摘要
第一部分：血小板衍化生长因子基因活化材料的制备及生物活性检测
目的：构建含有人血小板衍化生长因子-B (hPDGF-B)基因片段的真核表达质粒,并利用兔成纤维细胞做生物活性检测。
方法：应用基因重组技术和限制性内切酶双酶切构建并鉴定EGFP-N1-hPDGF-B真核表达载体,体外培养兔成纤维细胞,传代培养后用阳离子脂质体(LipofectAMINE):EGFP-N1-hPDGF-B为3uL：1μg的比例转染,G418筛选获得稳定转染的细胞。通过RT-PCR和荧光法检测PDGF-B表达水平；利用四甲基偶氮唑法检测PDGF-B的生物学活性绘制成纤维细胞生长曲线。
结果：成功构建含EGFP-N1-PDGF-B基因的真核表达载体,经RT-PCR检测了PDGF-B在转录水平mRNA的表达情况,荧光法检测目标基因成功转染至靶细胞,转染后的成纤维细胞呈强阳性表达,并持续4周以上。
结论：重组真核表达载体构建正确,并能在成纤维细胞中表达PDGF-B mRNA, PDGF-B基因转染可使成纤维细胞有效而稳定的表达活性PDGF-B而产生生物效应。对成纤维细胞有明显的促进生长作用。
第二部分：血小板衍化生长因子基因活化材料在体实验研究的组织学分析
目的：利用光镜和透射电镜等组织形态学观察手段,比较实验组与对照组在重建愈合过程中胶原表达情况和重建细胞显微结构的差异,以评价脂质体与携带血小板衍化生长因子基因片段的合成质粒共同构成的基因转染系统在兔前交叉韧带重建动物模型中对移植物愈合的促进作用。
方法：成年新西兰兔48只,随机分为实验组和对照组。取半腱肌肌腱替代前交叉韧带制备重建动物模型,实验组在重建部位给予基因转染材料,对照组给予盐水。术后1个月,3个月和6个月,取出重建前交叉韧带标本,对其进行光学显微镜和电镜观察,比较胶原表达和细胞超微结构的变化。
结果：光镜下观察发现,在不同时期,实验组的成纤维细胞数量、胶原含量、胶原纤维的直径以及胶原纤维的排列均好于对照组,实验组标本组织形态在6月时已十分接近正常前交叉韧带组织结构。电镜观察见各时期实验组的细胞增生现象比对照组活跃,提示修复细胞代谢更旺盛,纤维间隙内有较多基质成分,胞质中可见较为丰富的内质网结构和线粒体。
结论：PDGF生长因子基因转染的重建韧带组织结构愈合好于对照组,提示以脂质体为载体携带PDGF-B基因的质粒在重建前交叉韧带愈合局部具有良好的原位转染生物学效能。具有一定的实际应用前景。
第三部分血小板衍化生长因子基因活化材料在体实验生物力学测试
目的：观察脂质体-PDGF-B基因转染系统对兔半腱肌肌腱替代前交叉韧带动物模型中重建韧带愈合强度的促进作用。
方法：成年新西兰兔48只,随机分为实验组和对照组。取半腱肌肌腱替代前交叉韧带制备重建动物模型,实验组在重建部位给予基因转染材料,对照组给予盐水。术后1个月,3个月和6个月,取出重建前交叉韧带标本,对其作生物力学测试,记录最大断裂负荷(极限负荷)、断裂时最大拉伸位移,应力、应变及刚度,并作出以上数据随时间变化的关系图谱。
结果：1.实验标本大体观察无异常,对照组重建韧带断裂1例,实验组未发现重建韧带断裂,动物模型重建手术总体成功率为97.9%。
2.术后1个月时,两组标本的各项力学指标无显著性差异。术后第3月和第6月时,实验组重建韧带的最大断裂负荷和弹性模量两项性能显著大于对照组,p≤0.05,但两组的其它力学指标无显著性差异。
结论：脂质体-PDGF基因转染系统对兔重建前交叉韧带愈合有明显的正性促进作用,可以有效提高韧带移植物在重建塑性过程中的生物力学性能。
part 1:Synthesis of gene active material of human platelet-derived growth factor-B and it's bioactivity test
Objective:To construct the eukaryotic expression plasmid of human platelet-derived growth factor-B (hPDGF-B), and explore the ability of transfection of cultured rabbit Gingival Fibroblasts with platelet-derived growth factor-B in vitro.
Methods:To construct and verify the eukaryotic expression vector for EGFP-N1-hPDGF-B by recombinant DNA technique and restriction enzyme.
Rabbit gingival fibroblasts were cultured in vitro and the cultured gingival fibroblasts were transfected with the complexes of LipofectAMINE reagent and EGFP-N1-hPDGF-B in the ratio of 3:1 (μL:μg) in vitro. Positive clones were selected by G418 screening. The expression of hPDGF-B was detected by RT-PCR and fluorescence microscope. MTT test was done to examine the bioactivity of hPDGF-B and draw a growth curve of fibroblasts.
Results:The eukaryotic vector containing EGFP-N1-PDGF-B was constructed successfully. The expression of PDGF-B on the level of mRNA was tested by PCR. The process that the target cell was transfected by target gene can be observed under fluorescent microscope. Fibroblasts cells showed prominently elevated mRNA expression of hPDGF-B and lasted over 4 weeks.
Conclusions:The recombinant vector was constructed correctly, and the mRNA of rabbit hPDGF-B could be efficiently and stably expressed in Gingival Fibroblasts cells. Transfected Gingival Fibroblasts could effectively express the active hPDGF-B, take biological effects and apparently improve growth. fibroblast
Part 2:The histological evaluations of experiment of reconstructive anterior cruciate ligament with PDGF gene material in vivo
objective To determine the difference between two groups on collagen expression and microstructural observation according to histomorphological method, including light microscope and transmission electron microscope. To evaluate the function of PDGF-B with lipofectMINE gene transfected system to improve the healing of transplant in the rabbit anterior cruciate ligament reconstructive model.
Methods Twenty-four New Zealand adult rabbits were randomly divided into 2 groups, the experimental group and the control group. The rabbit anterior cruciate ligament reconstructive model was established according to the procedure as mentioned previously. PDGF-B was locally injected into the reconstructive location in the experimental group while only normal saline (NS) was administered in the control group. The samples were taken and studied under light microscope and electron microscope 1,3,6 months after operation respectively. Through which we can observe the changes of collagen expression and the microstrcture of cells.
Results 1.The amounts of fibroblasts and collagen, the diameter and arrangement of collagen fiber in the experimental group are all superior to those in the control group. And the histological morphology was approaching the normal structure 6 month after operation under light microscope.
2. The karyokinesis of cell miscrostructure was more active in the experimental group than that in the control group, indicating a prosperous recovery metabolism. There were plenty of endoplasm and mitochondria in cytoplast and copious extracellular matrix between fibers
Conclusion The reconstructive tendon transfected by PDGF was better than that in the control group, indicating that PDGF gene plasmids using lipofectMINE as vector have favorable transfected biological effects in the healing location of resconstructive anterior cruciate ligament and have a specific practical prospect.
Part 3:Biomechanical test of reconstructive anterior cruciate ligament graft with PDGF gene material in vivo
Objective:To observe the liposome-PDGF gene transfected system's function in improving the biomechanical strength of reconstructive ligament in animal models.
Methods:Twenty-four New Zealand adult rabbits were randomly divided into two groups, the experimental group and the control group. Each group concludes 12 animals. The rabbit anterior cruciate ligament reconstructive model was established, PDGF-B was locally injected into the reconstructive location in the experimental group while only NS was administered in the control group. Specimens were obtained 1 month,3 months and 6 months after operation, to observe the tendon graft in general configuration, test their biomechanical indexes including the maximum broken load (peak load), the displacement of maximum load, and calculated stress, strain and stiffness, and form a time-dependent relationship chart according to data above.
Results:1. The animals showed no obvious abnormality by observation. There is one case experiencing rupture of ligament in control group while no such case was found in the experimental group. The total successful rate of reconstructive surgery in animal models is 97.9%.
2. There was no apparent difference between two groups on each biomechanical index one month after operation. The maximum broken load and stiffness of tendon graft in the experimental group was significantly higher than that in the control group, p≤0.05, while other biomechanical indexes between two groups show no difference 3 months and 6 months after operation respectively.
Conclusion:Liposome-PDGF gene transfected system has a positive promoting effect in the wound healing of anterior cruciate ligament reconstruction in rabbits, and can enhance biomechanical efficiency in the process of reconstruction of ligament graft.
引文
1. Miyasaka KC, Daniel DM, Stone ML, et al. The incidence ofknee ligament injuries in the general population. Am J Knee Surg.1991,4(1):3-8
2. Kujala UM, Taimela S, Antti-Poika I, et al. Acute injuries in soccer, ice hockey, volleyball, basketball, judo, and karate: analysis of national registry data. BMJ.1995,2(311):1465-1468
3.敖英芳,田得祥,崔国庆,等.运动员前交叉韧带损伤的流行病学研究.体育科学.2000,20(4)：47-49
4. Markolf KL, Mensch JS, Amstutz HC. Stiffness and laxity of the knee--the contributions of the supporting structures. A quantitative in vitro study. J Bone Joint Surg Am.1976 Jul, 58(5):583-594
5. Amis AA, Dawkins GP. Functional anatomy of the anterior cruciate. Fibre bundle actions related To ligament replacements and injuries. J Bo ne Joint Surg Br.1991,73(2):260-267
6. Butler DL, Noyes FR, Grood ES. Ligamentous restraints to anterior-posterior drawer in the human knee. A biomechanical study. J Bone Joint Surg Am.1980 Mar,62(2):259-270
7.管于宁,D. LBulter,杨汉国,等.前交叉韧带及其纤维束对膝关节前错运动的约束作用。实验力学.1995,10(1)：27-30
8. Frank CB,Jackson DW. The science of reconstruction of the anterior cruciate ligament. J Bone Joint Surg (Am).1997,79:1556
9.陈加尔.人工韧带修复前交叉韧带的现状.中华外科杂志.1992,3:25
10. Mody BS, Howard L, Harding ML, et al. The ABC crab and polyester prosthetic ligament for ACL-deficient knees. J Bone Joint Surg (Br).1993,75:818
11. Roolker W, Patt TW, Van Dijk CN, et al.The Gore-Tex prosthetic ligament as a salvage procedure on deficient knees. Knee Surg Sports Traumatol Arthrosc.2000,8 (1):20
12. Jaureguito JW, Paulos LE. Why grafts fail. Clin Orthop. 1996,325:25-41
13. Arnoczky SP, Tarvin GB, Marshall JL. Anterior cruciate ligament replacement using patellar tendon. An evaluation of graft revascularization in the dog. J Bone and Joint Surg 1982,64:217-227
14. Evans CH, Ghivizzani SC, Robbins PD. Orthopaedic gene therapy. Clin Orthop.2004,429:316-329
15. DesRosiers EA, Yahia L, Rivard Ch. J Orthop Res.1996,14(2): 200-208
16. Murphy PC, Loitz BJ, Frank CB, et al. Iochem Cell Biol. 1993,71 (11-12):522-529
17. Amiel D, Nagineni CN, Choi SH. Med Sci Sports Exerc.1995, 27(6):824-851
18. Dahlgren LA, Mohammed HO, Nixon AJ. Temporal expression of growth factors and matrix molecules in healing tendon lesions. J Orthop Res.2005,23(1):84-92
19.唐毅,陈鸿辉,李斯明,等.透明质酸联合碱性成纤维细胞生长因子对韧带细胞增殖的影响.中国修复重建外科杂志.2001,15(3)：158161
20. Hori Y, Nakamura T, Matsumoto K, et al. Experimental study on in situ tissue engineering of the stomach by an acellular collagen sponge scaffold graft. ASAIO J.2001,47(3):206-210
21. Kanemaru S, Nakamura T, Omori K, et al. Regeneration of mastoid air cells in clinical applications by in situ tissue engineering. Laryngoscope.2005 Feb,115(2):253-258
22. Omori K, Nakamura T, kanemaru S, et al. Regenerative medicine of the trachea:the first human case. Ann Otol Rhinol Laryngol. 2005,114:429-433
23. Omori K, Nakamura T, kanemaru S, et al. Cricoid regeneration using in situ tissue engineering in canine latynx for the treatment of subglottic stenosis. Ann Otol Rhinol Laryngol. 2004,113:623-627
1. Alvarez R H, Kantar jian H M, Coes J E. Biology of platelet-drived growth factor and its involvement in disease [J]. Mayo Clin Proc, 2006,81(9):1241-1257
2. Nishita T, Takahasi M, Kasuya T, Matsui K, Ichihara N, Murakami M, Asari M. Measurement of erythrocyte carbonic anhydrase isozymes (CA-I and CA-II) in racehorses and riding horses. J Vet Med Sci.2005 Jan;67(1):63-7.
3. Bartold PM. Platelet-derived growth factor stimulates hyaluronate but not proteoglycan synthesis by human gingival fibroblasts in vitro. J Dent Res.1993 Nov;72(11):1473-80.
4. Wang HL, Pappert TD, Castelli WA, Chiego DJ Jr, Shyr Y, Smith BA. The effect of platelet-derived growth factor on the cellular response of the periodontium:an autoradiographic study on dogs. J Periodontol.1994 May;65(5):429-36.
5. Howell TH, Fiorellini JP, Paquette DW, Offenbacher S, Giannobile WV, Lynch SE. A phase Ⅰ/Ⅱ clinical trial to evaluate a combination of recombinant human platelet-derived growth factor-BB and recombinant human insulin-like growth factor-I in patients with periodontal disease. J Periodontol.1997 Dec;68(12):1186-93.
6.金向青曹金芳余杰等血小板衍化生长因子对人牙周韧带细胞在壳聚糖-磷酸三钙支架材料上附着和增殖的影响,牙体牙髓牙周病杂志200717(11)626-629.
7. Breitbart AS, Mason JM, Urmacher C, et al。 Gene-enhanced tissue engineering:applications for wound healing using cultured dermal fibroblasts transduced retrovirally with the PDGF-B gene. Ann Plast Surg.1999 Dec;43 (6):632-9.
8. Eming SA, Medalie DA, Tompkins RG, Genetically modified human keratinocytes overexpressing PDGF-A enhance the performance of a composite skin graft. Hum Gene Ther.1998 Mar 1;9(4):529-39.
9. Norton A, Peplinski GR, Tsung K. expression of secreted platelet derived growth factor b by recombinant nonreplicating and noncytopathic vaccinia virus。Ann Surg.1996 Oct;224(4):555-60
10. Liechty KW, Nesbit M, Herlyn M, Adenoviral-mediated overexpression of platelet-derived growth factor-B corrects ischemic impaired wound healing. J Invest Dermatol.1999 Sep;113(3):375-83.
11. Deguchi J, Namba T, Hamada H, Targeting endogenous platelet-derived growth factor B-chain by adenovirus-mediated gene transfer potently inhibits in vivo smooth muscle proliferation after arterial injury. Gene Ther.1999 Jun;6(6):956-65.
12. Doukas J, Chandler LA, Gonzalez AM, Matrix immobilization enhances the tissue repair activity of growth factor gene therapy vectors. Hum Gene Ther.2001 May 1;12 (7):783-98.
13. Ropert C. Liposomesasa gene delivery system. Braz J Med Viol Res,1999,32:163-169
14. PALMER GD, STEINERT A, PASCHER A, et al. Gene-Induced Chondrogenesis of Primary Mesenchymal Stem Cells in vitro [J].Mol Ther,2005,12(2):219-228.
15. Nishishita T, Lin PC. Angiopoietin 1, PDGF-B, and TGF-beta Gene regulation in endothelial cell and smooth muscle cell interaction [J]. J Cell Biochem,2004,91(3):584 -593
16.赵力,张雪松,刘程伟,等PDGF-B基因转染骨髓间充质干细胞的实验研究[J]黑龙江医药科学,2005,28(6)：16-17
17.闫国和,粟永萍,王军平,等。重组真核表达载体pEGFP-N1/PDGF-A的构建及真皮干细胞的转染[J]第三军医大学学报,2005,27(20)：2005-2008
18.王忠彪,孙逊,李勇,等。PDGF-B基因表达及其对成纤维细胞增生 和胶原合成的促进作用.[J]中华创伤杂志,1984,14:216-218
19. Robbins KC, Leal F。Pierce JH, et al. The V-sis/PDGF-B transforming gene product localizes to cell membranes but is not a secretory protein. EM BO J,1985,4(1) 257-288.
20. Westermark B, Sorg C. Biology of platelet-derived growth factor (Cytokines Vol.5). Switzerland:S. karger AG. Basel,1993, 13-18.
1. Gentleman E, Livesay GA, Dee KC, et al. Development of ligament-like structural organization and properties in cell-seeded collagen scaffolds in vitro. Ann Biomed Eng.2006, 34(5):726-736
2. Noyes FR. Functional Properties of knee ligaments and alterations induced by immobilization. Clin Orthop,1977, 123:210-242
3.Amiel D, Frank C,Harwood F, et al. Tendons and ligaments:a morphological and biochemical comparision. J Orthop Res.1984, 1:257-265
4.Cooper JAJr, Bailey LO, Carter JN, et al. Evaluation of the anterior cruciate ligament, medial collateral ligament, achilles tendon and patellar tendon as cell sources for tissue-engineered ligament. Biomaterials.2006,27(13): 2747-2754
5. Heckmann L, Schlenker HJ, Fiedler J, et al. Human mesenchymal progenitor cell responses to a novel textured poly(L-lactide) scaffold for ligament tissue engineering. J Biomed Mater Res Biomater.2006,21:234-239
6.Weiler A, Fo rster C, Hunt P, et al. The influence of locally appllied Platelet derived growth factor-BB on free tendon graft remodeling after anterior cruciate ligament reconstruction. Am J Sports Med.2004,32(4):881-891
7. Amiel D, Kleiner JB, Roux RD, et al. The phenomenon of "ligamentization":anterior cruciate ligament reconstruction with autogenous patellar tendon. J Orthop Res.1986,4(2):162-172
8. Blickenstaff KR, Grana WA, Egle D. Analysis of a semitendinosus autograft in a rabbit model. Am J Sports Med.1997 Jul-Aug,25 (4): 554-559
9. Papachristou G, Tilentzoglou A, Efstathopoulos N, Khaldi L. Reconstruction of anterior cruciate ligament using the doubled tendon graft technique:an experimental study in rabbits. Knee Surg Sports Traumatol Arthrosc.1998,6(4):246-252
10. Goradia VK, Rochat MC, Kida M, Grana WA. Natural history of a hamstring tendon autograft used for anterior cruciate ligament reconstruction in a sheep model. Am J Sports Med.2000 Jan-Feb, 28(1):40-46
11.薛海滨,敖英芳,于长隆,应用半腱肌腱重建前交叉韧带末端形成的特点中国运动医学杂志.2002,21:127-130
12. Sluss JR, Liberti JP, Jiranek WA, et al. pN collagen type III within tendon grafts used for anterior cruciate ligament reconstruction. J Orthop Res.2001,19(5):852-857
13. Riechert K, Labs K, Lindenhayn K, Sinha P. Semiquantitative analysis of types I and III collagen from tendons and ligaments in a rabbit model. J Orthop Sci.2001,6(1):68-74
14.王永健,敖英芳.自体半腱肌腱移植重建前交叉韧带移植物组织学变化的实验研究.中国运动医学杂志.2004,23(6):609-612
15. Watanabe N, Woo S L, Papageorgiou C. Fate of donor bone marrow cells in medial collateral ligament after simulated autologous transplantation. Microsc Res Tech.2002,58:39-44
16. Gill SS, Turner MA, Battaglia TC, et al. Semitendinosus regrowth:biochemical, ultrastructural, and physiological characterization of the regenerate tendon. Am J Sports Med. 2004,32(5):1173-1181
17. Bartold PM, Raben A. Growth factor modulation of f ibroblasts in simulated wound healing. Periodontal Res.1996,31(3):205-216
18.沈雁,陈鸿辉,李贤让,等.成纤维细胞生长因子和表皮生长因子及复合因子对兔ACL, MCL体外增殖作用.中国修复重建外科杂志.2005,19(3)：229-223
19. Kirchberg K, Lange TS, Klein EC, etal. Induction of betal integrinsynthesisbyrecombinant platelet-derived growth factor(PDGF-AB) correlates with an enhanced migratory response of humandermal fibroblasts to various extracel lularmatrix proteins. Exp Cell Res.1995,220(1):29-35
20. Chen Y, Rabinovitch P. Altered cell cycle responses to insuline like growth factor I, but not platelet-derived growth factor and epidermal growth factor, insenescing human frbroblasts. Cell Physiol.1990,144:18
21.Nakamura N, Hart DA, Boorman RS, et al. Decorin antisense gene therapy improves functional healing of early rabbit ligament scar with enhanced collagen fibrillogenesis in vivo. J Orthop Res.2000, 18(4):517-523
22. Popova SN, Rodriguez-Sanchez B, Liden A, et al. Themes enchymal alphall beteal integrinattenuates PDGF-BB-stimulated Chemotaxis of embryonic f ibro blasts on collagens. DevBiol.2004, 270(2):427-442
23. Kobayashi D, Kurosaka M,Yoshiya S, etal. Effect of basic fibroblast growth factor on the healing of defects in the canine anterior cruciate liagment. Knee Surg Sports Traumatol Arthrose.1997,5(3):189-194
24. Ries C, Petrides PE. Cytokine regulation of matrix metalloproteinases activity and its regulatory dysfunction in disease. Biol Chem.1995,376:345-355
25. Weiler A, Forster C, Hunt P, et al. The influence of locally applied platelet-derived growth factor-BB on free tendon graft remodeling after anterior cruciate ligament reconstruction. Am J Sports Med.2004,32(4):881-891
26. Yoshikawa T, Tohyama H, Katsura T,et al. Effects of local administration of vascular endothelial growth factor on mechanical characteristics of the semitendinosus tendon graft after anterior cruciate ligament reconstruction in sheep. Am J Sports Med.2006 Dec,34(12):1918-25
27. Nakamura N, Shino K, Matsuume T, et al. Early biological effect of in vivo gene transfer of platelet-derived growth factor(PDGF)-B into healing patella ligament. Gene Ther.1998,5(9):1165-1170
28. Petersen W, Pufe T, Zantop T, et al. Hypoxia and PDGF have a synergistic effect that increases the expression of the angiogenetic peptide vascular endothelial growth factor in Achilles tendon fibroblasts. Arch Orthop Trauma Surg.2003 Nov, 123(9):485-8
29.李峰,陈连旭,张继英,等.应用植入转染血小板衍化生长因子基因的骨髓间充质干细胞的灭活同种异体跟腱重建兔前交叉韧带效果的实验研究.中国运动医学杂志.2007,26(4):419-426
30. Dunn MG, Liesch JB, Tiku ML, et al. Development of fibroblast- seeded ligament analogs for ACL reconstruction. J Biomed Mater Res.1995 Nov,29(11):1363-1371
1. Goradia VK, Rochat MC, Grana WA, et al. Tendon-to-bone healing of a semitendinosus tendon autograft used for ACL reconstruction in a sheep model. Am J Knee Surg.2000,13(3):143-51
2. Weiler A, Peine R, Pashmineh-Azar A, Tendon healing in a bone tunnel. Part I:Biomechanical results after biodegradable interference fit fixation in a model of anterior cruciate ligament reconstruction in sheep. Arthroscopy.2002 Feb.18(2):113-124
3. Weiler A, Hoffmann RF, Bail HJ, et al. Tendon healing in a bone tunnel. PartⅡ:Histologic analysis after biodegradable interference fit fixation in a model of anterior cruciate ligament reconstruction in sheep. Arthroscopy.2002 Feb,18 (2):124-135
4. Yamakado K, Kitaoka K, Yamada H, etal.The influence of mechanical stress on graft healing in a bone tunnel. Arthroscopy. 2002 Jan,18(1):82-90
5. Buss DD, Warren RF, Wickiewicz TL, et al. Arthroscopically assisted reconstruction of the anterior cruciate ligament with use of autogenous patellar-ligament grafts. Results after twenty-four to forty-two months. J Bone Joint Surg Am.1993 Sep, 75(9):1346-1355
6. Gill SS, Turner MA, Battaglia TC, et al. Semitendinosus regrowth:biochemical, ultrastructural, and physiological characterization of the regenerate tendon. Am J Sports Med. 2004,32(5):1173-1181
7. Zhou JS, Zhang CC, Pan GP, et al. An experiment on transplantation of cryop reserved bone-anteri or cruciate ligament (ACL)-bone allograft. Chin J Orthop Trauma.2004,6: 546-549
8. Amiel D, Kleiner JB, Roux RD, Harwood FL, Akeson WH. The phenomenon of "ligamentization":anterior cruciate ligament reconstruction with autogenous patellar tendon. J Orthop Res. 1986,4(2):162-172
9.王永健,敖英芳.白体半腱肌腱一直重建前交叉韧带移植物组织学变化的实验研究.中国运动医学杂志.2004,23(6):609-612
10. Kuroda R, Kurosaka M, Yoshiya S, Mizuno K. Localization of growth factors in the reconstructed anterior cruciate ligament: immunohistological study in dogs. Knee Surg Sports Traumatol Arthrosc.2000,8(2):120-126
11. Petersen W, Unterhauser F, Pufe T, Zantop T, Sudkamp NP, Weiler A. The angiogenic peptide vascular endothelial growth factor (VEGF) is expressed during the remodeling of free tendon grafts in sheep. Arch Orthop Trauma Surg.2003 May,123(4):168-174
12. Hildebrand KA, Woo SL, Smith DW, Allen CR, Deie M, Taylor BJ, Schmidt CC. The effects of platelet-derived growth factor-BB on healing of the rabbit medial collateral ligament. An in vivo study. Am J Sports Med.1998 Jul-Aug,26(4):549-54
13. Letson AK, Dahners LE. The effect of combinations of growth factors on ligament healing. Clin Orthop Relat Res.1994 Nov, (308):207-12
14. Weiler A, Forster C, Hunt P, et al. The influence of locally applied platelet-derived growth factor-BB on free tendon graft remodeling after anterior cruciate ligament reconstruction. Am J Sports Med.2004,32(4):881-891
15. Nakamura N, Shino K, Natsuume T, et al. Early biological effect of in vivo gene transfer of platelet-derived growth factor (PDGF)-B into healing patellar ligament. Gene Ther.1998 Sep,5(9):1165-70
16. Petersen W, Pufe T, Zantop T, et al. Hypoxia and PDGF have a synergistic effect that increases the expression of the angiogenetic peptide vascular endothelial growth factor in Achilles tendon fibroblasts. Arch Orthop Trauma Surg.2003 Nov,123(9):485-488
17.李峰,陈连旭,张继英,等.应用植入转染血小板衍化生长因子基因的骨髓间充质干细胞的灭活同种异体跟腱重建兔前交叉韧带效果的实验研究.中国运动医学杂志.2007,26(4):419-426
18. Letson AK, Dahners LE. The effect of combinations of growth factors on ligament healing. Clin Orthop Relat Res.1994 Nov, (308):207-12
19. Panossian V, Liu SH, Lane JM, Finerman GA. Fibroblast growth factor and epidermal growth factor receptors in ligament healing. Clin Orthop Relat Res.1997 Sep, (342):173-180
20. Panossian V, Liu SH, Lane JM, Finerman GA. Fibroblast growth factor and epidermal growth factor receptors in ligament healing. Clin Orthop Relat Res.1997 Sep, (342):173-180
21. Hildebrand KA, Woo SL, Smith DW, The effects of platelet-derived growth factor-BB on healing of the rabbit medial collateral ligament. An in vivo study. Am J Sports Med.1998 Jul-Aug,26(4):549-554
22. Kobayashi D, Kurosaka M, Yoshiya S, Mizuno K. Effect of basic fibroblast growth factor on the healing of defects in the canine anterior cruciate ligament. Knee Surg Sports Traumatol Arthrosc. 1997,5(3):189-194
23. Kobayashi D, Kurosaka M, Yoshiya S, et al. Effect of basic fibroblast growth factor on the healing of defects in the canine anterior cruciate ligament. Knee Surg Sports Traumatol Arthrosc. 1997,5(3):189-194
24. Letson AK, Dahners L E. The effect of combinations of growth factors on ligament healing. Clin Orthop,1994,308:207
[1]. Wong MW, Qin L, Lee KM, et al. Healing of bone-tendon junction in a bone trough:a goat partial patellectomy model. Clin Orthop Relat Res,2003 Aug;413:291-302
[2].陈百成,栾广,张展翅。自体骨膜包裹肌腱对腱骨愈合的影响。中华骨科杂志,2006；26(2)：118-122
[3]. Hays PL, Kawamura S, Deng XH, et al. The role of macrophages in early healing of a tendon graft in a bone tunnel. Bone Joint Surg Am,2008 Mar;90(3):565-579
[4]. Eriksson E.Vascular ingrowth into ACL-grafts. Knee Surg Sports Traumatol Arthrosc,2008 Apr;16(4):341
[5]. Wen CY, Qin L, Lee KM, et al. Influence of bone adaptation on tendon-to-bone healing in bone tunnel after anterior cruciate ligament reconstruction in a rabbit model. J Orthop Res,2009 Nov;27(11):1447-1456
[6]. Hays PL, Kawamura S, Deng XH, et al. The role of macrophages in early healing of a tendon graft in a bone tunnel. J Bone Joint Surg Am,2008;90:565-579
[7]. Berg EE, Pollard ME, Kang Q.Interarticular bone tunnel healing. Arthroscopy,2001;17 (2):189-195
[8]. Fauno P, Kaalund S. Tunnel widening after hamstring anterior cruciate ligament reconstruction is influenced by the type of graft fixation used:a prospective randomized study. Arthroscopy,2005 Nov;21 (11):1337-1341
[9]. Kaeding C, Farr J, Kavanaugh T, et al. prospective randomized compairsion of bioresorbable and titanium anterior cruciate ligament interference screw. Arthroscopy,2005;21:147-151
[10]. Myers P, Logan M, Stokes A, et al.Bioabsorbable versus titanium interference screws with hamstring autograft in anterior cruciate ligament reconstruction:a prospective randomized trial with 2-year follow-up. Arthroscopy,2008;24 (7):817-823
[11]. Moisala AS, Jarvela T, Paakkala A, et al. Comparison of the bioabsorbable and metal screw fixation after ACL reconstruction with a hamstring autograft in MRI and clinical outcome:a prospective randomized study.Knee Surg Sports Traumatol Arthrosc,2008;16(12):1080-1086
[12]. Brand J Jr,Weiler A, Carbon DN, et al. Graft fixation of cruciate ligament reconstruction. Am J Sports Med,2000;28:761-774
[13]. Drogset JO, Gr(?)ntvedt T, Myhr G. Magnetic resonance imaging analysis of bioabsorbable interference screws used for fixation of bone-patellar tendon-bone autografts in endoscopic reconstruction of the anterior cruciate ligament. Am J Sports Med,2006 Jul;34(7):1164-1169
[14]. Frosch KH, Sawallich T, Schtitze G, et al. Magnetic resonance imaging analysis of the bioabsorbable Milagro interference screw for graft fixation in anterior cruciate ligament reconstruction. Strategies Trauma Limb Reconstr,2009 Oct;4(2):73-79
[15]. Warden WH, Chooljian D, Jackson DW.Ten-year magnetic resonance imaging follow-up of bioabsorbable poly-L-lactic acid interference screws after anterior cruciate ligament reconstruction. Arthroscopy,2009;24(3):371-373
[16]. Ahmad C S, Gardner TR, Groh M, et al. Mechanical properties of soft tissue fem oral fixation devices for anterior cruciate ligament reconstruction.Am J Sports Med,2004; 32(3):635-640
[17]. Harilainen A, Sandelin J, Jansson KA. Cross-pin femoral fixation versus metal interference screw fixation in anterior-cruciate ligament reconstruction with hamstring tendons:results of a controlled prospective randomized study with 2-year follow-up. Arthroscopy,2005 Jan;21(1):25-33
[18]. Rose T, Hepp P, Venus J, et al. Prospective randomized clinical comparison of femoral transfixation versus bioscrew fixation in hamstring tendon ACL reconstruction-a preliminary report. Knee Surg Sports Traumatol Arthrosc,2006;14(8):730-738
[19]. Robert H, E s-Sayeh J, Heymann D, et al.Ham string insertion site healing after anterior cruciate ligament reconstruction in patients with symptomatic hardware or repeat rupture:a histologic study in 12 patients. Arthroscopy,2003;19(9):948-954
[20]. Berg TL, Paulos LE. Endoscopic ACL reconstruction using stryker biosteon cross-pin femoral fixation and interlock cross-pin tibial fixation.Surg Technol Int,2004;12:239-244
[21]. Webster KE, Feller JA, Hameister KA. Bone tunnel enlargement following anterior cruciate ligament reconstruction:a randomized comparison of hamstring and patellar tendon grafts with 2-year followup.Knee Surg Sports Traumatol Arthrosc,2001;9:86-91
[22]. Dargel J, Koebke J, Bruggemann GP, et al. Tension degradation of anterior cruciate ligament grafts with dynamic flexion-extension loading:a biomechanical model in porcine knees. Arthroscopy,2009 Oct;25(10):1115-25
[23]. Shen PH, Lien SB, Shen HC, et al. Comparison of different sizes of bioabsorbable interference screws for anterior cruciate ligament reconstruction using bioabsorbable bead augmentation in a porcine model. Arthroscopy,2009 Oct;25(10):1101-1107
[24]. Tetsumura S, Fujita A, Nakajima M, et al. Biomechanical comparison of different fixation methods on the tibialside in anterior cruciate ligament reconstruction:a biomechanical study in porcine tibialbone. Orthop Sci,2006;11(3):278-282
[25]. Ninkovic S, Savic D, Stankovic M, et al. Comparison of clinical results of anterior cruciate ligament reconstruction using two different procedures. Acta Chir Iugosl, 2005;52(2):89-94
[26]. van Dijck RA, Saris DB, Willems JW, et al. Additional surgery after anterior cruciate ligament reconstruction:can we improve technical aspects of the initial procedure? Arthroscopy,2008 Jan;24(1):88-95
[27]. Denti M, Lo Vetere D, Bait C, et al. Revision anterior cruciate ligament reconstruction:causes of failure, surgical technique, and clinical results. Am J Sports Med,2008 Oct;36 (10):1896-1902
[28]. Ekdahl M,Nozaki M, Ferretti M, et al.The effect of tunnel placement on bone-tendon healing in anterior cruciate ligament reconstruction in a goat model. Am J Sports Med,2009; 37(8):1522-1530
[29].张培,刘泉,周建生,等。骨隧道大小对前交叉韧带止点转归的影响。中国修复重建外科杂志,2008；22(5)：528-532
[30]. Chen CH, Liu HW, Tsai CL,et al.Photoencapsulation of bone morphogenetic protein-2 and periosteal progenitor cells improve tendon graft healing in a bone tunnel. Am J Sports Med 2008:36:461-473
[31].吴海山。人工韧带与组织工程韧带研究进展。医师进修杂志,2005,28(5):1-3
[32]. Sasaki K, Kuroda R, Ishida K, et al.Enhancement of tendon-bone osteointegration of anterior cruciate ligament graft using granulocyte colony-stimulating factor. Am J Sports Med,2008;36 (8):1519-1527
[33]. Lim JK, Hui J,Li L,et al. Enhancement of tendon graft osteointegration using mesenchymal stem cells in a rabbit model of anterior cruciate ligament reconstruction. Arthroscopy,2004; 20(9):899-910
[34]. Karaoglu S, Celik C, Korkusuz P. The effects of bone marrow or periosteum on tendon-to-bone tunnel healing in a rabbit model. Knee Surg Sports Traumatol Arthrosc,2009;17:170-178
[35]. Qin L, Fok P, Lu H, et al. Low intensity pulsed ultrasound increase the matrix hardness of the healing tissues at bone-tendon insertion-a partial patellectomy model in rabbits. ClinBiomech(Bristol, Avon),2006;21(4):387-394
[36]. Lu H, Qin L, Fik P, et al.Low intensity pulsed accelerates bone tendon junction healing:a partial patellectomy model in rabbits. Am J Sports Med,2006; 34(8):1287-1296
[37]. Wang CJ, Wang FS, Yang KD, et al.The effect of sock wave treatment at the tendon-bone interface-an Histomorphological and biomechanical study in rabbits. Orthop Res,2005;23 (2):274-280
[38]. Kyung HS, Kim SY, Oh CW, et al. Tendon-to-bone tunnel healing in a rabbit model:the effect of periosteum augmentation at the tendon-to-bone interface. Knee Surg Sports Traumatol Arthrosc,2003;11 (1):9-15
[39]. Chen CH, Chen WJ, Shih CH, et al. Arthroscopic anterior cruciate ligament reconstruction with periosteum-enveloping hamstring tendon graft. Knee Surg Sports Traumatol Arthrosc,2004; 12 (5):398-405
[40]. Tien YC,Chih TT, Lin JH, et al. Augmentation of tendon-bone healing by the use of calcium-phosphate cement. J Bone Joint Surg Br,2004:86:1072-1076
[41]. Huangfu X, Zhao J. Tendon-bone healing enhancement using injectable tricalcium phosphate in a dog anterior cruciate ligament reconstruction model. Arthroscopy,2007;23:455-462
[42]. Gulotta LV, Kovacevic D, Ying L, et al. Augmentation of tendon-to-bone healing with a magnesium-based bone adhesive. Am J Sports Med,2008:36:1290-1297
[43]. Kobayashi M, Nakagawa Y, Suzuki T, et al.A retrospective review of bone tunnel enlargement after anterior cruciate ligament reconstruction with hamstring tendons fixed with a metal round cannulated interference screw in the femur. Arthroscopy,2006 Oct;22 (10):1093-1099
[44]. Iorio R, Vadala A, Argento G,et al.Bone tunnel enlargement after ACL reconstruction using autologous hamstring tendons:a CT study. Int Orthop,2007 Feb;31(1):49-55
[45]. Fules PJ, Madhav RT, Goddard RK, et al. Evaluation of tibial bone tunnel enlargement using MRI scan cross-sectional area measurement after autologous hamstring tendon ACL replacement. Knee,2003;10:87-91
[46]. Harilainen A, Linko E,Sandelin J.Randomized prospective study of ACL reconstruction with interference screw fixation in patellar tendon autografts versus femoral metal plate suspension and tibial post fixation in hamstring tendon autografts:5-year clinical and radiological follow-up results.Knee Surg Sports Traumatol Arthrosc,2006, Jun;14(6):517-528
[47]. Sakai H, Yajima H, Hiraoka H, et al.The influence of tibial fixation on tunnel enlargement after hamstring tendon anterior cruciate ligament reconstruction.Knee Surg Sports Traumatol Arthrosc.2004, Sep;12(5):364-370
[48]. Foldager C, Jakobsen BW, Lund B, et al. Tibial tunnel widening after bioresorbable poly-lactide calcium carbonate interference screw usage in ACL reconstruction. Knee Surg Sports Traumatol Arthrosc,2009,Jul;16
[49]. Zysk SP, Fraunberger P, Veihelmann A, et al. Tunnel enlargement and changes in synovial fluid cytokine p rofile following anterior cruciate ligament reconstruction with patellar tendon and hamstring tendon autografts. Knee Surg Sports Traumatol Arthrosc,2004;12:98-103
[1]. Noyes FR. Functional Properties of knee ligaments and alterations induced by immobilization[J]. Clin Orthop 1977,123:210-242.
[2]. Amiel D, Frank C,Harwood F, et al. Tendons and ligaments:a morphological and biochemical comparision. J Orthop Res,1984,1:257-265.
[3]. Evans CH, Ghivizzani SC, Robbins PD. Orthopaedic gene therapy. Clin Orthop,2004,429:316-329.
[4]. DesRosiers EA, YahiaL, Rivard CH, et al. Proliferative and matrix synthesis response of canine anterior cruciate ligament fibroblasts submitted to combined growth factors. J Orthop Res, 1996; 14 (2):200-208
[5]. Murphy PC, Loitz BJ, Frank CB, et al. Iochem Cell Biol, 1993:71(11-12):522-529
[6]. Amiel D, Nagineni CN, Choi SH. Med Sci Sports Exerc,1995;27(6):824-851
[7].张喜善,罗怀灿,鲁玉来,等。韧带愈合过程中转化生长因子β1及其Ⅰ型受体表达的实验研究.中国矫形外科杂志2001 8(5)：472-474
[8]. Murphy PG, Loitz BJ, Frank CB,et al. Influence of exogenous growth factors on the expression of plasminogen activators by explants of nomal and healing rabbit ligaments[J]. Biochem Cell Biol,1993,71(12):522-529
[9]. Dstosiers EA, Yahia L,Rivard CH. Proliferative and matrix synthesis response of canine anterior curciate ligament fibroblasts submitted to combined growth factors[J].J Orthop Res,1996,14(2):200-208.
[10]. Murphy PG, Hart DA. Influence of exogenous growth factors on the expression of plasminogen activators and plasminogen activator inhibitors by cells isolated from normal and healing rabbit ligaments[J].J Orthop Res.1994,12(4):564-575.
[11]. Sciore P, Boykiw R, Hart DA, et al. Semiquantitative reverse transcription-polymerase chain reaction analysis of mRNA for growth factors and growth factor receptors from normal and healing rabbit medial collateral ligament tissue[J]. J Orthop Res,1998,16:429-437.
[12]. Panossian V, Liu SH, Lane JM, et al. Fibroblast growth factor and epidermal growth factor receptors in ligament healing [J]. Clin Orthop,1997,342:173-180.
[13]. DesRosiers EA, Yahia L, Rivard C2H, et al. Proliferative and matrix synthesis response of canine anterior cruciate ligament fibroblasts submitted to combined growth factors [J] J Orthop Res,1996,14:200-208.
[14].李贤让,陈鸿辉,沈雁.等.酸性成纤维细胞生长因子及表皮生长因子对兔韧带细胞增殖的影响.中国矫形外科杂志,2003,11(10)：689-691.
[15]. SchmidtCC, Georgescu HI, Kwoh CK, et al。Effect of growth factors on the proliferation of fibroblasts from the medial collateral and anterior cruciate ligaments [J]. Orthop Res,1995,13 (2):184-190.
[16].唐毅,陈鸿辉,李斯明,等.透明质酸联合碱性成纤维细胞生长因子对韧带细胞增殖的影响.中国修复重建外科杂志,2001,15(3)：158-161.
[17]. Kabayashi D, Kurosaka M, Yoshiya S, et al. Effect of basic fibroblast growth factor on the healing of defect s in the canine anterior cruciate ligament. Knee Surg Sport s Traumatol Arthrosc, 1997,5 (3):189-194.
[18].张春礼,范宏斌,徐虎,等.碱性成纤维细胞生长因子促进冻干肌腱移植重建前交叉韧带后早期血管生成的组织学观察.中华创伤骨科 杂志,2006,8(2)：157-161
[19]. Higashinakagawa M. Biological effect s of basic fibroblast growth factor (bFGF) and osreogenic protein (OP-1/BMP-7) on bone marrow cells and development of cellhybrid artificial bone. J Stomatological Society J apan,1999,66:118-121.
[20]. Conti NA, Dahners L E. The effect of exogenous growth factors on the healing of ligaments [J]. Trans Orthop Res-Soc,1993,18:60.
[21]. Letson AK, Dahners L E. The effect of combinations of growth factors on ligament healing[J]. Clin Orthop,1994,308:207.
[22]. Hannafin JA, Attia E, warren RG, et al. The dffect of cytokines on the chemotactic migration of canine knee ligament fibroblasts[J]. Trans Orthop Res Soc,1997,22:50.
[23]. Lee J, Chamberlin TA, Schreck PJ, et al. In situ localization of growth factors during the early healing of knee ligaments[J]. Trans Orthop Res Soc,1995,20:158.
[24]. Dahlgren LA, Mohammed HO, Nixon AJ. Temporal expression of growth factors and matrix molecules in healing tendon lesions [J]. J Orthop Res,2005,23(1):84-92.
[25]. Fu SC,Wong YP, Cheuk YC, et al. TGF-betal reverses the effects of matrix anchorage on the gene expression of decorin and procollagen type Ⅰ in tendon fibroblasts [J]. Clin Orthop Relat Res,2005,431 (2):226-232.
[26]. Hildebrand KA, Deie M, Allen CR, et al. Early expression of marker genes in the rabbit medial collateral and anterior cruciate ligaments:the use of different viral vectors and the effects of injury [J]. J Orthop Res,1999,17(1):37-42.
[27]. Nakamura N, Hart DA, Boorman RS, et al. Decorin antisense gene therapy improves functional healing of early rabbit ligament scar with enhanced collagen fibrillogenesis in vivo [J]. J Orthop Res,2000,18(4):517-523.
[28]. Shimomura T, Jia F, Niyibizi C, et al. Antisense oligonucleotides reduce synthesis of procollagen alphal (Ⅴ) chain in human patellar tendon fibroblasts:potential application in healing ligaments and tendons[J]. Connect Tissue Res,2003,44(3):167-172.
[29].蒋青,林共周,曲绵域,等.前十字韧带重建早期生长因子的表达.中国运动医学杂志,2000,19(3)：249-250