中华创伤骨科杂志
中華創傷骨科雜誌
중화창상골과잡지
CHINESE JOURNAL OF ORTHOPAEDIC TRAUMA
2014年
8期
710-714
,共5页
乔瑞红%王东%孙海钰%高敏
喬瑞紅%王東%孫海鈺%高敏
교서홍%왕동%손해옥%고민
间充质干细胞%骨髓%壳聚糖%羟基磷灰石类%材料试验
間充質榦細胞%骨髓%殼聚糖%羥基燐灰石類%材料試驗
간충질간세포%골수%각취당%간기린회석류%재료시험
Mesenchymal stem cells%Bone marrow%Chitosan%Hydroxyapatites%Materials testing
目的 探讨负载去细胞基质的壳聚糖(CS)/纳米羟基磷灰石(nHA)复合微球与骨髓基质干细胞(BMSCs)联合培养的生物相容性.方法 制备负载去细胞基质的CS/nHA复合微球,分离、提纯SD大鼠的BMSCs.实验分为4组(每组每个时间点6个样本):空白组:单纯BMSCs培养,对照组1:去细胞基质与BMSCs共培养,对照组2:nHA与BMSCs共培养,实验组:负载去细胞基质的CS/nHA与BMSCs共培养.于培养14、21 d,采用茜素红染色观察BMSCs的成骨分化能力.于培养3、7、10、14、21 d,应用酶联免疫吸附测定试剂盒检测各组碱性磷酸酶(ALP)活性,应用逆转录聚合酶链式反应检测Ⅰ型胶原和骨桥蛋白的表达. 结果 负载去细胞基质的CS/nHA复合微球呈球形,粒径范围为3.52 ～ 29.65 μm.培养第21天,实验组内可见大量钙质沉积,茜素红染色呈阳性;对照组有少量钙质沉积;空白组钙质沉积更少.从培养第7天开始,实验组的ALP活性、Ⅰ型胶原表达量明显高于其他3组,对照组1、对照组2的ALP活性、Ⅰ型胶原表达量又明显高于空白组,差异均有统计学意义(P＜0.05).从培养第10天开始,实验组的骨桥蛋白表达量明显高于其他3组,对照组1、对照组2的骨桥蛋白表达量又明显高于空白组,差异均有统计学意义(P＜0.05).培养第21天,实验组和对照组ALP活性、Ⅰ型胶原及骨桥蛋白的表达均达到高峰. 结论 负载去细胞基质的CS/nHA复合微球与BMSCs具有良好的生物相容性.
目的 探討負載去細胞基質的殼聚糖(CS)/納米羥基燐灰石(nHA)複閤微毬與骨髓基質榦細胞(BMSCs)聯閤培養的生物相容性.方法 製備負載去細胞基質的CS/nHA複閤微毬,分離、提純SD大鼠的BMSCs.實驗分為4組(每組每箇時間點6箇樣本):空白組:單純BMSCs培養,對照組1:去細胞基質與BMSCs共培養,對照組2:nHA與BMSCs共培養,實驗組:負載去細胞基質的CS/nHA與BMSCs共培養.于培養14、21 d,採用茜素紅染色觀察BMSCs的成骨分化能力.于培養3、7、10、14、21 d,應用酶聯免疫吸附測定試劑盒檢測各組堿性燐痠酶(ALP)活性,應用逆轉錄聚閤酶鏈式反應檢測Ⅰ型膠原和骨橋蛋白的錶達. 結果 負載去細胞基質的CS/nHA複閤微毬呈毬形,粒徑範圍為3.52 ～ 29.65 μm.培養第21天,實驗組內可見大量鈣質沉積,茜素紅染色呈暘性;對照組有少量鈣質沉積;空白組鈣質沉積更少.從培養第7天開始,實驗組的ALP活性、Ⅰ型膠原錶達量明顯高于其他3組,對照組1、對照組2的ALP活性、Ⅰ型膠原錶達量又明顯高于空白組,差異均有統計學意義(P＜0.05).從培養第10天開始,實驗組的骨橋蛋白錶達量明顯高于其他3組,對照組1、對照組2的骨橋蛋白錶達量又明顯高于空白組,差異均有統計學意義(P＜0.05).培養第21天,實驗組和對照組ALP活性、Ⅰ型膠原及骨橋蛋白的錶達均達到高峰. 結論 負載去細胞基質的CS/nHA複閤微毬與BMSCs具有良好的生物相容性.
목적 탐토부재거세포기질적각취당(CS)/납미간기린회석(nHA)복합미구여골수기질간세포(BMSCs)연합배양적생물상용성.방법 제비부재거세포기질적CS/nHA복합미구,분리、제순SD대서적BMSCs.실험분위4조(매조매개시간점6개양본):공백조:단순BMSCs배양,대조조1:거세포기질여BMSCs공배양,대조조2:nHA여BMSCs공배양,실험조:부재거세포기질적CS/nHA여BMSCs공배양.우배양14、21 d,채용천소홍염색관찰BMSCs적성골분화능력.우배양3、7、10、14、21 d,응용매련면역흡부측정시제합검측각조감성린산매(ALP)활성,응용역전록취합매련식반응검측Ⅰ형효원화골교단백적표체. 결과 부재거세포기질적CS/nHA복합미구정구형,립경범위위3.52 ～ 29.65 μm.배양제21천,실험조내가견대량개질침적,천소홍염색정양성;대조조유소량개질침적;공백조개질침적경소.종배양제7천개시,실험조적ALP활성、Ⅰ형효원표체량명현고우기타3조,대조조1、대조조2적ALP활성、Ⅰ형효원표체량우명현고우공백조,차이균유통계학의의(P＜0.05).종배양제10천개시,실험조적골교단백표체량명현고우기타3조,대조조1、대조조2적골교단백표체량우명현고우공백조,차이균유통계학의의(P＜0.05).배양제21천,실험조화대조조ALP활성、Ⅰ형효원급골교단백적표체균체도고봉. 결론 부재거세포기질적CS/nHA복합미구여BMSCs구유량호적생물상용성.
Objective To explore the biocompatibility of bone marrow mesenchymal stem cells (BMSCs) and composite microspheres of decellularized matrix loaded chitosan/nanohydroxyapatite (DMCS/nHA) in co-culture.Methods After preparation of composite microspheres of DMCS/nHA and isolation and purification of BMSCs from SD rats,the culture was conducted in 4 groups (6 specimens at each time point for each group).BMSCs were cultured with the composite microspheres in the experimental group,with decellularized matrix in control group 1,with nHA in control group 2,and with nothing in the blank control group.The osteogenic differentiation capability of BMSCs in vitro was assessed by alizarin red staining at 14 and 21 days after culture.The activity of alkaline phosphate (ALP) was assessed by ELISA and the expression levels of type Ⅰ collagen and oesteopontin by RT-PCR at 3,7,10,14 and 21 days after culture.Results The composite microspheres were spherical in shape and the particle diameters ranged from 3.52 to 29.65 μm.At 21 days,the experimental group had a greater amount of calcified tubercle with positive alizarin red staining than both control groups and the blank control group.After 7 days,the expression levels of ALP and type Ⅰ collagen were significantly higher in the experimental group than in the other 3 groups,and in both control groups than in the blank control group (P ＜ 0.05).After 10 days,the expression of oesteopontin was significantly higher in the experimental group than in the other 3 groups,and in both control groups than in the blank control group (P ＜ 0.05).The expression of ALP,collagen Ⅰ and oesteopontin reached the peak values at 21 days in the experimental group and both control groups.Conclusion The composite microspheres of DMCS/nHA may have excellent biocompatibility with BMSCs.
