自体组织工程生长板修复兔长骨生长板损伤的实验研究
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摘要
由创伤、感染等引起的长骨生长板损伤，会引起骨骺与干骺端之间形成骨桥，由此导致的肢体短缩与成角畸形一直是小儿矫形外科医师面临的重大挑战，至今尚无可靠的治疗方案。1967年Lanlgenskiold首次报道用骨桥切除、脂肪填塞的方法治疗生长板部分早闭取得一定效果。自此，人们不断尝试将硅胶、骨蜡、脂肪、游离生长板及血管化生长板等不同移植物用于治疗生长板损伤，取得了一定效果。但这些材料及方法均存在不同的缺点及问题，游离软骨和血管化生长板移植修复存在移植物的来源受限，供、受体匹配难及移植物免疫排斥等问题，非软骨类组织如脂肪、肌肉、骨蜡和硅胶等不能再生修复生长板，只能防止小范围生长板损伤并肢体畸形，不能改善肢体生长发育。改进生长板损伤修复方法及寻找更理想的植入材料仍是小儿四肢矫形外科面临的一大难题。二十世纪九十年代以来，组织工程学科的兴起为治疗生长板损伤提供了契机。本实验采用组织工程技术，尝试用自体组织工程生长板治疗长骨生长板损伤，期望为临床解决生长板损伤修复难题奠定基础。
一 兔髂骨生长板细胞的培养及体内成软骨作用的研究
目的：建立大量生产髂骨生长板软骨细胞的培养及保存方法，观察髂骨生长板细胞在裸鼠体内的成软骨作用，为生长板组织工程提供种子细胞。方法：取二周龄兔髂骨生长板软骨，经机械剪切和化学消化作用后，接种、培养及液氮保存，通过显微镜观察其生物
第四军医大学硕士学位论文
学表现，并通过甲苯胺兰、碱性磷酸酶〔alkaline phosphatase，ALP)
染色及n型胶原染色对其生物学特性进行鉴定。将生长良好的第
二代细胞注入裸鼠背部，于第四周取材，进行组织学观察。结果:
细胞可在体外大量增殖并在六代内无明显反分化，但缺乏向肥大细
胞进一步分化的能力。冷冻复苏细胞存活率为92%。注射的细胞
悬液可在裸鼠体内形成软骨组织，并进一步向肥大细胞转化.结论:
成功建立了骼骨生长板细胞的培养及冻存方法，证明骼骨生长板细
胞可在体内进一步形成软骨组织并向肥大期细胞转化。
二兔骼骨生长板细胞和脱钙骨墓质共培养构建组织工程生长板
目的:探索兔骼骨生长板细胞和同种异体脱钙骨基质
(demineralized bone matrix，DBM)共培养构建组织工程生长板的可
行性。方法:取三周龄兔骼骨生长板软骨细胞经传代培养后作为种
子细胞与制备的脱钙去抗原同种异体DBM于体外联合培养，通过
对复合物行扫描电镜(seanning electronie mierosco讲，sEM)观察
及固定后切片“HE”染色，了解细胞在材料中的生长情况.结果:
体外复合培养24小时，软骨细胞即开始贴附于DBM网架上;复合培
养7天，分布于支架材料上的生长板细胞迅速分化增殖，分泌细胞外
基质:第21天，细胞长满支架，重叠生长。结论:同种异体DBM
和骼骨生长板细胞共同培养体外构建组织工程生长板是可行的。
三自体组织工程生长板治疗长骨生长板损伤的实脸研究
目的:探讨自体组织工程生长板在治疗生长板损伤中的作用。
方法:自三周龄兔骼蜻处切取部分铭骨生长板软骨经机械剪切和
n型胶原酶消化后培养生长板软骨细胞，体外单层培培养扩增后接
种到同种异体脱钙骨基质(demine司ized bone matrix，DBM)上，
混合培养一周后，植入兔自体右侧胫骨上端内侧生长板模型缺损处
(A组)，分别设单纯DBM植入(B组)及单纯破坏即生长板破
坏后无任何植入物(C组)作为对照组，左侧胫骨不作处理。术后
动态“X”线摄片测量下肢短缩及成角畸形改变，通过组织切片
第四军医大学硕士学位论文
“HE”染色及11型胶原免疫组化(imm~histochemistry)染色观
察自体组织工程生长板在体内的转归。结果:术后第二周三组右侧
胫骨均出现轻度畸形，A、B组较C组为轻，但三者之间无显著差
异(p>0.05)，此后B、C组兔右侧胫骨出现渐进性严重短缩及成
角畸形，而A组右侧胫骨畸形则无明显加重，各时间点A组胫骨
成角及缩短畸形与B、C组有显著差异(P<0.05)，术后第十六周
时组织切片显示，损伤区基本恢复正常生长板结构，H型胶原免疫
组化染色阳性，而B、C组损伤区则由新生骨组织修复。结论:自
体组织工程生长板植入可有效防止急性生长板损伤后肢体畸形的
发生，植入的组织工程生长板可产生柱状排列的结构，细胞可表达
n型胶原。
四人胚肋软骨静止区细胞的体外培养及生物学特性研究
目的:研究体外培养的人胚肋软骨静止区细胞的生物学特性
及分化规律，为组织工程生长板的研究提供种子细胞。方法:采用
11型胶原酶消化的方法获取人胚肋软骨静止区细胞，观察细胞形态
学变化;测定细胞生长曲线;借助特殊染色、免疫荧光染色的方法了
解糖胺聚糖、碱性磷酸酶(alkaline phosPhatase，ALP)、11型胶原
酶的合成情况。结果:贴壁后细胞呈多角形，从第六代开始绝大多
数细胞转变为成纤维细胞状，细胞培养至第二代开始分泌大量n
型胶原。结论:体外单层培养的人胚肋软骨静止区细胞具有正常的
生长增殖与分化能力，可分化至成熟期软骨细胞。
Bone bridge will form between epiphysis and metaphysis if the growth plate of long bone is injured by wound ,infection and tumor et al. The shortening and angulation of limbs caused by bone bridge is one of the big problems which many orthopedists encounters. In 1967,Langenskiold first reported that the partly early closure of the growth plate can be treated by the method of bone bridge cutting and fat packing .Since that ,people attempted to solve the problem by transplanting different grafts such as silica gel ,bone wax , fat and free growth plate et al. The above methods have some effects ,but they also have many defects. Immune expelling between the supply and the recipient is one of the dilemmas. Fat, muscle and bone wax only can prevent bone bridge forming caused by gentle injury instead of the growth plate regenerating, they also can not improve the growth and development of the limbs. In recent years, people try to solve the problem by the method of tissue engineering. We attempt to treat the injury
of long bone growth plate by using the tissue-engineered autologous growth plate established in vitro. The experiment aims to establish the foundation for the clinical solving of the tough problem. 1.The culture of the iliac growth-plate chondrocytes and
the study on its cartilage-formation role in vivo Objective:To establish a kind of iliac growth-plate chondrocyte culture and storage method so that we can obtain the seed cells in large quantity for the usage of engineered growth plate, tissue.To observe the cartilage formation of the cells in vivo. MethodsrThe cells were harvested from the iliac growth-plate cartilage of the two-week old rabbit and cultured. Their biological phenomenons were observed and their biological characteristics were identified by using toluidine blue, alkaline phosphatase staining and immunohistochemistry staining for type II collagen. The second generation cells were harvested and injected into the dorsum of nude mice. Specimens were harvested 4 weeks later for histological examination. Results:The cells could proliferate in vitro and have no apparent de-differentiation within the sixth generation, but they could not differentiate into the hypertrophic cells. The cells revived from being frozen(-196癈) was 92%. The cells injected into the body of the nude mice could form the cartilage-like tissue. The iliac growth-plate chondrocytes can differentiate into hypertrophic cells in vivo. Conclusion:We have established a method of culture and storage of iliac growth-plate chondrocytes and cartilage-formation of the cells in vivo was verified. The cells can differentiate into hypertrophic chondrocytes in vivo.
2.Experimental study on the establishment of tissue-engineered growth-plate by allograft demineralized bone matrix cocultured with the rabbit iliac growth-plate cells
Objective:To investigate the feasibility of establishing tissue-engineered growth-plate by allograft demineralized bone matrix cocultured with the rabbit iliac growth-plate cells. Methods: We obtain the growth plate cells from iliac crest epiphyseal cartilage of the three-week old New Zealand rabbits by dissection and sequential digestion with 0.2% collagenase(type II).After the cells proliferated with monolayer culture in vitro for three weeks.The cells were harvested and cocultured with the scaffold DBM.The cell-material complex was observed by "HE" staining and electronic scanning microscope in order to evaluate the interaction between cells and DBM. Results: Twenty-four hours after coculture, the cells adhered to DBM scaffolds; Seven days later, the growth-plate chondrocytes proliferated in the DBM network and secret the extracellular matrix; The cells grow all formed on the surface of the scaffold after three weeks. Conclusion: We can successfully establish the tissue-engineering growth-plate by allograft demineralized bone matrix cocultured with the rabbit iliac growth-plate cells .
3.The experimental study on the treatment of growth plate injuries with a tissue-engineered autologous growth plate Objective.
一 兔髂骨生长板细胞的培养及体内成软骨作用的研究
目的：建立大量生产髂骨生长板软骨细胞的培养及保存方法，观察髂骨生长板细胞在裸鼠体内的成软骨作用，为生长板组织工程提供种子细胞。方法：取二周龄兔髂骨生长板软骨，经机械剪切和化学消化作用后，接种、培养及液氮保存，通过显微镜观察其生物
第四军医大学硕士学位论文
学表现，并通过甲苯胺兰、碱性磷酸酶〔alkaline phosphatase，ALP)
染色及n型胶原染色对其生物学特性进行鉴定。将生长良好的第
二代细胞注入裸鼠背部，于第四周取材，进行组织学观察。结果:
细胞可在体外大量增殖并在六代内无明显反分化，但缺乏向肥大细
胞进一步分化的能力。冷冻复苏细胞存活率为92%。注射的细胞
悬液可在裸鼠体内形成软骨组织，并进一步向肥大细胞转化.结论:
成功建立了骼骨生长板细胞的培养及冻存方法，证明骼骨生长板细
胞可在体内进一步形成软骨组织并向肥大期细胞转化。
二兔骼骨生长板细胞和脱钙骨墓质共培养构建组织工程生长板
目的:探索兔骼骨生长板细胞和同种异体脱钙骨基质
(demineralized bone matrix，DBM)共培养构建组织工程生长板的可
行性。方法:取三周龄兔骼骨生长板软骨细胞经传代培养后作为种
子细胞与制备的脱钙去抗原同种异体DBM于体外联合培养，通过
对复合物行扫描电镜(seanning electronie mierosco讲，sEM)观察
及固定后切片“HE”染色，了解细胞在材料中的生长情况.结果:
体外复合培养24小时，软骨细胞即开始贴附于DBM网架上;复合培
养7天，分布于支架材料上的生长板细胞迅速分化增殖，分泌细胞外
基质:第21天，细胞长满支架，重叠生长。结论:同种异体DBM
和骼骨生长板细胞共同培养体外构建组织工程生长板是可行的。
三自体组织工程生长板治疗长骨生长板损伤的实脸研究
目的:探讨自体组织工程生长板在治疗生长板损伤中的作用。
方法:自三周龄兔骼蜻处切取部分铭骨生长板软骨经机械剪切和
n型胶原酶消化后培养生长板软骨细胞，体外单层培培养扩增后接
种到同种异体脱钙骨基质(demine司ized bone matrix，DBM)上，
混合培养一周后，植入兔自体右侧胫骨上端内侧生长板模型缺损处
(A组)，分别设单纯DBM植入(B组)及单纯破坏即生长板破
坏后无任何植入物(C组)作为对照组，左侧胫骨不作处理。术后
动态“X”线摄片测量下肢短缩及成角畸形改变，通过组织切片
第四军医大学硕士学位论文
“HE”染色及11型胶原免疫组化(imm~histochemistry)染色观
察自体组织工程生长板在体内的转归。结果:术后第二周三组右侧
胫骨均出现轻度畸形，A、B组较C组为轻，但三者之间无显著差
异(p>0.05)，此后B、C组兔右侧胫骨出现渐进性严重短缩及成
角畸形，而A组右侧胫骨畸形则无明显加重，各时间点A组胫骨
成角及缩短畸形与B、C组有显著差异(P<0.05)，术后第十六周
时组织切片显示，损伤区基本恢复正常生长板结构，H型胶原免疫
组化染色阳性，而B、C组损伤区则由新生骨组织修复。结论:自
体组织工程生长板植入可有效防止急性生长板损伤后肢体畸形的
发生，植入的组织工程生长板可产生柱状排列的结构，细胞可表达
n型胶原。
四人胚肋软骨静止区细胞的体外培养及生物学特性研究
目的:研究体外培养的人胚肋软骨静止区细胞的生物学特性
及分化规律，为组织工程生长板的研究提供种子细胞。方法:采用
11型胶原酶消化的方法获取人胚肋软骨静止区细胞，观察细胞形态
学变化;测定细胞生长曲线;借助特殊染色、免疫荧光染色的方法了
解糖胺聚糖、碱性磷酸酶(alkaline phosPhatase，ALP)、11型胶原
酶的合成情况。结果:贴壁后细胞呈多角形，从第六代开始绝大多
数细胞转变为成纤维细胞状，细胞培养至第二代开始分泌大量n
型胶原。结论:体外单层培养的人胚肋软骨静止区细胞具有正常的
生长增殖与分化能力，可分化至成熟期软骨细胞。
Bone bridge will form between epiphysis and metaphysis if the growth plate of long bone is injured by wound ,infection and tumor et al. The shortening and angulation of limbs caused by bone bridge is one of the big problems which many orthopedists encounters. In 1967,Langenskiold first reported that the partly early closure of the growth plate can be treated by the method of bone bridge cutting and fat packing .Since that ,people attempted to solve the problem by transplanting different grafts such as silica gel ,bone wax , fat and free growth plate et al. The above methods have some effects ,but they also have many defects. Immune expelling between the supply and the recipient is one of the dilemmas. Fat, muscle and bone wax only can prevent bone bridge forming caused by gentle injury instead of the growth plate regenerating, they also can not improve the growth and development of the limbs. In recent years, people try to solve the problem by the method of tissue engineering. We attempt to treat the injury
of long bone growth plate by using the tissue-engineered autologous growth plate established in vitro. The experiment aims to establish the foundation for the clinical solving of the tough problem. 1.The culture of the iliac growth-plate chondrocytes and
the study on its cartilage-formation role in vivo Objective:To establish a kind of iliac growth-plate chondrocyte culture and storage method so that we can obtain the seed cells in large quantity for the usage of engineered growth plate, tissue.To observe the cartilage formation of the cells in vivo. MethodsrThe cells were harvested from the iliac growth-plate cartilage of the two-week old rabbit and cultured. Their biological phenomenons were observed and their biological characteristics were identified by using toluidine blue, alkaline phosphatase staining and immunohistochemistry staining for type II collagen. The second generation cells were harvested and injected into the dorsum of nude mice. Specimens were harvested 4 weeks later for histological examination. Results:The cells could proliferate in vitro and have no apparent de-differentiation within the sixth generation, but they could not differentiate into the hypertrophic cells. The cells revived from being frozen(-196癈) was 92%. The cells injected into the body of the nude mice could form the cartilage-like tissue. The iliac growth-plate chondrocytes can differentiate into hypertrophic cells in vivo. Conclusion:We have established a method of culture and storage of iliac growth-plate chondrocytes and cartilage-formation of the cells in vivo was verified. The cells can differentiate into hypertrophic chondrocytes in vivo.
2.Experimental study on the establishment of tissue-engineered growth-plate by allograft demineralized bone matrix cocultured with the rabbit iliac growth-plate cells
Objective:To investigate the feasibility of establishing tissue-engineered growth-plate by allograft demineralized bone matrix cocultured with the rabbit iliac growth-plate cells. Methods: We obtain the growth plate cells from iliac crest epiphyseal cartilage of the three-week old New Zealand rabbits by dissection and sequential digestion with 0.2% collagenase(type II).After the cells proliferated with monolayer culture in vitro for three weeks.The cells were harvested and cocultured with the scaffold DBM.The cell-material complex was observed by "HE" staining and electronic scanning microscope in order to evaluate the interaction between cells and DBM. Results: Twenty-four hours after coculture, the cells adhered to DBM scaffolds; Seven days later, the growth-plate chondrocytes proliferated in the DBM network and secret the extracellular matrix; The cells grow all formed on the surface of the scaffold after three weeks. Conclusion: We can successfully establish the tissue-engineering growth-plate by allograft demineralized bone matrix cocultured with the rabbit iliac growth-plate cells .
3.The experimental study on the treatment of growth plate injuries with a tissue-engineered autologous growth plate Objective.
引文
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25. Pho RW, Patterson MH, Kour AK, Kumar VP. Free vascularised epiphyseal transplantation in upper extremity reconstruction. J Hand Surg(Am), 1988, 13: 440-447
26．杜晓冰，邰勇华，区士欢 骺板移植的实验形态学观察．中国临床解剖学杂志，1995，13：297-300
27. Glickman AM, Yang JP, Stevens DG, Bowen CV. Epiphyseal plate transplantatio n between sites of different growth potential. J Pediatr Orthop, 2000; 20(3): 289-295
28. Stevens DG, Boyer MI, Bowen CVA. Transplantation of epi-physeal plate allografts between animals of different ages. J Pediatr Orthop, 1999; 19(3): 398-403
29. Drzewiecki AE, Randolph MA, Hotchkiss RH, Weiland AJ. Vascu-larized growth plate transplantation: A comparative study in the rat. J Reconstr Mierosurg, 1992; 8(2): 93-100
30. Boyer MI, Danska JS, Nolan L, Kiral A, Bowen CV. Microvaseular trans-plantation of physeal atlografts. J Bone Joint Surg(Am), 1995; 77(B): 806-814
31. Boyer MI, Bowen CVA, Danska JS. Microvascular allo-graft growth plate transplantation. J Bone Joint Surg(Br), 1993; 75: supplⅢ: 245
32. Mohtai M, Hotokebuchi T, Arai K, Sugioka Y. Changes in growth plate morphology associated with rejection of rat limb allo-grafts. J Bone Joint Surg (Am), 1992; 74(9): 1375-1384
33. Rudolph B, Dallek M, Meenen NM, Jungbluth KH. Behavior of artificially-induced epiphyseal groove defects in rabbits. Part 2. Transplantation of autologous and homologous groove cartilage [J] Unfallchirurgie. 1993, 19(3): 131-137.
34. Dallek M, Meenen N M, Jungbluty K H. Behavior of artificially-induced epiphyseal groove defects. part
3: Transplantation of autologous and homologous rib cartilage in Gottingen miniplgs. Fingdings after a 16 week interval[J] Unfallchirurgie, 1995, 21(5): 219-226
35 Mikami H, Fresh allograft of intact growth plate into immature articular cartilage defects in rats [J]. Nippin Seikeigeka Gakkai Zasshi, 1992, 66(5): 539-547
36. Mohtai M, HotokebuChi T, Arai K, Sugioka Y. Changes in growth-plate morphology associated with rejection of rat-limb allografts [J] J Bone Joint Surg(Am), 1992, 74(9): 1375-1384
37. Barr SJ, Zaleske DJ. Physeal reconstruction with blocks of cartilage of varying developmental time[J]. J Pediatr Orthop, 1992, 12(6): 766-773
38. Hyc A, Malejczyk J, Osiecka A, Moskalewski S. Immunological response against allogeneic Chondrocytes Transplanted into joint surface defects in rats [J] Cell Transplant, 1997, 6(2): 119-124
39. Cundy PJ, Jofe M, Zaleske DJ, Ehrlich MG, Mankin HJ. Physeal reconstruction using tissue donated from early pastnatal limbs in a murine model [J], J Ortho Res[J]. 1991, 9(3): 360-366
40．李晓光，邢国强．邢岩胚胎骨骺移植修复关节而缺损的实验研究[J]．白求恩医科大学学报．1997，23(2)：160
41. Bowen CV, Bray PW, Boyer MI, Fowler JD, Nolan L. Short-term response of epiphyseal plate cell population following selectives devascularization and microsurgical revascularization[J], Microsurgery, 1994, 15(8): 555-562
42. Kato Y, Iwamoto M, Koike T, Suzuki F, Takano Y Terminal differentiation and calcification in rabbit chondrocyte cultures in centrifuge tubes: Regulation by transforming growth factor β and serum factors [J]. Proc Natl Acad Sci USA, 1988, 85(24): 9552.9556.
43. Yan WQ, Tong MH, Yang TS. Reorganization of isolated chondrocytes into growth plate-like tissue in pelleted cultures[J], J Bone Miner Metab, 1995, 13(2): 77-80
44. Ballock RT, Reddi AH. Thyroxine is the factor that regulates morphogenesis of columar cartilage from isolated chondrocyte in chemically defined medium [J]. J Cell Biol, 1994, 126(5): 1311-1318
45．周强，李起鸿，戴刚等，杨柳高数量骺板软骨细胞聚集培养生成软骨组织组织的观察[J]第三军医大学学报，2002，24(5) 556-558
46．周强，李起鸿，戴刚，杨柳 经离心管培养骺软骨异体修复兔骺板部分损伤的初步观察[J]第三军医大学学报，2001，23(10)：1139-1142
47. Ballock RT, Zhou X, Mink LM, Chen DH, Mita BC. Both retinoic acid and 1, 25(OH)2 vitamin D3 inhibit thyroid hormone-induced terminal defferentiation of growth plate chondrocytes[J]J Orthop Res, 2001, 19(1): 43-49,
48. Ballock RT, Zhou X, Mink LM, Chen DH, Mita BC, Stewart MC. Expression of cyclin-dependent kinase ingibitors in epiphyseal chondrocytes indeced to terminally defferentiate with thyoid hormone [J], Endocrinology, 2000, 141(12): 4552-4557
49. Estrada LE, Dodge GR, Richardson DW, Farole A, Jimenez SA. Characterization of a biomaterial with cartilage-like properties expressing type Xeollagen generated in vitro using neonatal porcine articular and growth palte chondroeytes[J]Osteoarthritis cartilage, 2001, 9(2): 169-177
50. Kawabe N, Yoshinao M. The repair of full-thickness articular cartilage defects. Immune responses to reparativetissue formed by allogeneic growth plate chondrocyte implants[J]. Clin Orthop, 1991, (268): 279-293
51. Freyria AM, Ronziere MC, Roche S, Rousseau CF, Herbage D. Regulation of growth, protein synthesis, and maturation of fetal bovineepiphyseal chondroeytes grown in high-density culture in the presence of ascorbic acid, retinoic acid, and dihydrocytochalasin B [J]. J cell Biochem, 1999, 76(1): 84-98
52．周勇刚，卢世璧，王继芳，张永刚，黄靖香，组织工程软
骨用于骨骺早闭治疗的实验研究中华外科杂志，2000，38，：742-744
53．周强，李起鸿，杨柳，戴刚骺板软骨细胞复合生物凝胶．培养生成软骨组织的观察[J]，第三军医大学学报，2002，24(5)：559-562
54. Roche S, Ronziere MC, Herbage D, Freyria AM. Native and DPPA cross-linked collagen sponges seeded with fetal bovine epiphyseal chondrocytes used for cartilage tissue engineering [J] Biomaterials, 2001, 22(1)9-18
55. Tobita M, Ochi M, Uchio YmMori R, Iwasa J, Katsube K, Motomura T. Treatment of growth plate injury with autogenous chondrocytes: a study in rabbits. Acta Orthop Scand 2002; 73(3): 352-358
56．杨志明，王建，秦延武，解慧琪 组织工程软骨移植修复兔生长板缺损中华骨科杂志2002，22(2)：103-107
57．刘建国，徐执扬，马卫华，齐欣，黄岚峰，崔林，徐莘香，生物活性PCL／PLA三维多孔块膜为载体的骺软骨细胞体外实验研究 骨与关节损伤杂志2001，16(5)：355-358
58. Lee EH, Chen F, Chan J, Bose K. Treatment of growth arrest by transfer of cultured chondrocytes into physeal defects[J], J Pediatr Orthop, 1998, 18(2): 155-160
59. Jouve JL, Mottet V, Cottalorda J, Frayssinet P, Bollini G. Reimplantation of growth plate chondrocyte cultures in central growth plate defects: Part Ⅰcharacterization of cultures[J], J Pediatr Orthop B, 1998, 7(2)167-173
60. Jouve JL, Cottalorda J, Mottet V, Frayssinet P, Petit P, Bollini G. Reimplantation of growth plate chondrocyte cultures in central growth plate defects: Part ⅡSurgecal experimentation in rabbits[J], J Pediatr Orthop B, 1998, 7(2)167-173
61. Lee CW, Martinek V, Usas A, Musgrave D, Pickvance EA, Robbins P, Moreland MS, Fu FH, Huard J. Muscle-Based Gene Thrapy and Tissue Engineering for Treatment of Growth Plate Injuries. Journal of Pediatric Orthopaedics
2002, 22: 565-572
62. Trippel SB, Wroblewski J, Makower AM, Whelan MC, Schoenfeld D, Doctrow SR. Regulation of growth-plate chondrocytes by insulin-like growth factor Iand basic fibroblast growth factor. J Bone Joint Surg[Am] 1993, 75: 177-189
63. Rbson H, Siebler T, Stever DA. Thyroid hormone acts directly on growth plate chondrocytes to promote hypertrophic differentiation and inhibit clonal expansion and cell proliferation. Endocrinology 2000, 14(10): 3887-97
64. Juppner H, Role of parathyroid hormone-related peptide and Indian hedgehog in skeletal development. Pediar Nephrol. 2000, 14: 606-611
65. Umehara T, Immunohistochemical localization of transforming growth factor-beta 1 in growth plate freshly transplanted into a full-thickness defectin articular cartilage [J], Nippon Seikeigeka Gakkai Zasshi, 1994, 65(9): 790-797
66. Foster BK, Hansen AL, Gibson GJ, Hopwood JJ, Binns GF, Wiebkin OW. Reimplantation of growth plate chondrocytes into growth plate defects in sheep. J Orthop Res 1990 Jul; 8(4): 555-64
67. Park JS, Ahn JI, Oh DI. Chondroeytes alllgraft transplantation for damaged growth plate reconstruction. Yousei Med J 1994 Dec: 35(4): 375-87
68．王建 杨志明 解慧琪 培养软骨移植修复兔生长板缺损中国修复重建外科杂志 2001，15(1)：(53-56)
69. Schwartz Z, Somers A, Mellonig JT, Carnes DL Jr, Wozney JM, Dean DD, Cochran DL, Boyan BD. Addition of human recombinant bone morphogenetic protein-2 to inactive commercial human demineralized freeze-dried bone allograft make an effective composite bone inductive implant material. J Periodontol. 1998, 69(4): 470
70. Darrouzet V, Fizet D, Deminiere C, Baquey C, Aran JM, Bebear JP. Xenogenie ossieular implants: An experimental
study of heterotopic, demineralized, lyophilized, porine implants in the guinea pig. Clin Otolaryngol. 1999; 24(3): 190
71. Bright RW.Operative correction of partial epiphyseal plate closure by osseous-bridge resection and silicone-rubber implant. An experimental study in dogs. J Bone Joint Surg[Am] 1974; 56: 655-664
72. Broughton NS, Dickens DR, Cole WG, Menelaus MB. Epiphysealysis for partial growth plate arrest. Results after four years or at maturity. J Bone Joint Surg [Br]1989; 71: 13-6
73. Foster BK, John B, Haster C. Free fat interpositional graft in acute physeal injuries: the anticipatory Langenskiold procedure. J Pediatr Orthop 2000; 20: 282-285
74. Bowen CV, Ethridge CP, O'Brien BM, Frykman GK, Gumley GJ. Experimental micro-vascular growth plate transfers. Part Ⅰ: investigation of vascularity. J Bone Joint Surg[Br] 1988; 70: 305-10
75. Kawabe N, Ehrich MG, Mankin HJ. Growth plate reconstruction use chondrocyte allograft transplants. J Pediatr Orthop 1987, 7: 381-388
2. Langenskiold A. Surgical treatment of partial closure of the growth plate. J Pediatr Orthop, 1981; 1(1): 3-11
3. Langenskiold A, Videman T, Nevalainen T. The fate of fat transplants in operations for partial closure of the growth plate: Clinical examples and an experimental study. J Bone Joint Sury(Br), 1986; 68(2): 234-238
4. Peterson HA. Partial growth arrest and its treatment. J Pediatr Orthop. 1984, 4: 246-258
5. Bright TW. Partial growth arrest: identification, classification and results of treatment. Orthop Trans 1982, 6: 65-66
6. Williamson RV, Staheli LT. Partial physeal growth arrest: Treatment bybridge resection and fat interposition. J Pediatr Orthop, 1990; 10(6): 769-776
7. Kershaw CJ, Kenwright J. Epiphyseal distraction for bony bridges: a biomechanical and morphologie study. J Pediatr Orthop, 1993, 13: 46-50
8. Bostock SH, Peach BGS. Spontaneous resolution of an osseous bridge affecting the distal tibial epiphysis. J Bone Joint Surg(Br), 1996, 78B: 662-663
9. Osterman K. Healing of large surgical defects of the epiphyseal plate. An experimental study. Clin Orthop, 1994; 300: 264-268
10. Cady RB, Spadaro JA, Fitzgerald JA, Pinkes J, Albanese SA. The effects of fat interplwition for central physeal defects. Clin Orthop, 1992, 282: 304-309
11. Lee EH, Gao GX, Bose K. Management of partial growth arrest: physis, fat or silastic? J Pediatr Orthop, 1993, 13: 368-372
12. Lee EH, Chen F, Chan JW. The effects of surgery on the iliac
apophysis:a n experimental study. J Pediatr Orthop, 1998, 18(3): 406-409
13. Martiana K, Low CK, Tan SK, Pang MW. Comparison of various interpositional materials in the prevention of transphyseal bone bridge formation. Clin Orthop, 1996; 325: 218-224
14. Olin A, Creasman C, Shapiro F. Free physeal transplantation in the rabbit. An experimental approach to focal lesions. J Bone Joint Surg Am, 1954; 66(1): 7-20
15. Takato T, Harii K, Komuro Y, Yonehara Y. Ecperimental study on growth of epiphysial plate: free graft in rabbits, Br J Plast Surg. 1993 46(5): 416-20.
16．范丁安，张建立．朱振华．骺部分早闭再开放自体骺软骨充填术．中华小儿外科杂志 1996，17：349-351
17. Savarese JJ 3rd, Brinken BW, Zaleske DJ. Epiphyseal replacement in a murine model. J Pediatr Orthop, 1995; 15(5): 682-690
18. Boyer MI, Bray PW, Boven CVA. Epiphyseal plate transplantation: an historical review. Br J Plast Surg. 1994 47(8): 563-9
19. Manfrini M, Randolph MA, Andrisano A, Weiland AJ. Orthotopic knee grafts in rats: A model for growth plate transplantation. Microsurgery, 1988; 9(4): 242-245
20. Bowen CV, O'Brien BM, Gumley GJ. Experimental microvas-cular growth plate transfers: Part2-Investigation of feasibility. J Bone Joint Surg(Br), 1988; 70(2): 311-314
21. Innocenti M, Ceruso M, Manfrini M, Angeloni R, Lauri G, Capanna R, Bufalini C. Free vascularized growth plate transfer after bone tumor resection in children. J Reconstr Microsurg, 1995; 14(2): 137-143.
22. Vilkki SK. Distraction and micro vascular epiphysis transfer for radialclubhand. J Hand Surg Br, 1995; 23(4): 445-452
23. Tsai TM, Ludwig L, Tonkin M. Vascularized fibular epiphyseal transfer: a clinical study. Clin Orthop, 1956, 210: 225-234
24. Nunley JA, Spiegl PV, Goldner RD, Urbaniak JR. Longitudinal epiphyseal growth after replantation and transplantation in children. J Hand Surg(Am). 1987, 12A: 274-279.
25. Pho RW, Patterson MH, Kour AK, Kumar VP. Free vascularised epiphyseal transplantation in upper extremity reconstruction. J Hand Surg(Am), 1988, 13: 440-447
26．杜晓冰，邰勇华，区士欢 骺板移植的实验形态学观察．中国临床解剖学杂志，1995，13：297-300
27. Glickman AM, Yang JP, Stevens DG, Bowen CV. Epiphyseal plate transplantatio n between sites of different growth potential. J Pediatr Orthop, 2000; 20(3): 289-295
28. Stevens DG, Boyer MI, Bowen CVA. Transplantation of epi-physeal plate allografts between animals of different ages. J Pediatr Orthop, 1999; 19(3): 398-403
29. Drzewiecki AE, Randolph MA, Hotchkiss RH, Weiland AJ. Vascu-larized growth plate transplantation: A comparative study in the rat. J Reconstr Mierosurg, 1992; 8(2): 93-100
30. Boyer MI, Danska JS, Nolan L, Kiral A, Bowen CV. Microvaseular trans-plantation of physeal atlografts. J Bone Joint Surg(Am), 1995; 77(B): 806-814
31. Boyer MI, Bowen CVA, Danska JS. Microvascular allo-graft growth plate transplantation. J Bone Joint Surg(Br), 1993; 75: supplⅢ: 245
32. Mohtai M, Hotokebuchi T, Arai K, Sugioka Y. Changes in growth plate morphology associated with rejection of rat limb allo-grafts. J Bone Joint Surg (Am), 1992; 74(9): 1375-1384
33. Rudolph B, Dallek M, Meenen NM, Jungbluth KH. Behavior of artificially-induced epiphyseal groove defects in rabbits. Part 2. Transplantation of autologous and homologous groove cartilage [J] Unfallchirurgie. 1993, 19(3): 131-137.
34. Dallek M, Meenen N M, Jungbluty K H. Behavior of artificially-induced epiphyseal groove defects. part
3: Transplantation of autologous and homologous rib cartilage in Gottingen miniplgs. Fingdings after a 16 week interval[J] Unfallchirurgie, 1995, 21(5): 219-226
35 Mikami H, Fresh allograft of intact growth plate into immature articular cartilage defects in rats [J]. Nippin Seikeigeka Gakkai Zasshi, 1992, 66(5): 539-547
36. Mohtai M, HotokebuChi T, Arai K, Sugioka Y. Changes in growth-plate morphology associated with rejection of rat-limb allografts [J] J Bone Joint Surg(Am), 1992, 74(9): 1375-1384
37. Barr SJ, Zaleske DJ. Physeal reconstruction with blocks of cartilage of varying developmental time[J]. J Pediatr Orthop, 1992, 12(6): 766-773
38. Hyc A, Malejczyk J, Osiecka A, Moskalewski S. Immunological response against allogeneic Chondrocytes Transplanted into joint surface defects in rats [J] Cell Transplant, 1997, 6(2): 119-124
39. Cundy PJ, Jofe M, Zaleske DJ, Ehrlich MG, Mankin HJ. Physeal reconstruction using tissue donated from early pastnatal limbs in a murine model [J], J Ortho Res[J]. 1991, 9(3): 360-366
40．李晓光，邢国强．邢岩胚胎骨骺移植修复关节而缺损的实验研究[J]．白求恩医科大学学报．1997，23(2)：160
41. Bowen CV, Bray PW, Boyer MI, Fowler JD, Nolan L. Short-term response of epiphyseal plate cell population following selectives devascularization and microsurgical revascularization[J], Microsurgery, 1994, 15(8): 555-562
42. Kato Y, Iwamoto M, Koike T, Suzuki F, Takano Y Terminal differentiation and calcification in rabbit chondrocyte cultures in centrifuge tubes: Regulation by transforming growth factor β and serum factors [J]. Proc Natl Acad Sci USA, 1988, 85(24): 9552.9556.
43. Yan WQ, Tong MH, Yang TS. Reorganization of isolated chondrocytes into growth plate-like tissue in pelleted cultures[J], J Bone Miner Metab, 1995, 13(2): 77-80
44. Ballock RT, Reddi AH. Thyroxine is the factor that regulates morphogenesis of columar cartilage from isolated chondrocyte in chemically defined medium [J]. J Cell Biol, 1994, 126(5): 1311-1318
45．周强，李起鸿，戴刚等，杨柳高数量骺板软骨细胞聚集培养生成软骨组织组织的观察[J]第三军医大学学报，2002，24(5) 556-558
46．周强，李起鸿，戴刚，杨柳 经离心管培养骺软骨异体修复兔骺板部分损伤的初步观察[J]第三军医大学学报，2001，23(10)：1139-1142
47. Ballock RT, Zhou X, Mink LM, Chen DH, Mita BC. Both retinoic acid and 1, 25(OH)2 vitamin D3 inhibit thyroid hormone-induced terminal defferentiation of growth plate chondrocytes[J]J Orthop Res, 2001, 19(1): 43-49,
48. Ballock RT, Zhou X, Mink LM, Chen DH, Mita BC, Stewart MC. Expression of cyclin-dependent kinase ingibitors in epiphyseal chondrocytes indeced to terminally defferentiate with thyoid hormone [J], Endocrinology, 2000, 141(12): 4552-4557
49. Estrada LE, Dodge GR, Richardson DW, Farole A, Jimenez SA. Characterization of a biomaterial with cartilage-like properties expressing type Xeollagen generated in vitro using neonatal porcine articular and growth palte chondroeytes[J]Osteoarthritis cartilage, 2001, 9(2): 169-177
50. Kawabe N, Yoshinao M. The repair of full-thickness articular cartilage defects. Immune responses to reparativetissue formed by allogeneic growth plate chondrocyte implants[J]. Clin Orthop, 1991, (268): 279-293
51. Freyria AM, Ronziere MC, Roche S, Rousseau CF, Herbage D. Regulation of growth, protein synthesis, and maturation of fetal bovineepiphyseal chondroeytes grown in high-density culture in the presence of ascorbic acid, retinoic acid, and dihydrocytochalasin B [J]. J cell Biochem, 1999, 76(1): 84-98
52．周勇刚，卢世璧，王继芳，张永刚，黄靖香，组织工程软
骨用于骨骺早闭治疗的实验研究中华外科杂志，2000，38，：742-744
53．周强，李起鸿，杨柳，戴刚骺板软骨细胞复合生物凝胶．培养生成软骨组织的观察[J]，第三军医大学学报，2002，24(5)：559-562
54. Roche S, Ronziere MC, Herbage D, Freyria AM. Native and DPPA cross-linked collagen sponges seeded with fetal bovine epiphyseal chondrocytes used for cartilage tissue engineering [J] Biomaterials, 2001, 22(1)9-18
55. Tobita M, Ochi M, Uchio YmMori R, Iwasa J, Katsube K, Motomura T. Treatment of growth plate injury with autogenous chondrocytes: a study in rabbits. Acta Orthop Scand 2002; 73(3): 352-358
56．杨志明，王建，秦延武，解慧琪 组织工程软骨移植修复兔生长板缺损中华骨科杂志2002，22(2)：103-107
57．刘建国，徐执扬，马卫华，齐欣，黄岚峰，崔林，徐莘香，生物活性PCL／PLA三维多孔块膜为载体的骺软骨细胞体外实验研究 骨与关节损伤杂志2001，16(5)：355-358
58. Lee EH, Chen F, Chan J, Bose K. Treatment of growth arrest by transfer of cultured chondrocytes into physeal defects[J], J Pediatr Orthop, 1998, 18(2): 155-160
59. Jouve JL, Mottet V, Cottalorda J, Frayssinet P, Bollini G. Reimplantation of growth plate chondrocyte cultures in central growth plate defects: Part Ⅰcharacterization of cultures[J], J Pediatr Orthop B, 1998, 7(2)167-173
60. Jouve JL, Cottalorda J, Mottet V, Frayssinet P, Petit P, Bollini G. Reimplantation of growth plate chondrocyte cultures in central growth plate defects: Part ⅡSurgecal experimentation in rabbits[J], J Pediatr Orthop B, 1998, 7(2)167-173
61. Lee CW, Martinek V, Usas A, Musgrave D, Pickvance EA, Robbins P, Moreland MS, Fu FH, Huard J. Muscle-Based Gene Thrapy and Tissue Engineering for Treatment of Growth Plate Injuries. Journal of Pediatric Orthopaedics
2002, 22: 565-572
62. Trippel SB, Wroblewski J, Makower AM, Whelan MC, Schoenfeld D, Doctrow SR. Regulation of growth-plate chondrocytes by insulin-like growth factor Iand basic fibroblast growth factor. J Bone Joint Surg[Am] 1993, 75: 177-189
63. Rbson H, Siebler T, Stever DA. Thyroid hormone acts directly on growth plate chondrocytes to promote hypertrophic differentiation and inhibit clonal expansion and cell proliferation. Endocrinology 2000, 14(10): 3887-97
64. Juppner H, Role of parathyroid hormone-related peptide and Indian hedgehog in skeletal development. Pediar Nephrol. 2000, 14: 606-611
65. Umehara T, Immunohistochemical localization of transforming growth factor-beta 1 in growth plate freshly transplanted into a full-thickness defectin articular cartilage [J], Nippon Seikeigeka Gakkai Zasshi, 1994, 65(9): 790-797
66. Foster BK, Hansen AL, Gibson GJ, Hopwood JJ, Binns GF, Wiebkin OW. Reimplantation of growth plate chondrocytes into growth plate defects in sheep. J Orthop Res 1990 Jul; 8(4): 555-64
67. Park JS, Ahn JI, Oh DI. Chondroeytes alllgraft transplantation for damaged growth plate reconstruction. Yousei Med J 1994 Dec: 35(4): 375-87
68．王建 杨志明 解慧琪 培养软骨移植修复兔生长板缺损中国修复重建外科杂志 2001，15(1)：(53-56)
69. Schwartz Z, Somers A, Mellonig JT, Carnes DL Jr, Wozney JM, Dean DD, Cochran DL, Boyan BD. Addition of human recombinant bone morphogenetic protein-2 to inactive commercial human demineralized freeze-dried bone allograft make an effective composite bone inductive implant material. J Periodontol. 1998, 69(4): 470
70. Darrouzet V, Fizet D, Deminiere C, Baquey C, Aran JM, Bebear JP. Xenogenie ossieular implants: An experimental
study of heterotopic, demineralized, lyophilized, porine implants in the guinea pig. Clin Otolaryngol. 1999; 24(3): 190
71. Bright RW.Operative correction of partial epiphyseal plate closure by osseous-bridge resection and silicone-rubber implant. An experimental study in dogs. J Bone Joint Surg[Am] 1974; 56: 655-664
72. Broughton NS, Dickens DR, Cole WG, Menelaus MB. Epiphysealysis for partial growth plate arrest. Results after four years or at maturity. J Bone Joint Surg [Br]1989; 71: 13-6
73. Foster BK, John B, Haster C. Free fat interpositional graft in acute physeal injuries: the anticipatory Langenskiold procedure. J Pediatr Orthop 2000; 20: 282-285
74. Bowen CV, Ethridge CP, O'Brien BM, Frykman GK, Gumley GJ. Experimental micro-vascular growth plate transfers. Part Ⅰ: investigation of vascularity. J Bone Joint Surg[Br] 1988; 70: 305-10
75. Kawabe N, Ehrich MG, Mankin HJ. Growth plate reconstruction use chondrocyte allograft transplants. J Pediatr Orthop 1987, 7: 381-388
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