CAJ | 학술논문

目的：探讨小肠黏膜下层（small intestinal submucosa，SIS）作为组织工程支架材料的生物安全性及生物相容性，以及SIS复合滑膜间质干细胞（synovial mesenchymal stem cells，SMSCs）体外成软骨诱导培养的可行性。方法常规方法自新西兰大白兔体内分离、培养SMSCs，物理方法及Abraham方法处理猪SIS，采用热源试验、皮肤致敏试验、全身毒性试验等方面检测SIS作为支架材料的生物安全性；将SMSCs与SIS复合进行体外成软骨诱导培养，观察SMSCs与SIS的组织相容性，及SMSCs在SIS支架上的增生及成软骨分化能力。结果经物理方法及Abraham方法处理后的SIS为表面光滑的浅乳白色半透明基膜，电镜下显示表面纤维组织交错形成疏松的三维网状立体结构，孔隙率约80%，孔径约100~200μm，且具有很好的生物安全性及生物相容性。扫描电镜显示SMSCs在SIS支架上黏附、生长及增殖活性良好，并能长入SIS的孔隙内，并分泌大量的细胞外基质，细胞间通过突起相互连接、或伸出伪足贴附于支架孔壁上；同时SMSCs与SIS复合物在体外具有良好的成软骨诱导分化能力，培养21 d后，免疫组织化学染色显示Ⅱ型胶原表达阳性。结论 SIS作为组织工程支架材料具有良好的生物安全性和生物相容性，对复合培养SMSCs的生长和定向诱导分化能力无抑制作用。
목적：탐토소장점막하층（small intestinal submucosa，SIS）작위조직공정지가재료적생물안전성급생물상용성，이급SIS복합활막간질간세포（synovial mesenchymal stem cells，SMSCs）체외성연골유도배양적가행성。방법상규방법자신서란대백토체내분리、배양SMSCs，물리방법급Abraham방법처리저SIS，채용열원시험、피부치민시험、전신독성시험등방면검측SIS작위지가재료적생물안전성；장SMSCs여SIS복합진행체외성연골유도배양，관찰SMSCs여SIS적조직상용성，급SMSCs재SIS지가상적증생급성연골분화능력。결과경물리방법급Abraham방법처리후적SIS위표면광활적천유백색반투명기막，전경하현시표면섬유조직교착형성소송적삼유망상입체결구，공극솔약80%，공경약100~200μm，차구유흔호적생물안전성급생물상용성。소묘전경현시SMSCs재SIS지가상점부、생장급증식활성량호，병능장입SIS적공극내，병분비대량적세포외기질，세포간통과돌기상호련접、혹신출위족첩부우지가공벽상；동시SMSCs여SIS복합물재체외구유량호적성연골유도분화능력，배양21 d후，면역조직화학염색현시Ⅱ형효원표체양성。결론 SIS작위조직공정지가재료구유량호적생물안전성화생물상용성，대복합배양SMSCs적생장화정향유도분화능력무억제작용。
Objective To investigate the biosecurity and biocompatibility of small intestinal submucosa (SIS) as scaffold for tissue engineering and to explore the possibility to induce synovial mesenchymal stem cells (SMSCs) differentiate into cartilage with SIS as the scaffold and SMSCs as the seed cells. Methods The SMSCs were isolated and cultured from the synovial mem-brane of knee joints of rabbits by a conventional method. The SIS was harvested from pigs according to the physical method and Abraham's method. 4 rabbits are divided into the experimental group and control group. The biosecurity of SIS as scaf-folds were investigated in pyrogen test, skin sensitization test and systemic toxicity test. The SMSCs and SIS were co-cultured in vitro and induced to differentiate into cartilage to explore the biocompatibility of SMSCs and SIS, the proliferation and differ-entiation ability of SMSCs on the scaffold of SIS. The varietyies were identified by the microscope. Results The SIS isolated with the physical method and Abraham's method is a milky and translucent membrane with a smooth surface. Under the electron microscope, SIS presented a porous Stereoscopic three-dimensional network structure, which is formed by fibrous tissues' intertex-ture. Its' porosity was about 80%and its aperture was 100-200μm. Meanwhile, the biosecurity and biocompatibility of SIS were also fine. In the trial that the SMSCs and SIS were co-cultured in vitro, the SMSCs can grow, adhere to and differentiate on the sur-face of SIS and into the hollows very well. It can also secrete extracellular matrix. Through scanning microscope observation, cells contact with each other by their neuritis, or adhered to the wall of hole by cellular protrution. On the surface of SIS, the SMSCs maintain the property that it can easily differentiate into the chondrogenic lineage in the chondrogenic medium. Immunochemistry staining showed positive expression of type II collagen post 21 days of co-cultrue. Conclusion SIS can be used as the scaffold to construct tissue engineering meniscus as it has good biosecurity and biocompatibility with SMSCs without disturbing the cell form or inhibiting the growth and function of SMSCs.