组织工程构建新型腹壁修复材料的实验研究
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摘要
目的:
(1)探讨种植骨骼肌成肌细胞于Ⅰ型胶原/聚乳酸/乙醇酸支架,模拟力学环境和组织血供,构建三维立体、具备有序肌纤维和收缩潜力的组织工程骨骼肌的可行性;
(2)探讨种植成纤维细胞于小肠粘膜下层,复加组织工程骨骼肌形成组织工程材料修复腹壁缺损的可行性;
(3)探讨组织工程材料与生物材料修复实验动物腹壁缺损的疗效优劣、再生组织差异,探讨进一步改进构建方法的途径。
方法:
(1)制备种子细胞和支架材料:①使用酶消化、差速贴壁法从乳鼠骨骼肌标本中分离纯化成肌细胞培养;②使用酶消化从乳鼠皮肤中分离纯化成纤维细胞培养;③按Abraham法制备猪小肠粘膜下层;④利用细胞形态学、免疫组化鉴定鉴定所制备种子细胞,利用免疫组化检查制备的小肠粘膜下层。
(2)构建组织工程材料,修复腹壁缺损:①成肌细胞接种于Ⅰ型胶原/PLGA混合凝胶,置于机械细胞刺激器中的力学环境,体外培养3周;②成纤维细胞接种于SIS,体外培养3周;③上述两者复合预构的网膜血管网,构建组织工程腹壁修复材料;④制备大鼠全层腹壁缺损,分别使用组织工程材料或生物材料(3层SIS)修复;⑤观察术后动物一般恢复情况,检查术后不同时期修复腹壁的电生理特性;⑥分批处死动物,测定术后不同时期两组修复后再生腹壁的腹壁厚度、抗张强度;⑦观察术后不同时期修复区中心腹壁的组织结构、细胞密度、细胞成分、血管化程度;确定组织工程材料修复后再生腹壁有否形态接近正常骨骼肌的肌肉层结构、新生肌纤维是否与宿主肌纤维融合、有否肌球蛋白重链表达;⑧统计分析。
结果:
(1)成功构建三维立体、肌纤维平行有序、具备收缩潜力的组织工程骨骼肌。
(2)成纤维细胞种植于SIS,复加组织工程骨骼肌构建的组织工程材料治疗腹壁缺损疗效优于生物材料;修复后的腹壁细胞密度更高、厚度更大、机械强度更高;具有纤维组织层和肌肉层结构、表达肌球蛋白重链、存在肌电活动。生物材料在术后2月完全被纤维组织代替。表现在
(3)术后3月,组织工程材料修复后腹壁厚度较2月时减少、肌电活动强度也减低。
结论:
(1)构建三维立体、具有类似正常骨骼肌形态和收缩潜力的组织工程骨骼肌是可行的。
(2)组织工程材料修复腹壁缺损疗效优于生物材料,且能弥补生物材料修复后再生腹壁主要由纤维组织再生构成、腹壁较正常腹壁薄、机械强度低、组织再生速度缓慢、血管化程度低等不足,有较好的发展前景。
(3)缺神经支配,组织工程材料在体内不能长久维持在最佳状态,需进一步改进构建方法。
创新点:
(1)经文献检索,本实验是国内外关于组织工程学技术应用于疝和腹壁外科的首次探索(附检索证明),为腹壁缺损修复的研究提供了新的思路。
(2)国内首次构建组织工程骨骼肌组织。
Objective:
(1)To study the feasibility of three-dimensional,contractile skeletal muscle with parallel muscle fiber constructed by myoblasts seeded on collagen I/PLGA under simulated mechanical circumstance and tissue blood supply.
(2)To study the feasibility of abdominal wall defect repaired by tissue engineered(TE) patch consist of small intestinal submucosa(SIS) with fibroblasts seeded on it and TE skeletal muscle.
(3)To compare the effect and regenerating organ of TE patch and SIS on repairing abdominal wall defect in animal models.To look for the potential approach to upgrade constructing.
Method:
(1)Preparation of seed cells and scaffold.①Utilize different methods,such as enzymatic digestion,adherence,flow cytometer and so on to isolate、purify,culture skeletal myoblasts from the skeletal muscles of neonate rat.②Utilize enzymatic digestion to isolate,purify, culture dermatic fibroblasts from neonate rats.③Manufacture porcine small intestinal submucosa following the method invented by Abraham.④Identify the myoblasts,fibroblasts, SIS through the observation of the shape of the cell with inverted microscope and immunocytochemistry.
(2)Construction of TE patch and repair abdominal wall defect in animal models.①skeletal myoblasts were suspended into a collagen/PLGA gel,under simulated mechanical circumstance inside mechanical cell stimulator for 3 weeks in vitro.②Suspend fibroblasts on SIS and co-cultivate for 3 weeks in vitro.③The above-mentioned two,combined with omental vasoganglion to form TE patch.④All animal underwent creation of a 3×2.5cm full-thickness abdominal wall defect and the defect was repaired by TE patch or biomaterial(3 layers of SIS).⑤Animal's recover information and electrophysiological characteristic of repaired abdominal wall postoperatively in two sets were recorded.⑥The mechanic strength of repaired abdominal wall postoperatively in two sets was measured.⑦The observation of tissue structure,cell density,cell component of the centre of repaired body wall was performed to confirm whether there was muscle layer similar to normal skeletal muscle in appearance,whether anagenetic muscle fiber could confluence with host muscle fiber,whether H-chain of myoglobulin could be detected.⑧Statistical analysis
Results:
(1) Three-dimensional skeletal muscles with parallel and ordered muscle fiber,potential to contract were constructed successfully.
(2)TE patch,consist of TE skeletal muscle and fibroblast seeded on SIS,was superior to biomaterial in the effect of repairing abdominal wall defect.in two sets were both execelent and similar.Anagenetic body wall in TE set was thicker,with fibrous tissue layer and muscle layer,displayed higher in cell density with better cellular infiltration.H-chain of myoglobulin was detected and electromyography evidenced single motor-unit potentials,sometimes grouped into arithmic discharges in t TE set.While Sixty days after surgery,biomaterials were completely replaced by fibrous tissue,no electric activity detected.
(3)The thickness of myoblast-seeded patches and intensity of their myoelectric activity were inferior at day 90 to 60 after implantation,suggesting a loss of contractile muscle fibers.
Conclusion:
(1)Construction of three-dimensional TE skeletal muscle,with similarity to normal skeletal muscle in appearance and potential to contract,is feasible.
(2)The therapeutic efficacy of TE patch to abdominal wall defect is superior to biomaterial.TE patch represent a very promising tool for body-wall defect repair and can retrieve biomaterial in scar replacing repaired body wall,low recovery rate,high long-term rate of heria,since they are repopulated by skeletal muscle cells and fibroblasts,show positive responses in the electrophysiological studies.
(3)Studies shouid be under way to identify strategies to improve and maintain the structural and functional integrity of implants for a longer time,especially in innervation.
Innovation:
(1)To our knowledge,this experiment is the first exploration of tissue engineering used in repairing of abdominal wall defect(certification affiliated).
(2)This experiment is also the first construction of tissue engineered skeletal muscle in China.
(1)探讨种植骨骼肌成肌细胞于Ⅰ型胶原/聚乳酸/乙醇酸支架,模拟力学环境和组织血供,构建三维立体、具备有序肌纤维和收缩潜力的组织工程骨骼肌的可行性;
(2)探讨种植成纤维细胞于小肠粘膜下层,复加组织工程骨骼肌形成组织工程材料修复腹壁缺损的可行性;
(3)探讨组织工程材料与生物材料修复实验动物腹壁缺损的疗效优劣、再生组织差异,探讨进一步改进构建方法的途径。
方法:
(1)制备种子细胞和支架材料:①使用酶消化、差速贴壁法从乳鼠骨骼肌标本中分离纯化成肌细胞培养;②使用酶消化从乳鼠皮肤中分离纯化成纤维细胞培养;③按Abraham法制备猪小肠粘膜下层;④利用细胞形态学、免疫组化鉴定鉴定所制备种子细胞,利用免疫组化检查制备的小肠粘膜下层。
(2)构建组织工程材料,修复腹壁缺损:①成肌细胞接种于Ⅰ型胶原/PLGA混合凝胶,置于机械细胞刺激器中的力学环境,体外培养3周;②成纤维细胞接种于SIS,体外培养3周;③上述两者复合预构的网膜血管网,构建组织工程腹壁修复材料;④制备大鼠全层腹壁缺损,分别使用组织工程材料或生物材料(3层SIS)修复;⑤观察术后动物一般恢复情况,检查术后不同时期修复腹壁的电生理特性;⑥分批处死动物,测定术后不同时期两组修复后再生腹壁的腹壁厚度、抗张强度;⑦观察术后不同时期修复区中心腹壁的组织结构、细胞密度、细胞成分、血管化程度;确定组织工程材料修复后再生腹壁有否形态接近正常骨骼肌的肌肉层结构、新生肌纤维是否与宿主肌纤维融合、有否肌球蛋白重链表达;⑧统计分析。
结果:
(1)成功构建三维立体、肌纤维平行有序、具备收缩潜力的组织工程骨骼肌。
(2)成纤维细胞种植于SIS,复加组织工程骨骼肌构建的组织工程材料治疗腹壁缺损疗效优于生物材料;修复后的腹壁细胞密度更高、厚度更大、机械强度更高;具有纤维组织层和肌肉层结构、表达肌球蛋白重链、存在肌电活动。生物材料在术后2月完全被纤维组织代替。表现在
(3)术后3月,组织工程材料修复后腹壁厚度较2月时减少、肌电活动强度也减低。
结论:
(1)构建三维立体、具有类似正常骨骼肌形态和收缩潜力的组织工程骨骼肌是可行的。
(2)组织工程材料修复腹壁缺损疗效优于生物材料,且能弥补生物材料修复后再生腹壁主要由纤维组织再生构成、腹壁较正常腹壁薄、机械强度低、组织再生速度缓慢、血管化程度低等不足,有较好的发展前景。
(3)缺神经支配,组织工程材料在体内不能长久维持在最佳状态,需进一步改进构建方法。
创新点:
(1)经文献检索,本实验是国内外关于组织工程学技术应用于疝和腹壁外科的首次探索(附检索证明),为腹壁缺损修复的研究提供了新的思路。
(2)国内首次构建组织工程骨骼肌组织。
Objective:
(1)To study the feasibility of three-dimensional,contractile skeletal muscle with parallel muscle fiber constructed by myoblasts seeded on collagen I/PLGA under simulated mechanical circumstance and tissue blood supply.
(2)To study the feasibility of abdominal wall defect repaired by tissue engineered(TE) patch consist of small intestinal submucosa(SIS) with fibroblasts seeded on it and TE skeletal muscle.
(3)To compare the effect and regenerating organ of TE patch and SIS on repairing abdominal wall defect in animal models.To look for the potential approach to upgrade constructing.
Method:
(1)Preparation of seed cells and scaffold.①Utilize different methods,such as enzymatic digestion,adherence,flow cytometer and so on to isolate、purify,culture skeletal myoblasts from the skeletal muscles of neonate rat.②Utilize enzymatic digestion to isolate,purify, culture dermatic fibroblasts from neonate rats.③Manufacture porcine small intestinal submucosa following the method invented by Abraham.④Identify the myoblasts,fibroblasts, SIS through the observation of the shape of the cell with inverted microscope and immunocytochemistry.
(2)Construction of TE patch and repair abdominal wall defect in animal models.①skeletal myoblasts were suspended into a collagen/PLGA gel,under simulated mechanical circumstance inside mechanical cell stimulator for 3 weeks in vitro.②Suspend fibroblasts on SIS and co-cultivate for 3 weeks in vitro.③The above-mentioned two,combined with omental vasoganglion to form TE patch.④All animal underwent creation of a 3×2.5cm full-thickness abdominal wall defect and the defect was repaired by TE patch or biomaterial(3 layers of SIS).⑤Animal's recover information and electrophysiological characteristic of repaired abdominal wall postoperatively in two sets were recorded.⑥The mechanic strength of repaired abdominal wall postoperatively in two sets was measured.⑦The observation of tissue structure,cell density,cell component of the centre of repaired body wall was performed to confirm whether there was muscle layer similar to normal skeletal muscle in appearance,whether anagenetic muscle fiber could confluence with host muscle fiber,whether H-chain of myoglobulin could be detected.⑧Statistical analysis
Results:
(1) Three-dimensional skeletal muscles with parallel and ordered muscle fiber,potential to contract were constructed successfully.
(2)TE patch,consist of TE skeletal muscle and fibroblast seeded on SIS,was superior to biomaterial in the effect of repairing abdominal wall defect.in two sets were both execelent and similar.Anagenetic body wall in TE set was thicker,with fibrous tissue layer and muscle layer,displayed higher in cell density with better cellular infiltration.H-chain of myoglobulin was detected and electromyography evidenced single motor-unit potentials,sometimes grouped into arithmic discharges in t TE set.While Sixty days after surgery,biomaterials were completely replaced by fibrous tissue,no electric activity detected.
(3)The thickness of myoblast-seeded patches and intensity of their myoelectric activity were inferior at day 90 to 60 after implantation,suggesting a loss of contractile muscle fibers.
Conclusion:
(1)Construction of three-dimensional TE skeletal muscle,with similarity to normal skeletal muscle in appearance and potential to contract,is feasible.
(2)The therapeutic efficacy of TE patch to abdominal wall defect is superior to biomaterial.TE patch represent a very promising tool for body-wall defect repair and can retrieve biomaterial in scar replacing repaired body wall,low recovery rate,high long-term rate of heria,since they are repopulated by skeletal muscle cells and fibroblasts,show positive responses in the electrophysiological studies.
(3)Studies shouid be under way to identify strategies to improve and maintain the structural and functional integrity of implants for a longer time,especially in innervation.
Innovation:
(1)To our knowledge,this experiment is the first exploration of tissue engineering used in repairing of abdominal wall defect(certification affiliated).
(2)This experiment is also the first construction of tissue engineered skeletal muscle in China.
引文
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24.Ko R,Kazacos EA,et al.Tensile strength comparison of small intestinal submucosa body wall repair.J Surg Res,2006;135(1):9-17.
25.Ansaloni L,Catena F,et al.Prospective randomized,double-blind,controlled trial comparing Lichtenstein's repair of inguinal hernia with polypropylene mesh versus Surgisis gold soft tissue graft:preliminary results.Acta Biomed,2003,74:10-14.
26.Burger JW,Halm JA,et al.Evaluation of new prosthetic meshes for ventral hernia repair.Surg Endosc,2006,20:1320-1323
27.Thomas PR,Benjamin KP,et al.A Comparative analysis of expanded polytetrafluoroethylene and small intestinal submucosa-implications for patch repair in ventral herniorrhaphy.J Surg Res,2007,143:44-49.
28.Badylak SE,Toll[us R,et al.The use of xenogenic small intestinal submucosa as a biomaterial for Achilles tendon repair in a dog model.J Biomed Mater Res,1995,29:977-980.
29.Baillie DR,Stawicki SP,et al.Use of human and porcine dermal-derived bioprostheses in complex abdominal wall reconstructions:a literature review and case report.Ostomy Wound Manage,2007;53(5):30-37.
30.Parker DM,Armstrong PJ,et al.Porcine dermal collagen(Permacol) for abdominal wall reconstruction.Curr Surg,2006;63(4):255-258.