Animal Center of the Army Medical University in China) and cultured in vitro. Brdu immunofluorescence assay was performed at 1, 3, and 7 days of culture. The particulated juvenile cartilage allograft/fibrin gel composites were subcutaneously transplanted into the SCID rats (provided by the Laboratory Animal Center of the Army Medical University). The specimens were taken for hematoxylin-eosin staining, safranin O staining and immunohistochemistry after 1 month. Cartilage defects of 8 mm in diameter were made in the knee joint of 10 adult Pitman-Moore strains (Laboratory Animal Center of the Army Medical University), and were randomized into two groups, which were then transplanted with the particulated juvenile cartilage allograft/fibrin gel composites (experimental group) or nothing(control group). The specimens were taken for hematoxylin-eosin staining, safranin O fast green staining, toluidine blue and immunohistochemistry at 3 months after transplantation. RESULTS AND CONCLUSION: Little Brdu incorporation was detected in juvenile cartilage fragments at 1 day of culture, some Brdu incorporation was defected at 3 days of culture. At 7 days of culture, a progressive increase in the Brdu signal was detected in chondrocytes within the cultured cartilage fragments, which seemed to localize along the tissue edge. At 1 month after subcutaneous transplantation, the particulated juvenile cartilage allograft still survived and were surrounded by few proliferative chondrocytes. There was no obvious tissue repair in the control group at 3 months after transplantation. In the experimental group, there was obvious tissue repair, the color of the newly formed tissues was similar to the normal cartilage tissue, which integrated well with the surrounding normal cartilage tissue, and the cells distributed evenly. These results imply that particulated juvenile cartilage allograft can achieve good results in repairing articular cartilage defects.
BACKGROUND: Particulated juvenile cartilage allograft is simple and easy to obtain, and relevant clinical studies are underway in the USA. However, how the transplanted juvenile cartilage fragments exert biological effects through biochemical mechanisms and genetic signal transduction is still unclear. There is as yet no report on this technology in China. OBJECTIVE: To explore the feasibility of articular cartilage defects repaired with particulated juvenile cartilage allograft. METHODS: The cartilage fragments were obtained from juvenile Pitman-Moore strains(provided by the Laboratory Animal Center of the Army Medical University in China) and cultured in vitro. Brdu immunofluorescence assay was performed at 1, 3, and 7 days of culture. The particulated juvenile cartilage allograft/fibrin gel composites were subcutaneously transplanted into the SCID rats (provided by the Laboratory Animal Center of the Army Medical University). The specimens were taken for hematoxylin-eosin staining, safranin O staining and immunohistochemistry after 1 month. Cartilage defects of 8 mm in diameter were made in the knee joint of 10 adult Pitman-Moore strains (Laboratory Animal Center of the Army Medical University), and were randomized into two groups, which were then transplanted with the particulated juvenile cartilage allograft/fibrin gel composites (experimental group) or nothing(control group). The specimens were taken for hematoxylin-eosin staining, safranin O fast green staining, toluidine blue and immunohistochemistry at 3 months after transplantation. RESULTS AND CONCLUSION: Little Brdu incorporation was detected in juvenile cartilage fragments at 1 day of culture, some Brdu incorporation was defected at 3 days of culture. At 7 days of culture, a progressive increase in the Brdu signal was detected in chondrocytes within the cultured cartilage fragments, which seemed to localize along the tissue edge. At 1 month after subcutaneous transplantation, the particulated juvenile cartilage allograft still survived and were surrounded by few proliferative chondrocytes. There was no obvious tissue repair in the control group at 3 months after transplantation. In the experimental group, there was obvious tissue repair, the color of the newly formed tissues was similar to the normal cartilage tissue, which integrated well with the surrounding normal cartilage tissue, and the cells distributed evenly. These results imply that particulated juvenile cartilage allograft can achieve good results in repairing articular cartilage defects.
引文
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[7]Kon E,Delcogliano M,Filardo G,et al.A novel nano-composite multi-layered biomaterial for treatment of osteochondral lesion:technique note and an early stability pilot clinical trial.Injury.2010;41(7):693-701.
[8]Gille J,Behrens P,Schulz AP,et al.Matrix-Associated Autologous Chondrocyte Implantation:A Clinical Follow-Up at 15 Years.Cartilage.2016;7(4):309-315.
[9]Karoubi G,Ormiston ML,Stewart DJ,et al.Single-cell hydrogel encapsulation for enhanced survival of human marrow stromal cells.Biomaterials.2009;30(29):5445-5455.
[10]Shimizu H,Ohashi K,Utoh R,et al.Bioengineering of a functional sheet of islet cells for the treatment of diabetes mellitus.Biomaterials.2009;30(30):5943-5949.
[11]Hayashi S,Kamei N,Ikuta Y,et al.Chondrocyte cell-sheet transplantation for treating monoiodoacetate-induced arthritis in rats.Tissue Eng Part C Methods.2017;23(6):346-356.
[12]Xu X,Shi D,Liu Y,et al.Synovium-derived mesenchymal stem cell sheet enhance autologous osteochondral transplantation in a rabbit momdel.Int J Clin Exp Med.2016;9(6):10322-10332.
[13]Shaari CM,Farber D,Brandwein MS,et al.Chracterizing the antigenic profile of the human trachea:Implications for tracheal transplantation.Head Neck.1998;20(6):522-527
[14]Liu K,Zhou GD,Liu W,et al.The dependence of in vivo stable ectopic chondrogenesis by human mesenchymal stem cells on chondrogenic differentiation in vitro.Biomaterials.2008;29(14):2183-2192.
[15]Ge Y,Gong YY,Xu Z,et al.The application of sheet technology in cartilage tissue engineering.Tissue Eng Part B Rev.2016;22(2):114-124.
[16]Mouthuy PA,Elsherbini Y,Cui Z,et al.Layering PLGA-based electrospum membranes and cell sheets for engineering cartilage-bone transition.Tissue Eng Regen Med.2016;10(4):263-274.
[17]Verdonk P,Dhollander A,Almqvist KF,et al.Treatment of osteochondral lesions in the knee using a cell-free scaffold.Bone Joint J.2015;97-B(3):318-323.
[18]Brix M,Kaipel M,Kellner R,et al.Successful osteoconduction but limited cartilage tissue quality following osteochondral repair by a cell-free multilayered nano-composite scaffold at the knee.Int Orthop.2016;40(3):625-632.
[19]Filardo G,Kon E,Di MA,et al.Treatment of knee osteochondritis dissecans with a cell-free biomimetic osteochondral scaffold:clinical and imaging evaluation at 2-year follow up.am J Sports Med.2013;41(8):1786-1793.
[20]Albrecht F,Roessner A,Zimmermann E.Closure of osteochondral lesions using chondral fragments and fibrin adhesive.Arch Orthop Trauma Surg.1983;101(3):213-217.
[21]Riboh JC,Cole BJ,Farr J.Particulated articular cartilage for symptomatic chondral defects of the knee.Curr Rev Musculoskelet Med.2015;11(1):21-34.
[22]Bonasia DE,Marmotti A,Rosso F,et al.Use of chondral fragments for one stage caitilage repair:a systematic review.World J Orthop.2015;6(11):1006.
[23]宁志刚,杨柳,王富友,等.保留钙化层结构的猪股骨滑车全厚软骨缺损模型建立[J].中国修复重建外科杂志,2012,26(5):527-531.
[24]Brittberg M,Aglietti P,Gambardella R,et al.ICRS Cartilage Injury Evaluation Package.2002:16-18.https://www.secot.es/uploads/descargas/formacion/escalas_valoracion/ICRS._TRAUMA_CARTaILAGO.pdf
[25]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 2(1):45-57.
[26]Kontturi LS,Jarvinen E,Muhonen V,et al.An injectable,insitu forming type IIcollagen/hyaluronic acid hydrogel vehicle for chondrocyte delivery in cartilage tissue engineering.Drug Deliv Transl Res.2014;4(2):149-158.
[27]Cole BJ,Farr J,Winalski CS,et al.Outcomes after a single-stage procedure for cell-based cartilage repair:a prospective clinical safety trial with 2-year follow-up.Am J Sports Med.2011;39(6):1170-1179.
[28]Kon E,Filardo G,Zaffagnini S,et al.Biodegradable polyurethane meniscal scaffold for isolated partial lesions or as combined procedure for knees with multiple comorbidities:clinical results at 2 years.Knee Surg Sports Traumatol Arthrosc.2014;22(1):128-134.
[29]Farr J,Yao JQ.Chondral Defect Repair with Particulated Juvenile Cartilage Allograft.Cartilage.2011;2(4):346-353.
[30]Farr J,Tabet SK,Margerrison E,et al.Clinical,Radiographic,and histological outcomes after cartilage repair with particulated juvenile articular cartilage:a 2-year prospective study.Am JSports Med.2014:42(6):1417.
[31]Mcmillan S,Light GT,Brtz C.All-arthroscopic implantation of minced juvenile chondral allograft for an isolated,full-thickness chondral lesion in the trochlea of an adult knee.Arthrosc Tech.2016;5(2):397-401.
[32]Arshi A,Wang D,Jones KJ.Combined particulated juvenile cartilage allograft transplantation and medial patellofemoral ligament reconstruction for symptomatic chondral defects in the setting of recurrent patellar instability.Arthrosc Tech.2016;5(5):1149-1154.
[33]Buckwalter JA,Bowman GN,Albright JP,et al.Clinical outcomes of patellar chondral lesions treated with juvenile particulated cartilage allografts.lowa Orthop J.2013;34(1)44-49.
[34]Kruse DL,Ng A,Paden M,et al.Athroscopic De Novo NT(R)juvenile allograft cartilage implantation in the talus:a case presentation.J Foot Ankle Surg.2012;51(2):218.
[35]Dunn JC,kusnezov N,Orr J,et al.Ostochondral defects of the upper extremity treated with particulated juvenile cartilage transfer.HAND.2015;10(4):683-687.
[36]Pascual-Garrido C,Hao J,Schrock J,et al.Arthroscopic juvenile allograft cartilage implantation for cartilage lesions of the hip.Arthrosc Tech.2016;5(4)929-933.
[37]Adkisson HD,Martin JA,Amendola RL,et al.The potential of human allogeneic juvenile chondrocytes for restoration of articular cartilage.Am J Sports Med.2010;38(7):1324-1333.
[38]Smeriglio P,Lai JH,Dhulipala L,et al.Comparative potential of juvenile and adult human articular chondrocytes for cartilage tissue formation in three-dimensional biomimetic hydrogels.Tissue Eng Part A.2015;21(1-2)147-155.
[39]Skaalure SC,Milligan IL,Bryant SJ.Age impacts extracellular matrix metabolism in chondrocytes encapsulated in degradable hydrogels.Biomed Mater.2012;7(2):024111.
[40]Bonasia DE,Martin JA,Marmotti A,et al.Cocultures of adult and juvenile chondrocytes compared with adult and juvenile chondral fragments:in vitro matrix production.Am J Sports Med.2011;39(11):2355-2361.
[41]Marmotti A,Bonasia DE,Bruzzone M,et al.Human cartilage fragments in a composite scaffold for single-stage cartilage repair:an in vitro study of the chondrocyte migration and the influence of TGF-beta1 and G-CSF.Knee Surg Sports Traumatol Arthrosc.2013;21(8):1819-1833.
[42]Adkisson HD,Milliman C,Zhang X,et al.Immune evasion by neocartilage-derived chondrocytes:Implications for biologic repair of joint articular cartilage.Stem Cell Res.2010;4(1):57-68.
[43]Tompkins M,Adkisson HD,Bonner KF.DeNovo NT Allograft.Oper Tech Sports Med.2013;21(2):82-89.
[2]Min BH,Choi WH,Lee YS,et al.Effect of different bone marrow stimulation techniques(BSTs)on MSCs mobilization.J Orthop Res.2013;31(11):1814-1819.
[3]Bedi A,Feeley BT,Williams RJ.Management of Articular Cartilage Defects of the Knee.J Bone Joint Surg Am.2010;92(4):994-1009.
[4]Perterson L,Minas T,Brittberg M,et al.Two-to-9-year outcome after autologous chondrocyte transplantation of the knee.Clin Orthop Relat Res.2000;374:212-214.
[5]Haddo O,Mahroof S,Higgs D,et al.The use of chondrogide membrane in autologous chondrocyte implantation.The knee.2004;(11)1:51-55.
[6]Filardo G,Kon E,Di Martino A,et al.Second-generation arthroscopic autologous chondrocyte implantation for the treatment of degenerative cartilage lesions.Knee Surg Sports Traumatol Arthrosc.2012;20(9):1704-1713.
[7]Kon E,Delcogliano M,Filardo G,et al.A novel nano-composite multi-layered biomaterial for treatment of osteochondral lesion:technique note and an early stability pilot clinical trial.Injury.2010;41(7):693-701.
[8]Gille J,Behrens P,Schulz AP,et al.Matrix-Associated Autologous Chondrocyte Implantation:A Clinical Follow-Up at 15 Years.Cartilage.2016;7(4):309-315.
[9]Karoubi G,Ormiston ML,Stewart DJ,et al.Single-cell hydrogel encapsulation for enhanced survival of human marrow stromal cells.Biomaterials.2009;30(29):5445-5455.
[10]Shimizu H,Ohashi K,Utoh R,et al.Bioengineering of a functional sheet of islet cells for the treatment of diabetes mellitus.Biomaterials.2009;30(30):5943-5949.
[11]Hayashi S,Kamei N,Ikuta Y,et al.Chondrocyte cell-sheet transplantation for treating monoiodoacetate-induced arthritis in rats.Tissue Eng Part C Methods.2017;23(6):346-356.
[12]Xu X,Shi D,Liu Y,et al.Synovium-derived mesenchymal stem cell sheet enhance autologous osteochondral transplantation in a rabbit momdel.Int J Clin Exp Med.2016;9(6):10322-10332.
[13]Shaari CM,Farber D,Brandwein MS,et al.Chracterizing the antigenic profile of the human trachea:Implications for tracheal transplantation.Head Neck.1998;20(6):522-527
[14]Liu K,Zhou GD,Liu W,et al.The dependence of in vivo stable ectopic chondrogenesis by human mesenchymal stem cells on chondrogenic differentiation in vitro.Biomaterials.2008;29(14):2183-2192.
[15]Ge Y,Gong YY,Xu Z,et al.The application of sheet technology in cartilage tissue engineering.Tissue Eng Part B Rev.2016;22(2):114-124.
[16]Mouthuy PA,Elsherbini Y,Cui Z,et al.Layering PLGA-based electrospum membranes and cell sheets for engineering cartilage-bone transition.Tissue Eng Regen Med.2016;10(4):263-274.
[17]Verdonk P,Dhollander A,Almqvist KF,et al.Treatment of osteochondral lesions in the knee using a cell-free scaffold.Bone Joint J.2015;97-B(3):318-323.
[18]Brix M,Kaipel M,Kellner R,et al.Successful osteoconduction but limited cartilage tissue quality following osteochondral repair by a cell-free multilayered nano-composite scaffold at the knee.Int Orthop.2016;40(3):625-632.
[19]Filardo G,Kon E,Di MA,et al.Treatment of knee osteochondritis dissecans with a cell-free biomimetic osteochondral scaffold:clinical and imaging evaluation at 2-year follow up.am J Sports Med.2013;41(8):1786-1793.
[20]Albrecht F,Roessner A,Zimmermann E.Closure of osteochondral lesions using chondral fragments and fibrin adhesive.Arch Orthop Trauma Surg.1983;101(3):213-217.
[21]Riboh JC,Cole BJ,Farr J.Particulated articular cartilage for symptomatic chondral defects of the knee.Curr Rev Musculoskelet Med.2015;11(1):21-34.
[22]Bonasia DE,Marmotti A,Rosso F,et al.Use of chondral fragments for one stage caitilage repair:a systematic review.World J Orthop.2015;6(11):1006.
[23]宁志刚,杨柳,王富友,等.保留钙化层结构的猪股骨滑车全厚软骨缺损模型建立[J].中国修复重建外科杂志,2012,26(5):527-531.
[24]Brittberg M,Aglietti P,Gambardella R,et al.ICRS Cartilage Injury Evaluation Package.2002:16-18.https://www.secot.es/uploads/descargas/formacion/escalas_valoracion/ICRS._TRAUMA_CARTaILAGO.pdf
[25]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 2(1):45-57.
[26]Kontturi LS,Jarvinen E,Muhonen V,et al.An injectable,insitu forming type IIcollagen/hyaluronic acid hydrogel vehicle for chondrocyte delivery in cartilage tissue engineering.Drug Deliv Transl Res.2014;4(2):149-158.
[27]Cole BJ,Farr J,Winalski CS,et al.Outcomes after a single-stage procedure for cell-based cartilage repair:a prospective clinical safety trial with 2-year follow-up.Am J Sports Med.2011;39(6):1170-1179.
[28]Kon E,Filardo G,Zaffagnini S,et al.Biodegradable polyurethane meniscal scaffold for isolated partial lesions or as combined procedure for knees with multiple comorbidities:clinical results at 2 years.Knee Surg Sports Traumatol Arthrosc.2014;22(1):128-134.
[29]Farr J,Yao JQ.Chondral Defect Repair with Particulated Juvenile Cartilage Allograft.Cartilage.2011;2(4):346-353.
[30]Farr J,Tabet SK,Margerrison E,et al.Clinical,Radiographic,and histological outcomes after cartilage repair with particulated juvenile articular cartilage:a 2-year prospective study.Am JSports Med.2014:42(6):1417.
[31]Mcmillan S,Light GT,Brtz C.All-arthroscopic implantation of minced juvenile chondral allograft for an isolated,full-thickness chondral lesion in the trochlea of an adult knee.Arthrosc Tech.2016;5(2):397-401.
[32]Arshi A,Wang D,Jones KJ.Combined particulated juvenile cartilage allograft transplantation and medial patellofemoral ligament reconstruction for symptomatic chondral defects in the setting of recurrent patellar instability.Arthrosc Tech.2016;5(5):1149-1154.
[33]Buckwalter JA,Bowman GN,Albright JP,et al.Clinical outcomes of patellar chondral lesions treated with juvenile particulated cartilage allografts.lowa Orthop J.2013;34(1)44-49.
[34]Kruse DL,Ng A,Paden M,et al.Athroscopic De Novo NT(R)juvenile allograft cartilage implantation in the talus:a case presentation.J Foot Ankle Surg.2012;51(2):218.
[35]Dunn JC,kusnezov N,Orr J,et al.Ostochondral defects of the upper extremity treated with particulated juvenile cartilage transfer.HAND.2015;10(4):683-687.
[36]Pascual-Garrido C,Hao J,Schrock J,et al.Arthroscopic juvenile allograft cartilage implantation for cartilage lesions of the hip.Arthrosc Tech.2016;5(4)929-933.
[37]Adkisson HD,Martin JA,Amendola RL,et al.The potential of human allogeneic juvenile chondrocytes for restoration of articular cartilage.Am J Sports Med.2010;38(7):1324-1333.
[38]Smeriglio P,Lai JH,Dhulipala L,et al.Comparative potential of juvenile and adult human articular chondrocytes for cartilage tissue formation in three-dimensional biomimetic hydrogels.Tissue Eng Part A.2015;21(1-2)147-155.
[39]Skaalure SC,Milligan IL,Bryant SJ.Age impacts extracellular matrix metabolism in chondrocytes encapsulated in degradable hydrogels.Biomed Mater.2012;7(2):024111.
[40]Bonasia DE,Martin JA,Marmotti A,et al.Cocultures of adult and juvenile chondrocytes compared with adult and juvenile chondral fragments:in vitro matrix production.Am J Sports Med.2011;39(11):2355-2361.
[41]Marmotti A,Bonasia DE,Bruzzone M,et al.Human cartilage fragments in a composite scaffold for single-stage cartilage repair:an in vitro study of the chondrocyte migration and the influence of TGF-beta1 and G-CSF.Knee Surg Sports Traumatol Arthrosc.2013;21(8):1819-1833.
[42]Adkisson HD,Milliman C,Zhang X,et al.Immune evasion by neocartilage-derived chondrocytes:Implications for biologic repair of joint articular cartilage.Stem Cell Res.2010;4(1):57-68.
[43]Tompkins M,Adkisson HD,Bonner KF.DeNovo NT Allograft.Oper Tech Sports Med.2013;21(2):82-89.