CAJ | 학술논문

目的：比较体外环境下循环动态拉力和静态非受力对衍生肌腱材料（tendon derivation biomaterials，TDBM）支架与肌腱细胞复合培养组织工程肌腱重塑化过程的影响。方法构建TDBM支架，自制单轴循环肌腱拉伸装置，分离并培养乳兔肌腱细胞，培养后将肌腱细胞与TDBM复合培养，并随机分为动态拉力组和静态非受力组。在培养的第3、7天对细胞-支架复合体行倒置显微镜和扫描电镜观察，在2、4、6周行大体、HE、Masson三染色并观察，RT-PCR技术每周检测各组样本中Ⅰ型和Ⅲ型胶原的基因表达情况，设置新鲜天然肌腱作为对照。结果倒置显微镜和扫描电镜观察肌腱细胞与TDBM相容性良好。动态拉力作用下，大体及组织学观察显示组织工程肌腱变细、变长，结构致密，胶原纤维排列整齐有序，形成特有的波纹结构；肌腱细胞位于胶原纤维层之间，其长轴与胶原纤维受力方向一致，细胞/基质比例逐渐降低，总体趋向于成熟肌腱结构特征；RT-PCR示Ⅰ、Ⅲ型胶原表达渐升高。静态非受力状态下培养的组织工程肌腱表面粗糙呈絮状，胶原基质排列相对无序且松散，趋向于分裂、降解。RT-PCR示Ⅰ、Ⅲ型胶原表达先升高后下降。结论动态拉力作用下TDBM支架与肌腱细胞复合培养可促进肌腱细胞分泌胶原基质及肌腱结构重塑，提高了组织工程肌腱的质量。
목적：비교체외배경하순배동태랍력화정태비수력대연생기건재료（tendon derivation biomaterials，TDBM）지가여기건세포복합배양조직공정기건중소화과정적영향。방법구건TDBM지가，자제단축순배기건랍신장치，분리병배양유토기건세포，배양후장기건세포여TDBM복합배양，병수궤분위동태랍력조화정태비수력조。재배양적제3、7천대세포-지가복합체행도치현미경화소묘전경관찰，재2、4、6주행대체、HE、Masson삼염색병관찰，RT-PCR기술매주검측각조양본중Ⅰ형화Ⅲ형효원적기인표체정황，설치신선천연기건작위대조。결과도치현미경화소묘전경관찰기건세포여TDBM상용성량호。동태랍력작용하，대체급조직학관찰현시조직공정기건변세、변장，결구치밀，효원섬유배렬정제유서，형성특유적파문결구；기건세포위우효원섬유층지간，기장축여효원섬유수력방향일치，세포/기질비례축점강저，총체추향우성숙기건결구특정；RT-PCR시Ⅰ、Ⅲ형효원표체점승고。정태비수력상태하배양적조직공정기건표면조조정서상，효원기질배렬상대무서차송산，추향우분렬、강해。RT-PCR시Ⅰ、Ⅲ형효원표체선승고후하강。결론동태랍력작용하TDBM지가여기건세포복합배양가촉진기건세포분비효원기질급기건결구중소，제고료조직공정기건적질량。
Objective To investigate the tissue remodeling and cell alignment of TDBM scaffolds seeded with rabbit tenocytes under the cycle dynamic tensile force or static tension-free culture in vitro. Methods TDBM were made by ourselves, and uniaxial cyclic tendon stretching device was designed and manufactured on our own. Primary tenocytes were isolated from the Achilles tendon of three-day-old New Zealand white rabbits and seeded into scaffolds, and were cultured collectively in DMEM in vitro. Samples were divided into two groups:dynamic tension-loaded group, and static tension-free group. Fresh natural tendons were used to be positive control. The experiment's time was six weeks. The scaffold-cell complexes were harvested at 3 and 7 days of culture for Inverted microscope and scanning electron micrograph (SEM) analysis. The morphological characters of the samples, including the general view, HE and Masson's dyeing, were observed at 2, 4 and 6 weeks. In addition, the gene expression of the I-type collagen and III-type collagen of the samples was detected by using Real time PCR at every week. Set fresh natural tendon as control. Results The inverted microscope and SEM showed that it was nice compatible condition between the tendon cells and TD-BM scaffold. In addition, the tendon of tension-loaded group revealed a structure of longitudinally aligned collagen fi bers and dense structure of collagen fibers arranged in orderly form a unique corrugated structure. Tenocytes layer located between the col-lagen fibers and aligned longitudinally along the force axis, with increased matrix deposition after the 3th week showed by RT-PCR. The cell/matrix ratio decreases. When cultured to 6 weeks, the tissue structure was very similar to that of fresh natural ten-don pattern. By contrast, HE and Masson's staining revealed the collagen fibro-tissue structure in tension-free groups with disorga-nized matrix structure and randomly distributed cells. Collagen fibers were gradually degraded with time. In tension-loaded group, Real-time PCR showed that gene expression of I-type collagen and III-type collagen gradually increase, but in tension-free group, the relative gene expression of I-type collagen and III-type collagen was highest at 3rd week, and from that time the relative expres-sion gradually decrease. Conclusion Under the dynamic stress, the TDBM scaffolds seeded with rabbit tenocytes can promote extra-cellular matrix biosynthesis and tendon structure remodeling, which is a promising method for tendon tissue engineering.