CAJ | 학술논문

目的探讨灌注法去细胞技术制备去细胞全喉支架及利用骨髓间充质干细胞使喉肌细胞再生的可行性.方法新西兰大白兔作为喉支架供体,通过双侧颈总动脉顺行灌注去离子剂,构建出喉肌去细胞外基质及活性软骨的全喉支架,取部分灌注后标本进行观察,其余用于下一步实验.青紫蓝兔为受体兔,取其骨髓问充质干细胞诱导培养为骨骼肌细胞后注入全喉支架的去细胞基质中.体外培养1 d后异位移植入受体兔大网膜.8周取出标本进行观察.结果大体观察发现喉在经去离子剂灌注后2 h已经呈透明状态,组织学及扫描电镜显示喉肌去细胞基质内无细胞残留物,纤维网状结构存在,孔隙率80.4%±3.2%(x±s,下同);软骨细胞存活率86.9%±1.5%.移植至大网膜的喉体4周和8周取出时大体观察发现喉支架完整,其表面有血管形成.组织学显示肌束存在.免疫组织化学检测α-肌动蛋白表达阳性.结论诱导后的骨髓间充质干细胞注入经去细胞技术处理的全喉支架可以构建出保留软骨支架并含有再生肌肉组织的喉,植人大网膜内生长有助于其血管化.
목적 탐토관주법거세포기술제비거세포전후지가급이용골수간충질간세포사후기세포재생적가행성.방법 신서란대백토작위후지가공체,통과쌍측경총동맥순행관주거리자제,구건출후기거세포외기질급활성연골적전후지가,취부분관주후표본진행관찰,기여용우하일보실험.청자람토위수체토,취기골수문충질간세포유도배양위골격기세포후주입전후지가적거세포기질중.체외배양1 d후이위이식입수체토대망막.8주취출표본진행관찰.결과 대체관찰발현후재경거리자제관주후2 h이경정투명상태,조직학급소묘전경현시후기거세포기질내무세포잔류물,섬유망상결구존재,공극솔80.4%±3.2%(x±s,하동);연골세포존활솔86.9%±1.5%.이식지대망막적후체4주화8주취출시대체관찰발현후지가완정,기표면유혈관형성.조직학현시기속존재.면역조직화학검측α-기동단백표체양성.결론유도후적골수간충질간세포주입경거세포기술처리적전후지가가이구건출보류연골지가병함유재생기육조직적후,식인대망막내생장유조우기혈관화.
Objective To prepare a deeelhilarized whole laryngeal scaffold by utilizing a perfusion-decellularized technique, reseed cells on it, and construct recellularized laryngeal muscles. Methods Perfusion decelluarized larynxes were obtained by common carotid arterious perfusion with detergents. Then they were performed by macroscopic view, histological examination, scanning electron microscopy (SEM) and cartilage viability. Decellularized laryngeal scaffold were then reseeded with inducted mesenchymal stem cells (MSCs). Composites were transferred into greater omentums of rabbits after one day' s adherence and harvested after eight weeks. Macroscopic view, histological examination and immunohistochemistry were performed. Results Perfusion larynxes became transparent after two hours. Histology and SEM indicated that perfnsion method shewed better deculluarized effect. More ventages and collagen fibers but no intact cell or anclei were retained in the decellularized martrix. Porosity measured by Image pro plus 6. 0 was 80. 4% ± 3.2% (x ± s). Chondrocyte vitality assay indicated chondrocyte vitality rate in the perfusion group was 86. 9% ± 1.5% . After eight weeks, vascularization formed and integrated cartilage frameworks still remained. Histological examination could clearly show the presence of muscle bundles and vessels. Immunohistoehemical examination indicated that sarcomeric-α actin expressed positively in corresponding areas. Conclusions It is feasible to reseed MSCs into the decellularized laryngeal muscle matrix for constructing tissue-engineered laryngeal muscles. This in vivo maturation into the omentum could be the first step before in situ implantation of the construct.