EDC交联的脱细胞近交系微型猪肌腱修复兔跟腱缺损的实验研究
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摘要
目的观察碳化二亚胺(EDC)交联改性的脱细胞版纳近交系微型猪肌腱移植修复兔跟腱缺损的疗效,探讨其作为异种肌腱移植支架材料的可行性,为异种肌腱移植的进一步研究提供实验依据。
方法1.EDC交联改性的脱细胞版纳近交系微型猪肌腱的制备及生物学特性检测：无菌条件下取云南农业大学近交系微型猪肌腱后,肌腱按不同处理方法分为三组：新鲜肌腱组(fresh tendon, FT)、单纯脱细胞肌腱组(simply decellulatized tendon, SDT)和EDC交联改性的脱细胞肌腱组(EDC cross-linked acellular tendon, EAT),通过对比各组肌腱的大体、组织学特点,体外降解特性,免疫原性和生物力学性能,评价EDC交联改性的脱细胞版纳近交系微型猪肌腱作为肌腱支架材料的应用价值。
2.将40只日本大白兔雌雄参半,体重在2.3-2.6kg,随机分为A、B两组,每组20只兔子。A组为碳化二亚胺(EDC)交联改性的脱细胞版纳近交系微型猪肌腱移植组,B组为自体肌腱移植组。制作兔双后肢跟腱缺损实验动物模型。A组缺损的肌腱用EDC交联改性的脱细胞版纳近交系微型猪肌腱修复,用3-0肌腱线改良HEMI-KESSLER进行端端吻合,B组缺损的肌腱用自体肌腱修复,用3-0肌腱线改良HEMI-KESSLER法进行端端原位吻合。3.移植术前3d、术后3d、1w、2w外周血白细胞分类计数、C反应蛋白检测。4.移植术后1、4、8、12周处死动物取材。检测分析取材标本中羟脯氨酸含量(吸收光谱法测定)、进行生物力学测定(抗拉强度生物材料力学多功能试验机)、组织学观察(HE和Movat-stained染色样本切片观察总细胞数、炎症细胞含量,排斥反应,血管分布,成熟胶原量及新生胶原沉积量)。
结果40只兔子均进入结果分析。1、移植术后2周内,EDC交联改性的脱细胞版纳近交系微型猪肌腱(EAT)组(A组)与自体肌腱(AT)移植组(B组)在白细胞分类计数和C反应蛋白量检测结果中,尽管EDC交联改性的脱细胞版纳近交系微型猪肌腱移植组在术后3d的白细胞数和C反应蛋白量均明显高于术前3d,但自体移植组的白细胞数和C反应蛋白量也分别高于术前3d,两组白细胞数的增加值间是无差别的,两组C反应蛋白量的增加值间差异无统计学意义,这和术后应激反应有关。术后1周及2周,两组之间的白细胞数和C反应蛋白量差异无统计学意义(p>0.05)。2、A、B两组移植术后局部反应小,伤口一期愈合,屈踝功能恢复正常。3.组织学检查均未见明显淋巴细胞浸润；肌腱缝合处胶原纤维相互衔接。4.生物力学测定证实术后1周A组肌腱与B组肌腱相比,拉伸强度低,差异有统计学意义(p<0.05)。术后4周及后期同期A组和B组相比,拉伸强度差别无著性意义(P>0.05)。
结论EDC交联改性的脱细胞版纳近交系微型猪肌腱其微结构更加紧密、腱束排列有序、形成大小形状相似的孔隙结构,提高了脱细胞肌腱的抗降解能力、拉伸强度、弹性模量,降低断裂伸长率,无明显细胞毒性并能降低脱细胞肌腱的免疫原性；EDC交联改性的脱细胞版纳近交系微型猪肌腱能成功修复兔跟腱缺损,且具有组织相容性好、移植排斥反应轻、生物力学性能强的优点。实验结果表明EDC交联改性的脱细胞版纳近交系微型猪肌腱有望成为修复肌腱缺损的异种肌腱移植材料。
Object:Ideal tendon repair materials combine minimal donor-site morbidity and ready availability with excellent healing and postoperative function. Bioengineered EDC cross-linked acellular tendon of Banna mini-pig inbred-lines was compared with tendon autografts as a potential human flexor tendon graft substitute.To investigate the effect of substitution of 1-ethyl-3-3-dimethy laminopropyl arbodiimide hydrochloride(EDC) cross-linked acellular tendon of Banna mini-pig inbred-lines for rabbit Achiles tendon, then evaluate the feasibility and validity of porcine-derived xenogeneic graft.
Methods:(1) Extensor digitorum tendons harvested from Banna mini-pig inbred-lines were divided into fresh tendon group,deep-freezed tendon group,simply decellularized tendon group and EDC cross-linked acellular tendon group.The histological and structural changes treated by different methods were obserbved via HE staining and scaning electron microscopy and compared between groups to evaluated the histological and structural influence of EDC cross-linking on tendons; 1-ethyl-3-3-dimethy laminopropyl arbodiimide hydrochloride EDC cross-linked acellular tendon of Banna mini-pig inbred-lines was taken out and processed. (2)40 Rabbits were divided into two groups randomly, group A was xenograft transplanting group,group B was autograft transplanting group and were used as controls. After transplanting, followed by unrestricted motion. White blood cell (WBC) differential count and CRP count at 3 days preoperation,3 days postoperation,and 1 week and 2 weeks postoperation. Animals were killed at 1、4、8and 12weeks. Specimens were analyzed for hydroxyproline content (absorption spectroscopy) and tensile strength. Hematoxylineosin and Movat-stained sections of the central graft and distal repair site were semiquantitatively scored for total cellularity, inflammatory cell content, foreign-body reaction,vascularity, mature collagen content, and new collagen deposition. histological observation, immunology and biomechanics were performed regularly.
Results:There were little imflammation, well healing and normal phalanx flextion postoperation. There was no proof of immigration of lymphoid cell. The collagen fiberswere connected each other in suture area, thus suggusting EDC cross-linked acellular tendon of Banna mini-pig inbred-lines was ingrowth and connected with normal tendon. There was no difference in bioscience between two groups eight weeks postoperation.
Conclutions:The EDC cross-linked acellular tendon of Banna mini-pig inbred-lines,with good histocompatibility, faint antigenicity and adequate bio-mechanical strength after xenografts.
方法1.EDC交联改性的脱细胞版纳近交系微型猪肌腱的制备及生物学特性检测：无菌条件下取云南农业大学近交系微型猪肌腱后,肌腱按不同处理方法分为三组：新鲜肌腱组(fresh tendon, FT)、单纯脱细胞肌腱组(simply decellulatized tendon, SDT)和EDC交联改性的脱细胞肌腱组(EDC cross-linked acellular tendon, EAT),通过对比各组肌腱的大体、组织学特点,体外降解特性,免疫原性和生物力学性能,评价EDC交联改性的脱细胞版纳近交系微型猪肌腱作为肌腱支架材料的应用价值。
2.将40只日本大白兔雌雄参半,体重在2.3-2.6kg,随机分为A、B两组,每组20只兔子。A组为碳化二亚胺(EDC)交联改性的脱细胞版纳近交系微型猪肌腱移植组,B组为自体肌腱移植组。制作兔双后肢跟腱缺损实验动物模型。A组缺损的肌腱用EDC交联改性的脱细胞版纳近交系微型猪肌腱修复,用3-0肌腱线改良HEMI-KESSLER进行端端吻合,B组缺损的肌腱用自体肌腱修复,用3-0肌腱线改良HEMI-KESSLER法进行端端原位吻合。3.移植术前3d、术后3d、1w、2w外周血白细胞分类计数、C反应蛋白检测。4.移植术后1、4、8、12周处死动物取材。检测分析取材标本中羟脯氨酸含量(吸收光谱法测定)、进行生物力学测定(抗拉强度生物材料力学多功能试验机)、组织学观察(HE和Movat-stained染色样本切片观察总细胞数、炎症细胞含量,排斥反应,血管分布,成熟胶原量及新生胶原沉积量)。
结果40只兔子均进入结果分析。1、移植术后2周内,EDC交联改性的脱细胞版纳近交系微型猪肌腱(EAT)组(A组)与自体肌腱(AT)移植组(B组)在白细胞分类计数和C反应蛋白量检测结果中,尽管EDC交联改性的脱细胞版纳近交系微型猪肌腱移植组在术后3d的白细胞数和C反应蛋白量均明显高于术前3d,但自体移植组的白细胞数和C反应蛋白量也分别高于术前3d,两组白细胞数的增加值间是无差别的,两组C反应蛋白量的增加值间差异无统计学意义,这和术后应激反应有关。术后1周及2周,两组之间的白细胞数和C反应蛋白量差异无统计学意义(p>0.05)。2、A、B两组移植术后局部反应小,伤口一期愈合,屈踝功能恢复正常。3.组织学检查均未见明显淋巴细胞浸润；肌腱缝合处胶原纤维相互衔接。4.生物力学测定证实术后1周A组肌腱与B组肌腱相比,拉伸强度低,差异有统计学意义(p<0.05)。术后4周及后期同期A组和B组相比,拉伸强度差别无著性意义(P>0.05)。
结论EDC交联改性的脱细胞版纳近交系微型猪肌腱其微结构更加紧密、腱束排列有序、形成大小形状相似的孔隙结构,提高了脱细胞肌腱的抗降解能力、拉伸强度、弹性模量,降低断裂伸长率,无明显细胞毒性并能降低脱细胞肌腱的免疫原性；EDC交联改性的脱细胞版纳近交系微型猪肌腱能成功修复兔跟腱缺损,且具有组织相容性好、移植排斥反应轻、生物力学性能强的优点。实验结果表明EDC交联改性的脱细胞版纳近交系微型猪肌腱有望成为修复肌腱缺损的异种肌腱移植材料。
Object:Ideal tendon repair materials combine minimal donor-site morbidity and ready availability with excellent healing and postoperative function. Bioengineered EDC cross-linked acellular tendon of Banna mini-pig inbred-lines was compared with tendon autografts as a potential human flexor tendon graft substitute.To investigate the effect of substitution of 1-ethyl-3-3-dimethy laminopropyl arbodiimide hydrochloride(EDC) cross-linked acellular tendon of Banna mini-pig inbred-lines for rabbit Achiles tendon, then evaluate the feasibility and validity of porcine-derived xenogeneic graft.
Methods:(1) Extensor digitorum tendons harvested from Banna mini-pig inbred-lines were divided into fresh tendon group,deep-freezed tendon group,simply decellularized tendon group and EDC cross-linked acellular tendon group.The histological and structural changes treated by different methods were obserbved via HE staining and scaning electron microscopy and compared between groups to evaluated the histological and structural influence of EDC cross-linking on tendons; 1-ethyl-3-3-dimethy laminopropyl arbodiimide hydrochloride EDC cross-linked acellular tendon of Banna mini-pig inbred-lines was taken out and processed. (2)40 Rabbits were divided into two groups randomly, group A was xenograft transplanting group,group B was autograft transplanting group and were used as controls. After transplanting, followed by unrestricted motion. White blood cell (WBC) differential count and CRP count at 3 days preoperation,3 days postoperation,and 1 week and 2 weeks postoperation. Animals were killed at 1、4、8and 12weeks. Specimens were analyzed for hydroxyproline content (absorption spectroscopy) and tensile strength. Hematoxylineosin and Movat-stained sections of the central graft and distal repair site were semiquantitatively scored for total cellularity, inflammatory cell content, foreign-body reaction,vascularity, mature collagen content, and new collagen deposition. histological observation, immunology and biomechanics were performed regularly.
Results:There were little imflammation, well healing and normal phalanx flextion postoperation. There was no proof of immigration of lymphoid cell. The collagen fiberswere connected each other in suture area, thus suggusting EDC cross-linked acellular tendon of Banna mini-pig inbred-lines was ingrowth and connected with normal tendon. There was no difference in bioscience between two groups eight weeks postoperation.
Conclutions:The EDC cross-linked acellular tendon of Banna mini-pig inbred-lines,with good histocompatibility, faint antigenicity and adequate bio-mechanical strength after xenografts.
引文
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