中华骨科杂志
中華骨科雜誌
중화골과잡지
CHINESE JOURNAL OF ORTHOPAEDICS
2013年
8期
855-862
,共8页
孙明林%安博%孙铭泽%朱雷%张春秋
孫明林%安博%孫銘澤%硃雷%張春鞦
손명림%안박%손명택%주뢰%장춘추
软骨细胞%灌流%生物反应器%组织工程
軟骨細胞%灌流%生物反應器%組織工程
연골세포%관류%생물반응기%조직공정
Chondrocytes%Perfusion%Bioreactors%Tissue engineering
目的 采用静电纺丝聚已内酯(polycaprolactone,PCL)支架与软骨细胞复合培养,比较静态和灌流生物反应器培养条件下对细胞增殖及基质分泌的影响.方法 构建PCL支架,自制灌流生物反应器,分离兔软骨细胞,培养后接种于PCL支架,分为灌流培养组和静态培养组.在培养第3、7、14天对支架-细胞复合体行扫描电镜观察,DNA、糖胺聚糖和总胶原定量检测；在培养第14天分析软骨特异性基因表达并观察软骨基质分泌情况.结果 电镜观察PCL支架纤维直径(1.67±0.76) μm,孔径(17.65土7.11)μm,可见支架中软骨细胞黏附生长良好,灌流培养条件下细胞增殖快,且较好地保持了软骨细胞特征形态.在培养第7天,灌流培养组DNA定量高于静态培养组；在培养第3、7和14天,灌流培养组糖胺聚糖定量均高于静态培养组,灌流培养组糖胺聚糖/DNA比值均高于静态培养组.在培养第14天,灌流培养组Ⅱ型胶原、蛋白聚糖基因表达增加；软骨分化指数高于静态培养组.在培养第14天,组织学染色可见灌流培养促进细胞的增殖和渗透生长,提高了软骨基质的分泌,并见软骨陷窝样结构.结论 在灌流生物反应器培养条件下,静电纺丝PCL支架与软骨细胞复合培养可促进软骨细胞的增殖和基质的分泌,提高了组织工程软骨的质量.
目的 採用靜電紡絲聚已內酯(polycaprolactone,PCL)支架與軟骨細胞複閤培養,比較靜態和灌流生物反應器培養條件下對細胞增殖及基質分泌的影響.方法 構建PCL支架,自製灌流生物反應器,分離兔軟骨細胞,培養後接種于PCL支架,分為灌流培養組和靜態培養組.在培養第3、7、14天對支架-細胞複閤體行掃描電鏡觀察,DNA、糖胺聚糖和總膠原定量檢測；在培養第14天分析軟骨特異性基因錶達併觀察軟骨基質分泌情況.結果 電鏡觀察PCL支架纖維直徑(1.67±0.76) μm,孔徑(17.65土7.11)μm,可見支架中軟骨細胞黏附生長良好,灌流培養條件下細胞增殖快,且較好地保持瞭軟骨細胞特徵形態.在培養第7天,灌流培養組DNA定量高于靜態培養組；在培養第3、7和14天,灌流培養組糖胺聚糖定量均高于靜態培養組,灌流培養組糖胺聚糖/DNA比值均高于靜態培養組.在培養第14天,灌流培養組Ⅱ型膠原、蛋白聚糖基因錶達增加；軟骨分化指數高于靜態培養組.在培養第14天,組織學染色可見灌流培養促進細胞的增殖和滲透生長,提高瞭軟骨基質的分泌,併見軟骨陷窩樣結構.結論 在灌流生物反應器培養條件下,靜電紡絲PCL支架與軟骨細胞複閤培養可促進軟骨細胞的增殖和基質的分泌,提高瞭組織工程軟骨的質量.
목적 채용정전방사취이내지(polycaprolactone,PCL)지가여연골세포복합배양,비교정태화관류생물반응기배양조건하대세포증식급기질분비적영향.방법 구건PCL지가,자제관류생물반응기,분리토연골세포,배양후접충우PCL지가,분위관류배양조화정태배양조.재배양제3、7、14천대지가-세포복합체행소묘전경관찰,DNA、당알취당화총효원정량검측；재배양제14천분석연골특이성기인표체병관찰연골기질분비정황.결과 전경관찰PCL지가섬유직경(1.67±0.76) μm,공경(17.65토7.11)μm,가견지가중연골세포점부생장량호,관류배양조건하세포증식쾌,차교호지보지료연골세포특정형태.재배양제7천,관류배양조DNA정량고우정태배양조；재배양제3、7화14천,관류배양조당알취당정량균고우정태배양조,관류배양조당알취당/DNA비치균고우정태배양조.재배양제14천,관류배양조Ⅱ형효원、단백취당기인표체증가；연골분화지수고우정태배양조.재배양제14천,조직학염색가견관류배양촉진세포적증식화삼투생장,제고료연골기질적분비,병견연골함와양결구.결론 재관류생물반응기배양조건하,정전방사PCL지가여연골세포복합배양가촉진연골세포적증식화기질적분비,제고료조직공정연골적질량.
Objective To investigate the chondrocyte proliferation and extracellular matrix biosynthesis of electrospun PCL scaffolds seeded with rabbit chondrocytes under flow perfusion culture in vitro.Methods Nonwoven PCL microfiber mats were fabricated,and contra-aperture cylindrical glass equipment as a perfusion bioreactor was designed and manufactured on our own.The experiment included peffusion culture group and static culture group.Primary chondrocytes were isolated from the knee joints of two-month-old New Zealand white rabbits and seeded into scaffolds.The scaffold-cell complexes were harvested at 3,7,and 14 days of culture for scanning electron micrograph (SEM) analysis,biochemical assay,real-time PCR and histology analysis.Results Electrospun PCL scaffolds were composed of microfibers with a diameter of 1.67±0.76 μm and pores with a diameter of 17.65±7.11 μm.SEM showed a better cell proliferation with typical morphology of chondrocytes under perfusion culture.At 7 days of culture,DNA content in perfusion culture group was higher than in static culture group.At 3,7 and 14 days of culture,compared with the static culture group,glycosaminoglycan (GAG) content and GAG/DNA ratio in perfusion culture group were higher,and the differences were statistically significant.At 14 days of culture,real-time PCR showed aggrecan and collagen type Ⅱ gene expression and collagen type Ⅱ to collagen type Ⅰ ratio were higher in perfusion culture group than in static culture group; HE and safranin O staining showed a significant cell proliferation,infiltration,as well as extracellular matrix biosynthesis in perfusion culture group.Conclusion Under flow perfusion culture,the electrospun PCL scaffolds seeded with rabbit chondrocytes can enhance chondrocyte proliferation and extracellular matrix biosynthesis,which is a promising method for cartilage tissue engineering.
