TBP化学萃取法去细胞同种异体肌腱制备方法的实验研究
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摘要
目的:肌腱损伤是手外科中的多发损伤,肌腱缺损的修复是手外科临床常见病之一,常用的修复方法是自体肌腱移植、转位、延长及自体筋膜替代缺损肌腱,但自体肌腱来源有限,多条肌腱缺损的治疗仍是临床上一个亟待解决的难题。随着同种异体肌腱的研究制备,为肌腱缺损的治疗开创了一条新的途径;同种异体肌腱来源充足、取材方便、手术时间短,减少患者新的损伤及供区的功能影响。但是同种异体肌腱移植后发生的粘连问题一直是困扰临床广泛应用的最大难题。肌腱是一个少血供、少细胞的组织,有相对较低的抗原性。胶原并不表现抗原性,肌腱的抗原性主要存在于其腱细胞成分,主要是组织相容性抗原(HLA)。本课题采用TBP化学萃取法去细胞同种异体肌腱,制备出肌腱细胞去除完全,肌腱的生物力学无改变的去细胞同种异体肌腱移植物,从而更好的去除同种异体肌腱的免疫原性,防止肌腱外源性愈合,促进肌腱内源性愈合,减轻移植后的肌腱的粘连。
方法:选用3个月龄健康雄性白色Leghorn鸡48只,平均1.9±0.053Kg。随机分为A、B、C三组,A组为TBP化学萃取法组、B组为深低温冷冻处理组,C组为空白对照组。本实验选用Lenghorn鸡双足第三趾为实验模型趾。取足趾掌侧纵行切口,长约10cm,切开皮肤后沿跖侧腱鞘浅层锐性分离,向侧方翻起皮瓣,暴露跖侧腱鞘,沿腱鞘正中纵行切开,长约10cm,于趾间关节伸直位趾深屈肌腱止点处横断趾深屈肌腱,分别将长、短腱纽于肌腱连接处离断,勿损伤肌腱端,向近端延长切口,仔细分离腱联合,游离出第三趾屈趾深肌腱,将其于小腿间关节部离断。取出肌腱,生理盐水漂洗,去除肌腱外膜。A组肌腱:40ml Tris缓冲溶液,PH=8.0,包括5mM EDTA,1%(v/v)TBP浸泡肌腱48小时,每24小时更换一次液体。再用40ml中性液体漂洗24小时,70%乙醇冲洗24小时(上述步骤均在振荡器上室温下进行)。肌腱保存在保温箱(37℃,5%CO2)和组织培养基中,包括10%胎牛血清,1%HEPES(羟乙基哌嗪乙磺酸),1%谷氨酰胺,0.4%艮他霉素,盘尼西林G200U/ml,链霉素200ug/ml,两性霉素B0.5ug/ml,备用;B组肌腱:浸泡于MEM液(含10%甘油)10-15分钟,无菌容器封装,贴标签记录取材日期、肌腱类型等项目,置于可控深低温冰箱内,逐步降温至-80℃,10d后测试;C组肌腱为空白对照组。对进行上述处理的三组肌腱分别行①形态组织学观察:分别在大体、HE染色及电镜三方面进行形态学观察分析;②生物力学测试:用Tytron-250生物力学试验机对三组肌腱分别行肌腱最大拉伸断裂强度(Pmax),肌腱最大拉伸断裂功耗(Wmax),及肌腱拉伸断裂延伸率(δmax)测定。③混合淋巴细胞培养测定:用含0.2%胶原酶RPM I1640液分别消化切碎的A、B、C组肌腱,分离获取细胞成分,调节浓度为1×106/ml,作为刺激细胞;同时采取受体鸡静脉血,用淋巴细胞分离液分离获取淋巴细胞,调节浓度为1×106/ml作为反应细胞.将刺激细胞分别等量与反应细胞混合培养,各组做3个复孔,混合培养5天后,吸取细胞悬液离心取沉淀制推片,瑞姬氏染色,光镜下计总淋巴细胞数(约500个)及转化细胞数,计算转化率如下:淋巴细胞转化率=转化细胞数/淋巴细胞总数×100%。计算各组3个复孔转化率均值,作为此组转化率.分别取10只供体鸡趾屈腱及10只受体鸡静脉血试验。所有统计结果采用方差分析。
结果:①形态组织学观察:大体上:化学萃取法处理后肌腱呈瓷白色,表面有光泽,质柔软,韧性好,肌腱完整无缺损;深低温冷冻处理组肌腱为乳白色,表面无光泽,质柔软,韧性好,肌腱完整无缺损;空白对照组肌腱肌腱为乳白色,表面无光泽,质柔软,韧性好,肌腱完整无缺损。HE染色:化学萃取法处理的肌腱腱束间可见疏松结缔组织,胶原纤维平行密集排列整成束,束间无腱细胞存在;深低温冷冻处理的肌腱腱束间可见疏松结缔组织,胶原纤维平行密集排列成束,束间可见腱细胞排列成串;空白对照组肌腱腱束间可见疏松结缔组织,胶原纤维平行密集排列成束,束间可见腱细胞排列成串。透射电镜观察:化学萃取法处理的肌腱:无残留细胞,胶原原纤维排列规整,多呈波浪状,纤维间相互交联,纤维上有明暗交替的周期性横纹。深低温冷冻处理的肌腱:细胞无肿胀,细胞膜不清晰,胞核无变性,核膜完整,胞浆少,粗面内质网、线粒体无明显肿胀;胶原原纤维排列规整,多呈波浪状,纤维间相互交联,部分纤维肿胀,纤维间隙增大,纤维上有明暗交替的周期性横纹,横纹不清。空白对照组肌腱:细胞无肿胀,细胞膜清晰,胞核无变性,核膜完整,粗面内质网、线粒体无肿胀.胶原原纤维排列规整、紧密,纤维粗细均匀,多呈波浪状,纤维间相互交联,纤维上有明暗交替的周期性横纹,横纹清晰。②生物力学测试:肌腱最大拉伸断裂强度(Pmax):三组间差异无统计学意义(P=0.527);肌腱最大拉伸断裂功耗(Wmax):三组间差异无统计学意义(P=0.275);肌腱拉伸断裂延伸率(δmax):三组间差异无统计学意义(P=0.545)。综合三方面测试结果认为三组肌腱生物力学方面无显著差异。③混合淋巴细胞培养测定:实验结果显示:三组间差异有统计学意义(F=4.652,P=0.018)。化学萃取法处理组淋巴细胞转化率(1.85±0.63%),显著低于较深低温冷冻法处理组淋巴细胞转化率(6.89±1.05%)及未处理组淋巴细胞转化率(14.96±1.81%)(p值<0.05);深低温冷冻法处理组淋巴细胞转化率也低于未处理组淋巴细胞转化率(p值<0.05)。说明化学萃取法几乎消除了肌腱的免疫源性,深低温冷冻法显著降低了肌腱的免疫源性。
结论:TBP化学萃取法处理肌腱去除腱细胞完全,并且安全不破坏细胞间的基质,几乎完全消除异体肌腱免疫原性,为异体肌腱的移植提供了新的方法。
TBP化学萃取法处理同种异体肌腱较深低温冷冻法能够更加完全的去除肌腱免疫原性,而且对细胞基质及胶原纤维没有影响,生物力学性能无改变。更有利于体外培养的肌腱细胞回植于肌腱支架上,可以促进移植后的肌腱的内源性愈合,减少外源性愈合,从而减轻粘连的发生。
Objective: Tendon injury is a common injury in the department of Hand Surgery. Reconstruction of tendon defects is a common clinical disease of Hand Surgery. Autograft tendon transplantation, transposition, extension and replacement with autograft fascia for tendon defects are commonly used, but the source of autograft tendon is limited. Multitude tendon defects in the clinical treatment is still an urgent problem to solve. As the study and preparation of allograft tendon blaze a new trail for tendon defects reconstruction with the following advantages: wide source, convenient drawing, shortened operation time, reduced new injury upon the patients and functional influences upon the donor site. However, the biggest problem confronting the wide clinical application of allograft tendon is the post-transplantation adhesion. Tendon is an issue with few blood supply and few cell bodies and a relatively low antigenicity. Collagen does not show the antigenicity and antigenicity of the tendon mainly exists in its cell components, i.e. histocompatibility antigen (HLA). The paper employs TBP chemical extraction to prepare the allograft tendon of which cells have been completely removed without changing its biomechanics. Immunity of the allograft tendon is better removed; exogenous healing is prevented and endogenous healing is promoted; post-transplantation adhesion is reduced.
Methods: Totally 48 healthy male white 3-month-old Leghorn chickens with body mass of 1.9±0.053 Kg were randomly divided into three groups of A, B, C: Group A for TBP chemical extraction treatment, Group B for deep cryogenic treatment, Group C for blank controller. The III toes of both feet in the selected Leghorn chickens were employed to establish the tendon injury model. The incision of about 10cm was made longitudinally on toe volar.The tendons were sharply separated along the lateral plantar tendon sheath of shallow after skin opening. The flap was turned over at lateral side and exposed lateral plantar tendon sheath. The incision was longitudinal along the median in tendon sheath, about 10cm. The flexor digitorum profundus was cut at insertion of The flexor digitorum profundus under unbend position of interphalangeal joints. Long and short tendon vincula tendinum was respectively cut at the tendon junction and did not damage tendon end. Extend the incision to proximal end, carefully separated tendon symphysis, dissociated the III flexor digitorum profundus, cut it at the joints of its legs. Removed the tendons, rinsed in saline, and removed outer membrane. Group A of tendons: immersed in approximately 40 mL of an extraction solution (pH 8.0) consisting of Tris buffer, 5 mM EDTA, and 1% (v / v) TBP for 48 hours. All solutions were refreshed every 24 h. And the tendons were rinsed in deionized water (40 mL) for 24 h and finally washed in 70% ethyl alcohol (40 mL) for 24 h. (All steps were conducted at room temperature and with constant agitation.) Tendons were maintained in a standard tissue culture incubator (370C, 5% CO2) and bathed in tissue culture medium with fetal bovine serum [10%], HEPES [1%], Lglutamine [1%], gentamicin [0.4%], penicillin G [200U/mL], streptomycin [200 mg/mL], and amphotericin B[0.5 mg/mL]); Group B tendons: immersed in MEM solution (containing 10% glycerol) 10-15 minutes, packaged in sterile container, labeled the derived date and tendons type of items. Placed in a refrigerator controlled deep hypothermia, gradually cooling down to -80℃, 10 days later post-test; group C tendons: blank controller. The above-mentioned three groups of tendons were tested by the following three methods.①morphological and histological observation: respectively In general, HE staining and electron microscopy observation of the three aspects of morphological analysis②biomechanical testing: A Tytron-250 bio-mechanical test machine to the three groups were treated by maximum tensile breaking strength (Pmax), the maximum tensile tendon rupture power (Wmax), and the tendon tensile elongation at break (δmax) determination.③measured mixed lymphocyte culture: RPM I1640 liquid containing 0.2% collagenase digested three groups tendons which were chopped. Separated access to cellular components.Regulated the concentration of 1×106/ml, as an incentive cells. while taked receptors chicken vein blood. separated lymphocytes Used lymphocyte separation medium. Regulated the concentration of 1×106/ml as a reaction cell. The stimulate cells were mixed with the same as reaction cell culture. Each group made three double-pored, mixed culture 5 days. Drawed cell suspension and centrifuged to push the deposition films, Sweden Giemsa staining. Calculated the number of total lymphocyte count (about 500) and conversion cells in light microscope. Conversion rates are as follows: lymphocyte transformation rate = the number of transformed cells / total lymphocyte cells×100%. Calculated the mean of three holes each group conversion rate as this group conversion rate.10 toe flexor tendon were collected from the chicken, and 10 receptor chicken blood were tested. All statistical results used analysis of variance.
Results:①Morphological and histological observation: general situation: chemically extracted tendon was porcelain white, glossy on surface, soft, tenacious, intact and non-defect; deep frozen tendon was milky white, non-glossy on surface, soft, tenacious, intact and non-defect; The blank controller was milky white, non-glossy on surface, soft, tenacious, intact and non-defect; HE staining: between the tendon bundles of loose connective tissues were seen in chemical extraction treatment. Collagen fibers paralleled with each other in bundles without tendon cells. Between the tendon bundles of loose connective tissues were seen in deep frozen tendons. Collagen fibers paralleled with each other in bundles. Tendon cells were seen between the clusters arrangement. Between the tendon bundles of loose connective tissues were seen in blank controller. Collagen fibers arranged in parallel in bundles. Between the clusters arrangement of tendon cells were seen. TEM: Chemically extracted tendons: no residual cells, collagen fibers neatly arranged, multi-wave-shaped fiber cross-linked with each other, fiber on the cyclical nature of light and dark stripes alternating. Deep frozen tendons: no swollen cells, the unclear membrane, no degenerated nucleus, intact nuclear membrane, cytoplasmic little, rough endoplasmic reticulum, no obviously swollen mitochondria. collagen fibers neatly arranged, mostly wavy fibers cross-linked with each other, some swollen fibers, increased fiber gap, the fiber has a cyclical alternating light and dark stripes and stripes unclear. Blank controller tendons: No swollen cells, clear membrane, no degenerated nucleus, intact nuclear membrane, rough endoplasmic reticulum and no swollen mitochondria. Collagen fibers arranged neat, close. Fiber thickness uniformity, mostly wavy, cross-linked with each other, on the cyclical nature of alternating light and dark stripes and a clear cross striations.②Biomechanical test: The maximum tensile tendon rupture strength (Pmax): differences among the three groups show no statistical significance (P = 0.527); The maximum tensile tendon rupture power (Wmax): difference among the three groups show no statistical significance (P = 0.275); Tendon tensile elongation at break (δmax): differences among the three groups have no statistical significance (P = 0.545). Comprehensive three-pronged test results that differences among the three groups of tendon biomechanics have no statistical significance.③Mixed lymphocyte culture test: Experimental results suggested difference among the three group the statistical significance (F = 4.652, P = 0.018). The lymphocyte transformation rate of chemical extraction treatment method (1.85±0.63%) significantly lower than those of deep cryogenic treatment group (6.89±1.05%) and untreated group (14.96±1.81%) (p value<0.05); lymphocyte transformation rate of deep cryogenic treatment group is also lower than that of untreated group (p value<0.05). Chemical extraction treatment method virtually eliminates the tendon immunogenicity, and the deep freezing treatment method significantly reduces the tendon immunity.
Conclusion: TBP chemical extraction treatment method can completely remove tendon cells without destroying the intercellular matrix. It almost eradicates the allograft tendon immunity as a new approach for tendon allograft transplantation.
TBP chemical extraction treatment method can remove the tendon immunogenicity more efficiently than the deep freezing treatment method without affecting its cell matrix and collagen fibers. It is more conducive to reimplant cultured tendon cells on the scaffold.It promotes post-transplantation endogenous tendon healing and reduces exogenous healing to reduce the adhesion occurrence.
方法:选用3个月龄健康雄性白色Leghorn鸡48只,平均1.9±0.053Kg。随机分为A、B、C三组,A组为TBP化学萃取法组、B组为深低温冷冻处理组,C组为空白对照组。本实验选用Lenghorn鸡双足第三趾为实验模型趾。取足趾掌侧纵行切口,长约10cm,切开皮肤后沿跖侧腱鞘浅层锐性分离,向侧方翻起皮瓣,暴露跖侧腱鞘,沿腱鞘正中纵行切开,长约10cm,于趾间关节伸直位趾深屈肌腱止点处横断趾深屈肌腱,分别将长、短腱纽于肌腱连接处离断,勿损伤肌腱端,向近端延长切口,仔细分离腱联合,游离出第三趾屈趾深肌腱,将其于小腿间关节部离断。取出肌腱,生理盐水漂洗,去除肌腱外膜。A组肌腱:40ml Tris缓冲溶液,PH=8.0,包括5mM EDTA,1%(v/v)TBP浸泡肌腱48小时,每24小时更换一次液体。再用40ml中性液体漂洗24小时,70%乙醇冲洗24小时(上述步骤均在振荡器上室温下进行)。肌腱保存在保温箱(37℃,5%CO2)和组织培养基中,包括10%胎牛血清,1%HEPES(羟乙基哌嗪乙磺酸),1%谷氨酰胺,0.4%艮他霉素,盘尼西林G200U/ml,链霉素200ug/ml,两性霉素B0.5ug/ml,备用;B组肌腱:浸泡于MEM液(含10%甘油)10-15分钟,无菌容器封装,贴标签记录取材日期、肌腱类型等项目,置于可控深低温冰箱内,逐步降温至-80℃,10d后测试;C组肌腱为空白对照组。对进行上述处理的三组肌腱分别行①形态组织学观察:分别在大体、HE染色及电镜三方面进行形态学观察分析;②生物力学测试:用Tytron-250生物力学试验机对三组肌腱分别行肌腱最大拉伸断裂强度(Pmax),肌腱最大拉伸断裂功耗(Wmax),及肌腱拉伸断裂延伸率(δmax)测定。③混合淋巴细胞培养测定:用含0.2%胶原酶RPM I1640液分别消化切碎的A、B、C组肌腱,分离获取细胞成分,调节浓度为1×106/ml,作为刺激细胞;同时采取受体鸡静脉血,用淋巴细胞分离液分离获取淋巴细胞,调节浓度为1×106/ml作为反应细胞.将刺激细胞分别等量与反应细胞混合培养,各组做3个复孔,混合培养5天后,吸取细胞悬液离心取沉淀制推片,瑞姬氏染色,光镜下计总淋巴细胞数(约500个)及转化细胞数,计算转化率如下:淋巴细胞转化率=转化细胞数/淋巴细胞总数×100%。计算各组3个复孔转化率均值,作为此组转化率.分别取10只供体鸡趾屈腱及10只受体鸡静脉血试验。所有统计结果采用方差分析。
结果:①形态组织学观察:大体上:化学萃取法处理后肌腱呈瓷白色,表面有光泽,质柔软,韧性好,肌腱完整无缺损;深低温冷冻处理组肌腱为乳白色,表面无光泽,质柔软,韧性好,肌腱完整无缺损;空白对照组肌腱肌腱为乳白色,表面无光泽,质柔软,韧性好,肌腱完整无缺损。HE染色:化学萃取法处理的肌腱腱束间可见疏松结缔组织,胶原纤维平行密集排列整成束,束间无腱细胞存在;深低温冷冻处理的肌腱腱束间可见疏松结缔组织,胶原纤维平行密集排列成束,束间可见腱细胞排列成串;空白对照组肌腱腱束间可见疏松结缔组织,胶原纤维平行密集排列成束,束间可见腱细胞排列成串。透射电镜观察:化学萃取法处理的肌腱:无残留细胞,胶原原纤维排列规整,多呈波浪状,纤维间相互交联,纤维上有明暗交替的周期性横纹。深低温冷冻处理的肌腱:细胞无肿胀,细胞膜不清晰,胞核无变性,核膜完整,胞浆少,粗面内质网、线粒体无明显肿胀;胶原原纤维排列规整,多呈波浪状,纤维间相互交联,部分纤维肿胀,纤维间隙增大,纤维上有明暗交替的周期性横纹,横纹不清。空白对照组肌腱:细胞无肿胀,细胞膜清晰,胞核无变性,核膜完整,粗面内质网、线粒体无肿胀.胶原原纤维排列规整、紧密,纤维粗细均匀,多呈波浪状,纤维间相互交联,纤维上有明暗交替的周期性横纹,横纹清晰。②生物力学测试:肌腱最大拉伸断裂强度(Pmax):三组间差异无统计学意义(P=0.527);肌腱最大拉伸断裂功耗(Wmax):三组间差异无统计学意义(P=0.275);肌腱拉伸断裂延伸率(δmax):三组间差异无统计学意义(P=0.545)。综合三方面测试结果认为三组肌腱生物力学方面无显著差异。③混合淋巴细胞培养测定:实验结果显示:三组间差异有统计学意义(F=4.652,P=0.018)。化学萃取法处理组淋巴细胞转化率(1.85±0.63%),显著低于较深低温冷冻法处理组淋巴细胞转化率(6.89±1.05%)及未处理组淋巴细胞转化率(14.96±1.81%)(p值<0.05);深低温冷冻法处理组淋巴细胞转化率也低于未处理组淋巴细胞转化率(p值<0.05)。说明化学萃取法几乎消除了肌腱的免疫源性,深低温冷冻法显著降低了肌腱的免疫源性。
结论:TBP化学萃取法处理肌腱去除腱细胞完全,并且安全不破坏细胞间的基质,几乎完全消除异体肌腱免疫原性,为异体肌腱的移植提供了新的方法。
TBP化学萃取法处理同种异体肌腱较深低温冷冻法能够更加完全的去除肌腱免疫原性,而且对细胞基质及胶原纤维没有影响,生物力学性能无改变。更有利于体外培养的肌腱细胞回植于肌腱支架上,可以促进移植后的肌腱的内源性愈合,减少外源性愈合,从而减轻粘连的发生。
Objective: Tendon injury is a common injury in the department of Hand Surgery. Reconstruction of tendon defects is a common clinical disease of Hand Surgery. Autograft tendon transplantation, transposition, extension and replacement with autograft fascia for tendon defects are commonly used, but the source of autograft tendon is limited. Multitude tendon defects in the clinical treatment is still an urgent problem to solve. As the study and preparation of allograft tendon blaze a new trail for tendon defects reconstruction with the following advantages: wide source, convenient drawing, shortened operation time, reduced new injury upon the patients and functional influences upon the donor site. However, the biggest problem confronting the wide clinical application of allograft tendon is the post-transplantation adhesion. Tendon is an issue with few blood supply and few cell bodies and a relatively low antigenicity. Collagen does not show the antigenicity and antigenicity of the tendon mainly exists in its cell components, i.e. histocompatibility antigen (HLA). The paper employs TBP chemical extraction to prepare the allograft tendon of which cells have been completely removed without changing its biomechanics. Immunity of the allograft tendon is better removed; exogenous healing is prevented and endogenous healing is promoted; post-transplantation adhesion is reduced.
Methods: Totally 48 healthy male white 3-month-old Leghorn chickens with body mass of 1.9±0.053 Kg were randomly divided into three groups of A, B, C: Group A for TBP chemical extraction treatment, Group B for deep cryogenic treatment, Group C for blank controller. The III toes of both feet in the selected Leghorn chickens were employed to establish the tendon injury model. The incision of about 10cm was made longitudinally on toe volar.The tendons were sharply separated along the lateral plantar tendon sheath of shallow after skin opening. The flap was turned over at lateral side and exposed lateral plantar tendon sheath. The incision was longitudinal along the median in tendon sheath, about 10cm. The flexor digitorum profundus was cut at insertion of The flexor digitorum profundus under unbend position of interphalangeal joints. Long and short tendon vincula tendinum was respectively cut at the tendon junction and did not damage tendon end. Extend the incision to proximal end, carefully separated tendon symphysis, dissociated the III flexor digitorum profundus, cut it at the joints of its legs. Removed the tendons, rinsed in saline, and removed outer membrane. Group A of tendons: immersed in approximately 40 mL of an extraction solution (pH 8.0) consisting of Tris buffer, 5 mM EDTA, and 1% (v / v) TBP for 48 hours. All solutions were refreshed every 24 h. And the tendons were rinsed in deionized water (40 mL) for 24 h and finally washed in 70% ethyl alcohol (40 mL) for 24 h. (All steps were conducted at room temperature and with constant agitation.) Tendons were maintained in a standard tissue culture incubator (370C, 5% CO2) and bathed in tissue culture medium with fetal bovine serum [10%], HEPES [1%], Lglutamine [1%], gentamicin [0.4%], penicillin G [200U/mL], streptomycin [200 mg/mL], and amphotericin B[0.5 mg/mL]); Group B tendons: immersed in MEM solution (containing 10% glycerol) 10-15 minutes, packaged in sterile container, labeled the derived date and tendons type of items. Placed in a refrigerator controlled deep hypothermia, gradually cooling down to -80℃, 10 days later post-test; group C tendons: blank controller. The above-mentioned three groups of tendons were tested by the following three methods.①morphological and histological observation: respectively In general, HE staining and electron microscopy observation of the three aspects of morphological analysis②biomechanical testing: A Tytron-250 bio-mechanical test machine to the three groups were treated by maximum tensile breaking strength (Pmax), the maximum tensile tendon rupture power (Wmax), and the tendon tensile elongation at break (δmax) determination.③measured mixed lymphocyte culture: RPM I1640 liquid containing 0.2% collagenase digested three groups tendons which were chopped. Separated access to cellular components.Regulated the concentration of 1×106/ml, as an incentive cells. while taked receptors chicken vein blood. separated lymphocytes Used lymphocyte separation medium. Regulated the concentration of 1×106/ml as a reaction cell. The stimulate cells were mixed with the same as reaction cell culture. Each group made three double-pored, mixed culture 5 days. Drawed cell suspension and centrifuged to push the deposition films, Sweden Giemsa staining. Calculated the number of total lymphocyte count (about 500) and conversion cells in light microscope. Conversion rates are as follows: lymphocyte transformation rate = the number of transformed cells / total lymphocyte cells×100%. Calculated the mean of three holes each group conversion rate as this group conversion rate.10 toe flexor tendon were collected from the chicken, and 10 receptor chicken blood were tested. All statistical results used analysis of variance.
Results:①Morphological and histological observation: general situation: chemically extracted tendon was porcelain white, glossy on surface, soft, tenacious, intact and non-defect; deep frozen tendon was milky white, non-glossy on surface, soft, tenacious, intact and non-defect; The blank controller was milky white, non-glossy on surface, soft, tenacious, intact and non-defect; HE staining: between the tendon bundles of loose connective tissues were seen in chemical extraction treatment. Collagen fibers paralleled with each other in bundles without tendon cells. Between the tendon bundles of loose connective tissues were seen in deep frozen tendons. Collagen fibers paralleled with each other in bundles. Tendon cells were seen between the clusters arrangement. Between the tendon bundles of loose connective tissues were seen in blank controller. Collagen fibers arranged in parallel in bundles. Between the clusters arrangement of tendon cells were seen. TEM: Chemically extracted tendons: no residual cells, collagen fibers neatly arranged, multi-wave-shaped fiber cross-linked with each other, fiber on the cyclical nature of light and dark stripes alternating. Deep frozen tendons: no swollen cells, the unclear membrane, no degenerated nucleus, intact nuclear membrane, cytoplasmic little, rough endoplasmic reticulum, no obviously swollen mitochondria. collagen fibers neatly arranged, mostly wavy fibers cross-linked with each other, some swollen fibers, increased fiber gap, the fiber has a cyclical alternating light and dark stripes and stripes unclear. Blank controller tendons: No swollen cells, clear membrane, no degenerated nucleus, intact nuclear membrane, rough endoplasmic reticulum and no swollen mitochondria. Collagen fibers arranged neat, close. Fiber thickness uniformity, mostly wavy, cross-linked with each other, on the cyclical nature of alternating light and dark stripes and a clear cross striations.②Biomechanical test: The maximum tensile tendon rupture strength (Pmax): differences among the three groups show no statistical significance (P = 0.527); The maximum tensile tendon rupture power (Wmax): difference among the three groups show no statistical significance (P = 0.275); Tendon tensile elongation at break (δmax): differences among the three groups have no statistical significance (P = 0.545). Comprehensive three-pronged test results that differences among the three groups of tendon biomechanics have no statistical significance.③Mixed lymphocyte culture test: Experimental results suggested difference among the three group the statistical significance (F = 4.652, P = 0.018). The lymphocyte transformation rate of chemical extraction treatment method (1.85±0.63%) significantly lower than those of deep cryogenic treatment group (6.89±1.05%) and untreated group (14.96±1.81%) (p value<0.05); lymphocyte transformation rate of deep cryogenic treatment group is also lower than that of untreated group (p value<0.05). Chemical extraction treatment method virtually eliminates the tendon immunogenicity, and the deep freezing treatment method significantly reduces the tendon immunity.
Conclusion: TBP chemical extraction treatment method can completely remove tendon cells without destroying the intercellular matrix. It almost eradicates the allograft tendon immunity as a new approach for tendon allograft transplantation.
TBP chemical extraction treatment method can remove the tendon immunogenicity more efficiently than the deep freezing treatment method without affecting its cell matrix and collagen fibers. It is more conducive to reimplant cultured tendon cells on the scaffold.It promotes post-transplantation endogenous tendon healing and reduces exogenous healing to reduce the adhesion occurrence.
引文
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22周继辉,曲彦隆,李宗虎,等.骨髓基质干细胞与肌腱修复.国际骨科学杂志,2007,28(1):123-125
23 ALEXANDER H, WEISS A B, PARSONS J R, et al. Ligament and tendonrepair with an absorbable polymer-coated carbon fiber stent. Bull Hosp J t Dis Orthop Inst, 1986, 46 (2):155-173
24 Fini M, Torricelli P, Giavaresi G, et al. In vitro study comparing two ollageneous membranes in view of their clinical application for rotator uff tendon regeneration. Orthop Res 2007,25(1):98-107
25 Ge H, Xu J, Zhou X. Decision support for tendon tissue engineering. Med Eng Technol, 2006,30(2):69-72
26 OUYANG H W, GOH J C, THAMBYAH A, et al. Knitted poly-lactideco- glycolide scaffold loaded with bone marrow stromal cells in repair and regeneration of rabbit achilles tendon. Tissue Engineering, 2003,9(3): 431-439
27 SAHOO S, OUYANG H W. Characterization of a novel polymeric scaffold for potential app lication in tendon/ligament tissue engineering.Tissue Engineering, 2006,12 (1):91-99
28曲彦隆,杨志明,解慧琪,等.组织工程化肌腱对T淋巴细胞亚群及其受体的影响.中国修复与重建杂志,2001,15(2):113-115
29邓万祥,赵胡瑞,董晖,等.异体肌腱移植在临床修复中的应用.中国修复与重建杂志,2005,19(8):666-668
30 Pinkowski J L, Rodrigo J L, Sharkey N A, et al. Immune response to nonspecific and altered tissue antigens in soft tissue allografts. Clin Orthop, 1996,32(6):80-85
31 Guidos C, Sinhua A A, Lee K C. Functional difference and complementation between dendritic cells and macrophages in cell activation. Immunoligy, 1987,61(3):269-276
32 Rtchie J R, Rarker R D. Graftselection in anterior cruciate ligament revision surgery. Clin Orthop, 1996,32(5):66-68
33 Aho A J, Eskola J, Ekfors T, et al. Immune responeses and clinical outcome of messive human osteroarticular allografts. Clin Orthop, 1998,34(6):196-206
34 Jackson D W, Sinmon TM, Kurzweil P. Survival of cells after intraarticular transplantation of fresh allografts of the patellar and anterior cruciate ligaments. Bone Joint Surg, 1992,74A:112
35 Amoczky S, Warren R, Ashlock M. Replacement of the antenior crucidte ligament using a patellar tendon allografts. Bone Joint Surg 1985,68A:376-382
36 Sharma P, Maffulli N, et al. Biology of tendon injury: healing modeling and remodeling.Musculoskelet Neuronal Interact. 2006, 6(2):181-190
37 Tsuzaki M, Brigman B, Yammamoto J, et al. Insulin-like growth factor-I is exprrssed by an avain flexor tendon cells. Orthop res, 2000,18(4):546-556
38 Bered Jik , Lian Pk. Biologic aspects of flexor tendon laceration and repair.Jone Joint surg. Am, 2003,85(3):539-550
39 Chang J, Thunder R, Most D, et al. Studies in flexor tendon wound healing: neutralizing antraling antibody to TGF-betal increases post-operativerange of motion. Plast Reconstr Surg, 2000,105:148-155
40耿树岩,王宏光,张文强,等. VEGF抗体对兔同种异体肌腱移植作用的实验研究.实用手外科, 2007,6 (21):93-95
41 Zhang AY, Pham H, Ho F, et al. Inhibition of TGF-beta-in-duced collagen production in rabbiy flexor tendons. Hand surg, 2004,29(2):230-235
42 Wang XT, Liu PY, Xin KQ, et al. Tendon healing in vitro: bFGF gene transfer to tenocytes by adeno-associated viral vectors promotes expression of collagen genes. Hand Surg Am, 2005,30(6):1255-1261
43 Lou J, Tu Y, Buras M, et al. BMP-12 gene transfer sugmentation of lacerted tendon repair. Orthop Res, 2001,19(6):1199-1202
44 Dai Q, Manfield L, Wang Y, et al. Adenovirus-mediated gene transfer to healing tendon-enhanced efficiency using a gelatin sponge. Orthop Res 2003;21(4):604-609
45 Chun feng Zhao, Hsiao-Feng Chieh, Karim Bakri, et al. The effects of bone marrow stromal cell transplants on tendon healing in vitro. Medical Engineering &Physics, 2009,30(5):1572-1577
46程昌志,张正治,潘险峰,等.肌腱愈合及粘连形成机制的研究:胰岛素样生长因子1基因真核体的构建及表达.中国临床康复, 2004,8(20):3984-3986
47 Goomer RS, Maris TM, Gelberman R, et al. Nonviral in vivo gene therapy for tissue engineering of articular cartilage and tendon repair. Clin Orthop, 2000,379(suppl):189-200
48 Nakamurs N, Hart DA, Boorman RS, et al. Decrin antisense gene therapy improves functional healing of early rabbit ligment scar with enhanced collagen fibrillogenesis in vivo. Orthop Res, 2000,18(4):517-523
49 Screen HR, Sbelton JC, Bader DL, et al. Cyelic tensile strain upregulates collagen synthesis in isolated tendon fascicles. Biochem Biophys Res Commun, 2005,336(2):424-429
50 Altman G H, Horan R L, Martin I, et al. Cell differentiation by mechanical stress. FASEB J, 2002,16:424-429
51 Zeichen J, Van Grienseven M. Bosch U The proliferative response ofis01ated human tendon fibroblasts to biaxial mechanical strain. Am Jsports Med, 2002,28(6),888-892
52曹德君,翟华玲,刘伟,等.体外构建组织工程化肌腱的初步研究.中华外科杂志, 2004,42(2):46-49
53夏长所,宏光祥,李红,等.几丁糖对肌腱腱鞘和腱外膜以及腱内膜细胞增殖和胶原产生的影响.中华显微外科杂志,2005,28(2):154-156
54罗静聪,杨志明,李秀群,等.生物衍生羊膜对大鼠跟腿粘连影响的实验研究.中华修复重建外科杂志,2004,18(5):431-434
55时志斌,汪德芬,张晓峰等.舒筋汤对肌腱粘连防治的生物力学和生化研究。中国矫形外科杂志,2002;9(2):151-153
56 Stevenson J H, Pang C Y, Lindsay W K, et al. Functional mechanical and biochemical assessment of ultrasound therapy on tendon healing in the chicken toe. PlastReconstr Surg, 1986,77(6):965-972
57田德虎,郭明珂,米立新,等.分米波防治屈肌腱粘连机制的实验研究.中华物理医学与康复杂志,2003,25(4):646-649
58陈兵,丁小邦,祝联,等.自体肌腱细胞介导修复肌腱缺损的实验研究.中华手外科杂志,2003,19(1):34-36
59邓丹,刘伟,曹谊林.组织工程化肌腱种子细胞的研究.国际生物医学工程杂志,2006,23(4):245-246
60邓丹,刘伟,杨漾,等.人皮肤成纤维细胞与PGA进行组织构建的适宜接种浓度.组织工程与重建外科,2006,2(3):345-347
61许锋,翟华玲,吴娟娟,等.体外培养的不同个体人肌腱细胞与皮肤成纤维细胞中胶原表达水平的比较研究.组织工程与重建外科,2006, 2(3) :367-368
62邓丹,武晓莉,许锋,等.MTT法检测国产PGA支架对人成纤维细胞增殖的影响.组织工程与重建外科,2006,2(1):67-69
2 Costa MA, Wu C, Pham BV, et al. Tissue engineering of flexor tendons: optimization of tenocyte proliferation useing growth factor supplementation. Tissue Eng 2006, 12(7):1937-1943
3耿树岩,王成琪.同种异体肌腱移植的研究成果.实用手外科杂志,2006,20(3):163-165
4鄂群,朱蓓,王东林,等.兔肌腱组织石蜡切片的制备.修复重建外科杂志,2007,21(4):424-425
5曲彦隆,杨志明,解慧琪,等.组织工程化肌腱对T淋巴细胞亚群及其受体的影响,中国修复与重建杂志,2001,15(2):113-115
6 Mina A, Ishi S, Agino T, et al. Effect of the immunological antigenicity of the allogencie tendons on tendon grdting. The hand,1982,14:111-119
7邓万祥,赵胡瑞,董晖,等.异体肌腱移植在临床修复中的应用.中国修复重建外科杂志, 2005,19(8):666-668
8 Pinkowski JL, Rodrigo JL, Sharkey NA, et al. Immune response to non- specific and altered tissue antigens in soft tissue allografts. Clin Orthop,1996,326:80-85
9 Zimmerman MC, Contiliano JH, Parsone JR, et al. Effect of freeze drying or irradiation on remodeling of tendon allograft in a rat model. Orthop Rex,1997,15(2):292-294
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15王宣生,张旭强,李庆泰.同种异体肌腱在肌腱缺损治疗的临床应用.现代临床医学,2006,12(6):24-25
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17唐林俊,雷军,方光荣,等.超深低温处理的异体肌腱移植.中华手外科杂志,2001,17(l):123
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19 Charles E, Laurel M, Vincent R. A compositive nerve graft system:Extracted rat perpheral nerve injected with cultural schwann cells. Muscle&Nerve,1996,19:97-99
20 Sondel1 M, Lundborg G, Kanje M. Rengeneration of the rat SCiatic nerve into allografts made acellular through chemical extraction. Brain Research, 1998,795:44-54
21王建云,刘小林,朱家凯,等.化学萃取同种异体神经种植雪旺细胞的体外研究.中华显微外科杂志,2002,25(3):189-191
22朱庆棠,朱家凯.去细胞组织工程化神经支架的制备和形态学研究.中华显微外科杂志,2004,27(1):35-37
23 Cartmell JS, Dunn MG. Effect of chemical treatments on tendon cellularity and mechanical properties. Biomed MaterRes, 2000,49(1):134-140
24 Cartmell Js, Dunn MG. Development of cell-seeded patellar tendonallografts for anterior cruciate ligment reconstruction. Tissue Eng, 2004, 10(7-8):1065-75
25 Majewski M, Betz O, Ochsner PE, et al. Ex vivo adenoviral transfer of bone morphogenetic protein 12 (BMP-12) cDNA improves Achilles tendon healing in a rat model. Gene Ther. 2008,15(16):1139-46
26 Chong AK, Ang AD, Goh JC, et al. Bone marow-derived mesenchymal stem cells influence early tendon-healing in a rabbit Achilles tendon model. Bone Joint Surg Am, 2007,8(9):74-81
27 Grant R L, Acosta, D. Prolonged adverse effects of benzalkonium chlori- e and sodium dodecyl sulfate in a primary culture system of rabbit corneal ep- ithelial cells. Fundam Appl Toxicol. 1996,33:71
28 Auletta C S, Kotkoskie L A, Saulog,T,et al. A dietary on cogenicity study of tributyl phosphate in the CD-1 mouse. Toxicology 1998,128:135
29 Auletta C S, Weiner M L, Richter W R. A dietary toxicity/on cogenicity s- tudy of tributyl phosphate in the rat. Toxicology 1998,128:125
30 Healy C E, Beyrouty P C, Broxup B R. Acute and subchronic neuroto- xicity studies with tri-n-butyl phosphate in adult Sprague-Dawley rats. Am In- dustr Hygiene Assoc. 1995,56:349
31 Noda T, Yamano T, Shimizu M, et al. Effects of TRI-n-butyl ph- osphate on pregnancy in rats. Food Chem Toxicol. 1994,32:1031
32 Tsuzaki M, Brigman B, Yammamoto J, et al. Insulin-like growth factor-I is expressed by an avain flexor tendon cells. Orthop res, 2000,18(4):546-556
1张友乐,杨克非,高新生,等.冷冻干燥异体肌腱移植的实验研究及临床应用.中华骨科杂志,1997,17(1):59-62
2 Mina A, Ishi S, Agino T, et al. effect of the im-munological an-tigenicity of the allogenic tendons on tendon grdting. The Hand,1982,14:111-119
3唐林俊,陈秉礼,丁钦蓉.脱氧鸟苷培养处理的同种异体肌腱移植的实验研究.中华骨科杂志,1996,16(3):175
4 Peterson RK, Shelton WR, Bomboy AL, et al. Allograft versus auto-graft patellar tendon anterior cruciate ligament reconstruction:A5-year follow-up. Arthroseopy, 2001,17(1):9-13
5 Gerber C, Lambert SM. Allograft reconstruetion of segmental defects of the humeral head for the treatment of chronic locked posterior dislocationof the shoulder. Bone Joint Surg Am,1996,78(3):376-382
6 Cao Y, Vacanty JP, Ma X, et al. Genenation of neo-tendon using synthetic polymers seeded wih tenocytes. Transp l Proc, 1994,26 (6):3390-3391
7张兆锋,商庆新,曹谊林.自体组织工程化肌腱预制的初步研究.中华手外科杂志,2002,18 (2):125-127
8 Webb K, Hitchcock R W, Smeal RM, et al. Cyclic strain increasesfibroblast proliferation matrix accumulation and elastic modulus of fibroblast-seeded polyurethane constructs. Biomech, 2006,39(6): 1136-1144
9高新生,余大庆,余瑞侠,等.同种异体肌腱移植和肌腱库的建立.中华手外科杂志,1996,12(2):89
10 Zimmerman M C, Contiliano J H,Parsone J R,et al. Effect of freeze drying or irradiation on remodeling of tendon allograft in a rat model. Orthop Rex, 1997,15(2):292-297
11 Cartmell J S, Dunn M G. Development of cell-seeded patellar tendon allografts for anterior cruciate ligament reconstruction. Tissue Eng. 2004 ,10(7-8):1065-1075
12吴富章,卜海富,赵刚,张保.玻璃化法保存异体肌腱移植的实验研究.安徽医科大学学报,2008,43(1):20-23
13 Fideler B M, Vangsness J K, Moore J, et al. Effects of Gamma irradination on the Human immunodeficiency Virus. Bone joint sung AM, 1994,76-A,1032-1038
14 Gwinner W, Uenne J, Lonnemann G, et al. Life-threatening comp lications of extracorporeal treatment in patients with severe eosinophilia. The International Journal of A rtificial Organs, 2005,28(12):1224-1227
15 Pekkarinen T, H ietalal O, Jamsa T, et al. Gamma irradiation and ethylene oxide in the sterilization of native reindeer bone morphogenetic protein extract. Scand J Surg, 2005,94(1):67-70
16 Smith RA, Ingels J, Lochemes J J, et al. Gamma irradiation of HIV-1. Journal of O rthopaedic Research, 2001,19:815-819
17 Hernigou G, Gras G, Marinello, et al. Influence of irradiation on the risk oftransmission of HIV in bone grafts obtained from app roppriately screened donors and followed by radiation sterilization. Cell and Tissue Banking, 2000,1:279-289
18 Rappe M, Horodyski M, Meister K, et al. Nonirradiated versus irradiated Achilles allograft: in vivo failure comparison. Am J Sports Med 2007, 35(10):1653-1658
19 King W, Grieb T, Forng R, et al. Pathogen inactivation of soft tissue allografts using high dose gamma irradiation with early clinical results. American Academy of rthopaedic Surgeons 2004 San Francisco California, 2004:571-578
20 Teri A, Grieb, Ren-Yo F, et al. High dose gamma irradiation for soft tissue allografts: high margin of safety with biomechanical integrity. Journal of rthopaedic Research, 2006, 24 (5):1011-1018
21孙官文,温树正.肌腱缺损治疗的现状及研究.医学综述, 2007, 13(20):1590-1592
22周继辉,曲彦隆,李宗虎,等.骨髓基质干细胞与肌腱修复.国际骨科学杂志,2007,28(1):123-125
23 ALEXANDER H, WEISS A B, PARSONS J R, et al. Ligament and tendonrepair with an absorbable polymer-coated carbon fiber stent. Bull Hosp J t Dis Orthop Inst, 1986, 46 (2):155-173
24 Fini M, Torricelli P, Giavaresi G, et al. In vitro study comparing two ollageneous membranes in view of their clinical application for rotator uff tendon regeneration. Orthop Res 2007,25(1):98-107
25 Ge H, Xu J, Zhou X. Decision support for tendon tissue engineering. Med Eng Technol, 2006,30(2):69-72
26 OUYANG H W, GOH J C, THAMBYAH A, et al. Knitted poly-lactideco- glycolide scaffold loaded with bone marrow stromal cells in repair and regeneration of rabbit achilles tendon. Tissue Engineering, 2003,9(3): 431-439
27 SAHOO S, OUYANG H W. Characterization of a novel polymeric scaffold for potential app lication in tendon/ligament tissue engineering.Tissue Engineering, 2006,12 (1):91-99
28曲彦隆,杨志明,解慧琪,等.组织工程化肌腱对T淋巴细胞亚群及其受体的影响.中国修复与重建杂志,2001,15(2):113-115
29邓万祥,赵胡瑞,董晖,等.异体肌腱移植在临床修复中的应用.中国修复与重建杂志,2005,19(8):666-668
30 Pinkowski J L, Rodrigo J L, Sharkey N A, et al. Immune response to nonspecific and altered tissue antigens in soft tissue allografts. Clin Orthop, 1996,32(6):80-85
31 Guidos C, Sinhua A A, Lee K C. Functional difference and complementation between dendritic cells and macrophages in cell activation. Immunoligy, 1987,61(3):269-276
32 Rtchie J R, Rarker R D. Graftselection in anterior cruciate ligament revision surgery. Clin Orthop, 1996,32(5):66-68
33 Aho A J, Eskola J, Ekfors T, et al. Immune responeses and clinical outcome of messive human osteroarticular allografts. Clin Orthop, 1998,34(6):196-206
34 Jackson D W, Sinmon TM, Kurzweil P. Survival of cells after intraarticular transplantation of fresh allografts of the patellar and anterior cruciate ligaments. Bone Joint Surg, 1992,74A:112
35 Amoczky S, Warren R, Ashlock M. Replacement of the antenior crucidte ligament using a patellar tendon allografts. Bone Joint Surg 1985,68A:376-382
36 Sharma P, Maffulli N, et al. Biology of tendon injury: healing modeling and remodeling.Musculoskelet Neuronal Interact. 2006, 6(2):181-190
37 Tsuzaki M, Brigman B, Yammamoto J, et al. Insulin-like growth factor-I is exprrssed by an avain flexor tendon cells. Orthop res, 2000,18(4):546-556
38 Bered Jik , Lian Pk. Biologic aspects of flexor tendon laceration and repair.Jone Joint surg. Am, 2003,85(3):539-550
39 Chang J, Thunder R, Most D, et al. Studies in flexor tendon wound healing: neutralizing antraling antibody to TGF-betal increases post-operativerange of motion. Plast Reconstr Surg, 2000,105:148-155
40耿树岩,王宏光,张文强,等. VEGF抗体对兔同种异体肌腱移植作用的实验研究.实用手外科, 2007,6 (21):93-95
41 Zhang AY, Pham H, Ho F, et al. Inhibition of TGF-beta-in-duced collagen production in rabbiy flexor tendons. Hand surg, 2004,29(2):230-235
42 Wang XT, Liu PY, Xin KQ, et al. Tendon healing in vitro: bFGF gene transfer to tenocytes by adeno-associated viral vectors promotes expression of collagen genes. Hand Surg Am, 2005,30(6):1255-1261
43 Lou J, Tu Y, Buras M, et al. BMP-12 gene transfer sugmentation of lacerted tendon repair. Orthop Res, 2001,19(6):1199-1202
44 Dai Q, Manfield L, Wang Y, et al. Adenovirus-mediated gene transfer to healing tendon-enhanced efficiency using a gelatin sponge. Orthop Res 2003;21(4):604-609
45 Chun feng Zhao, Hsiao-Feng Chieh, Karim Bakri, et al. The effects of bone marrow stromal cell transplants on tendon healing in vitro. Medical Engineering &Physics, 2009,30(5):1572-1577
46程昌志,张正治,潘险峰,等.肌腱愈合及粘连形成机制的研究:胰岛素样生长因子1基因真核体的构建及表达.中国临床康复, 2004,8(20):3984-3986
47 Goomer RS, Maris TM, Gelberman R, et al. Nonviral in vivo gene therapy for tissue engineering of articular cartilage and tendon repair. Clin Orthop, 2000,379(suppl):189-200
48 Nakamurs N, Hart DA, Boorman RS, et al. Decrin antisense gene therapy improves functional healing of early rabbit ligment scar with enhanced collagen fibrillogenesis in vivo. Orthop Res, 2000,18(4):517-523
49 Screen HR, Sbelton JC, Bader DL, et al. Cyelic tensile strain upregulates collagen synthesis in isolated tendon fascicles. Biochem Biophys Res Commun, 2005,336(2):424-429
50 Altman G H, Horan R L, Martin I, et al. Cell differentiation by mechanical stress. FASEB J, 2002,16:424-429
51 Zeichen J, Van Grienseven M. Bosch U The proliferative response ofis01ated human tendon fibroblasts to biaxial mechanical strain. Am Jsports Med, 2002,28(6),888-892
52曹德君,翟华玲,刘伟,等.体外构建组织工程化肌腱的初步研究.中华外科杂志, 2004,42(2):46-49
53夏长所,宏光祥,李红,等.几丁糖对肌腱腱鞘和腱外膜以及腱内膜细胞增殖和胶原产生的影响.中华显微外科杂志,2005,28(2):154-156
54罗静聪,杨志明,李秀群,等.生物衍生羊膜对大鼠跟腿粘连影响的实验研究.中华修复重建外科杂志,2004,18(5):431-434
55时志斌,汪德芬,张晓峰等.舒筋汤对肌腱粘连防治的生物力学和生化研究。中国矫形外科杂志,2002;9(2):151-153
56 Stevenson J H, Pang C Y, Lindsay W K, et al. Functional mechanical and biochemical assessment of ultrasound therapy on tendon healing in the chicken toe. PlastReconstr Surg, 1986,77(6):965-972
57田德虎,郭明珂,米立新,等.分米波防治屈肌腱粘连机制的实验研究.中华物理医学与康复杂志,2003,25(4):646-649
58陈兵,丁小邦,祝联,等.自体肌腱细胞介导修复肌腱缺损的实验研究.中华手外科杂志,2003,19(1):34-36
59邓丹,刘伟,曹谊林.组织工程化肌腱种子细胞的研究.国际生物医学工程杂志,2006,23(4):245-246
60邓丹,刘伟,杨漾,等.人皮肤成纤维细胞与PGA进行组织构建的适宜接种浓度.组织工程与重建外科,2006,2(3):345-347
61许锋,翟华玲,吴娟娟,等.体外培养的不同个体人肌腱细胞与皮肤成纤维细胞中胶原表达水平的比较研究.组织工程与重建外科,2006, 2(3) :367-368
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