CAJ | 학술논문

目的探讨应用骨髓基质干细胞(BMSCs)复合光接枝改性的聚羟基丁酸-羟基异戊酯(PHBV)构建组织工程化软骨的可行性. 方法将3代绵羊骨髓基质细胞接种于光接枝改性的三维PHBV支架材料上,24 h后以成软骨诱导液培养,3周后,复合培养物行扫描电镜观察,组织学观察,测定糖胺聚糖(GAG)含量.并将复合物埋植于绵羊腹部皮下,4周后取出,行大体及组织学观察. 结果经成软骨诱导后,扫描电镜下,BMSCs的突触逐渐变短,由长梭形向扁平形转变,分泌基质量亦明显增多.组织学观察见细胞支架复合物阿利新蓝、番红O、Ⅱ型胶原免疫组化染色均呈阳性.GAG含量测定示诱导3周后,细胞分泌的GAG量(1306.7±192.3)明显高于未经诱导的BMSCs(205.0±26.2)(P<0.001),但仍低于正常软骨细胞(1969.2±235.3)(P<0.001).复合物埋植于皮下4周后,其内炎症细胞浸润明显,但番红O、Ⅱ型胶原免疫组化染色均呈阳性. 结论以BMSCs为种子细胞,复合改性的PHBV,可于体外构建出软骨样组织,但该组织的理化特点与正常软骨仍有差距.
목적 탐토응용골수기질간세포(BMSCs)복합광접지개성적취간기정산-간기이무지(PHBV)구건조직공정화연골적가행성. 방법 장3대면양골수기질세포접충우광접지개성적삼유PHBV지가재료상,24 h후이성연골유도액배양,3주후,복합배양물행소묘전경관찰,조직학관찰,측정당알취당(GAG)함량.병장복합물매식우면양복부피하,4주후취출,행대체급조직학관찰. 결과 경성연골유도후,소묘전경하,BMSCs적돌촉축점변단,유장사형향편평형전변,분비기질량역명현증다.조직학관찰견세포지가복합물아리신람、번홍O、Ⅱ형효원면역조화염색균정양성.GAG함량측정시유도3주후,세포분비적GAG량(1306.7±192.3)명현고우미경유도적BMSCs(205.0±26.2)(P<0.001),단잉저우정상연골세포(1969.2±235.3)(P<0.001).복합물매식우피하4주후,기내염증세포침윤명현,단번홍O、Ⅱ형효원면역조화염색균정양성. 결론 이BMSCs위충자세포,복합개성적PHBV,가우체외구건출연골양조직,단해조직적이화특점여정상연골잉유차거.
Objective To explore the feasibility of building tissue engineered cartilage by bone marrow stromal cells and pbotografting modified copolymers of 3-hydroxybutymte and 3-hydroxyvalerate.Methods Sheep BMSCs were seeded in three-dimensional photografting modified PHBV scaffoids.Twenty-four hours later.composites were cultured with ehondrogenically inductive medium(DMEM)containing TGF-B(10 ng/m1),IGF-1(150 ng/m1)and 20% fetal bovine serum.Three weeks later,the constructs were evaluated by scanning electron microscopy(SEM)and light microscopy with alcian blue,safrine 0 and type Ⅱ collage immunohistochemical staining.GAG contents of constructs were determined by DMB(1,9-dimethylmethylene blue)binding assay at weekly intervals up to 3 weeks.The composites were implanted subcutaneously in sheep abedoml and were evaluated macroscopically and bistologically at 4 weeks postoperatively.Results SEM photograph showed.after one week culture,cell morphology changed from fibroblast-like elongated spindle to the flat rounded like chondrocytes,and the extra cellular matrix also increased obviousl~.Furthmore,with the culture time extension,this change were more evident.HE staining showed that cells filled all the inter-connected pores in the constructs.And more cells were observed in the outer layer of the constructs.ECM(extraeellular matrix)Was strongly positive by Aleian blue,Safrine O staining and type Ⅱ collage immunohistechemical staining.DMB binding assay revealed that the induced BMSCs GAG secretion(1306.7±192.3)wag significantly higher than BMSCs(205.0±26.2)(P<0.001),but it was significantly lower than passage 2 ehondrocytes(1969.2±235.3)(P<0.001).Saltine O and type Ⅱ collage immunohistochemical staining were positive in constructs implanted subcutaneously.Conclusion Tissue engineered cartilage could be obtained using BMSCs and photografting modified PHBV,but there are still gaps physiologically between the constructs and the nature cartilage.