CAJ | 학술논문

目的：评估以丝素蛋白为材料构建的一体化纤维环-髓核双相支架作为组织工程椎间盘支架的可行性。方法以丝素蛋白溶液为原料，分别采用石蜡球致孔法和相分离法制备三维多孔一体化纤维环-髓核支架。采用体视显微镜、扫描电镜观察支架内部结构，测定双相支架纤维环相和髓核相的孔径、孔隙率及一体化压缩弹性模量；分离培养兔纤维环细胞和髓核细胞，接种至双相支架的相应部位，体外培养48 h，扫描电镜、死活（Live/dead）细胞染色评价支架与细胞的生物相容性；CCK-8检测细胞的增殖活性。结果体视显微镜和扫描电镜可见双相支架纤维环相和髓核相均呈相互连通的多孔结构，孔隙高度连通，纤维环髓核交接区域结合紧密；纤维环相孔径为（220.0±23.1）μm，髓核相孔径为（90.0±17.8）μm；孔隙率分别为91%和93%；一体化支架压缩弹性模量为（150.7±6.8）kPa。扫描电镜可见均匀地黏附在支架表面，细胞周围有细胞外基质分泌；Live/dead染色显示细胞在支架上活性良好，无死细胞；CCK-8增殖分析显示纤维环细胞和髓核细胞均具有良好的增殖活性。结论以天然丝素蛋白构建一体化纤维环-髓核双相支架，具有良好的孔径、孔隙率和细胞相容性，一体化支架两部分结合紧密，并且具有优越的力学性能，是构建组织工程椎间盘的理想支架载体。
목적：평고이사소단백위재료구건적일체화섬유배-수핵쌍상지가작위조직공정추간반지가적가행성。방법이사소단백용액위원료，분별채용석사구치공법화상분리법제비삼유다공일체화섬유배-수핵지가。채용체시현미경、소묘전경관찰지가내부결구，측정쌍상지가섬유배상화수핵상적공경、공극솔급일체화압축탄성모량；분리배양토섬유배세포화수핵세포，접충지쌍상지가적상응부위，체외배양48 h，소묘전경、사활（Live/dead）세포염색평개지가여세포적생물상용성；CCK-8검측세포적증식활성。결과체시현미경화소묘전경가견쌍상지가섬유배상화수핵상균정상호련통적다공결구，공극고도련통，섬유배수핵교접구역결합긴밀；섬유배상공경위（220.0±23.1）μm，수핵상공경위（90.0±17.8）μm；공극솔분별위91%화93%；일체화지가압축탄성모량위（150.7±6.8）kPa。소묘전경가견균균지점부재지가표면，세포주위유세포외기질분비；Live/dead염색현시세포재지가상활성량호，무사세포；CCK-8증식분석현시섬유배세포화수핵세포균구유량호적증식활성。결론이천연사소단백구건일체화섬유배-수핵쌍상지가，구유량호적공경、공극솔화세포상용성，일체화지가량부분결합긴밀，병차구유우월적역학성능，시구건조직공정추간반적이상지가재체。
Objective To assess the prospect of integrated biphasic silk fibroin scaffold made by annulus fibrosus-nu?cleus pulposus tissue engineering in application as integrated intervertebral disc(IVD). Methods An integrated annulus fi brosus-nucleus pulposus(AF-NP)biphasic scaffold was made by silk fi broin using two different uncomplicated methods which were paraffin spheres-leaching method(outer AF phase)and phase separation method(inner NP phase). The scaf?fold was investigated by general observation, stereomicroscope and scanning electron microscopy(SEM). Its pore size, poros?ity, and compressive elastic modulus were determined. AF and NP cells were isolated from rabbit IVD and seeded into the corresponding phase of the scaffold respectively. The cell-scaffold complex was cultured for 48 hours. The biocompatibility of the scaffold was evaluated by SEM, live/dead staining while CCK-8 assay was used to assess cell proliferation. Results Stereomicroscope and SEM showed that AF phase and NP phase integrated perfectly without cross-linking. Both phases pos?sessed highly interconnected porous structure [pore size of AF and NP phase were(220.0±23.1)μm and(90.0±17.8)μm, re?spectively] and highly porosity(AF and NP phase were respectively 91%and 93%). In addition, this silk biphasic scaffold had impressive mechanical properties(150.7 ± 6.8)kPa. SEM revealed that disc cells attached to regions of pore walls, dis?tributed uniformly and secreted extracellular matrix. Live/Dead staining and cell count kit-8(CCK-8)analysis showed that the silk composite scaffold was non-cytotoxic to disc cells. Conclusion This silk biphasic AF-NP scaffold has satisfied pore size, porosity, biomechanical properties and biocompatibility, so it is ideal candidate for IVD tissue engineering.