CAJ | 학술논문

目的探讨不同初始接种密度的骨髓基质干细胞(BMSCs)接种至磷酸钙骨水泥(CPC)支架上对细胞增殖和成骨分化效果的影响,并验证BMSCs生长在CPC上的生物黏附性能.方法培养小型猪的BMSCs至第4代,按1、5、25 mol/mL 3种细胞接种密度接种至CPC支架上,成骨诱导培养1、4、8d时采用扫描电镜观察细胞黏附形态及性能,EdU法检测细胞增殖率,活/死细胞染色试剂盒检测活细胞百分比及黏附性能、单位面积活细胞数,茜素红染色法检测矿物质合成,实时聚合酶链式反应(RT-PCR)检测成骨基因碱性磷酸酶(ALP)、骨钙素(OC)、Ⅰ型胶原(Col-Ⅰ)、RUNT相关转录因子-2(Runx-2)的mRNA的表达.结果 BMSCs在CPC支架表面黏附扩展良好,细胞接种密度从l mol/mL上升至25 mol/mL,活细胞百分比、单位面积活细胞数、矿物质合成、成骨分化基因也相应升高.ALP表达量在4d时达峰值,5 mol/mL组和25 mol/mL组的ALP表达量均高于1 mol/mL组,差异有统计学意义(P＜0.05),5 mol/mL组与25 mol/mL组之间差异无统计学意义(P＞0.05).OC、Col-Ⅰ、Runx-2的mRNA表达量在培养8d时明显增高,此时5 mol/mL组与25 mol/mL组差异均无统计学意义(P＞0.05).培养4、8d时5 mol/mL组与25 mol/mL组的活细胞百分比、单位面积活细胞数比较差异均无统计学意义(P＞0.05).结论 BMSCs的初始接种密度并非越高越好,在5 mol/mL至25mol/mL之间时,细胞接种至复合生物功能化CPC支架上,更利于细胞的增殖、分化和细胞外基质的合成,且具有良好的生物黏附性能.
목적 탐토불동초시접충밀도적골수기질간세포(BMSCs)접충지린산개골수니(CPC)지가상대세포증식화성골분화효과적영향,병험증BMSCs생장재CPC상적생물점부성능.방법 배양소형저적BMSCs지제4대,안1、5、25 mol/mL 3충세포접충밀도접충지CPC지가상,성골유도배양1、4、8d시채용소묘전경관찰세포점부형태급성능,EdU법검측세포증식솔,활/사세포염색시제합검측활세포백분비급점부성능、단위면적활세포수,천소홍염색법검측광물질합성,실시취합매련식반응(RT-PCR)검측성골기인감성린산매(ALP)、골개소(OC)、Ⅰ형효원(Col-Ⅰ)、RUNT상관전록인자-2(Runx-2)적mRNA적표체.결과 BMSCs재CPC지가표면점부확전량호,세포접충밀도종l mol/mL상승지25 mol/mL,활세포백분비、단위면적활세포수、광물질합성、성골분화기인야상응승고.ALP표체량재4d시체봉치,5 mol/mL조화25 mol/mL조적ALP표체량균고우1 mol/mL조,차이유통계학의의(P＜0.05),5 mol/mL조여25 mol/mL조지간차이무통계학의의(P＞0.05).OC、Col-Ⅰ、Runx-2적mRNA표체량재배양8d시명현증고,차시5 mol/mL조여25 mol/mL조차이균무통계학의의(P＞0.05).배양4、8d시5 mol/mL조여25 mol/mL조적활세포백분비、단위면적활세포수비교차이균무통계학의의(P＞0.05).결론 BMSCs적초시접충밀도병비월고월호,재5 mol/mL지25mol/mL지간시,세포접충지복합생물공능화CPC지가상,경리우세포적증식、분화화세포외기질적합성,차구유량호적생물점부성능.
Objective To investigate the effects of cell seeding density on proliferation and osteodifferentiation of bone marrow mesenchymal stem cells (BMSCs) on calcium phosphate cement (CPC) scaffolds and on the bioadhesive property of BMSCs on CPC as well.Methods BMSCs were isolated from minipigs and cultured.The fourth passage BMSCs were seeded on CPC scaffolds at 3 different densities (1,5,25 mol/mL) for osteodifferentiation.On days 1,4 and 8,scanning electron microscopy was used to detect cell bioadhesive property and extension,EdU assay to detect cell proliferation,live/dead fluorescent staining assay to detect cell viability,and RT-PCR to detect mRNA expression of alkaline phosphatase (ALP),osteocalcin (OC),collagen type Ⅰ (Col-Ⅰ),and Runt-related transcription factor 2 (Runx-2).Results The BMSCs seeded on the CPC scaffolds showed fine bioadhesive property and extension.When the cell seeding density was increased from 1 mol/mL to 25 mol/mL,the percentage of live cells,osteodifferentiation,and bone mineral synthesis also increased.On day 4 when ALP gene peaked,ALP expression levels in the 5 mol/mL and 25 mol/mL groups were significantly higher than in the 1 mol/mL group (P ＜ 0.05),but there was no significant difference between the 5 mol/mL and 25 mol/mL groups (P ＞ 0.05).The expression levels of OC,Col-Ⅰ,and Runx-2 were significantly higher on day 8,but the differences between the 5 mol/mL and 25 mol/mL groups were not significant (P ＞ 0.05).There were no significant differences on day 4 or 8 between the 5 mol/mL and 25 mol/mL groups regarding the percentage of live cells or the number of live cells per specimen surface area (P ＞ 0.05).Conclusions BMSCs seeded on the CPC have good bioadhesive property.A too high seeding density may not be advantageous,for an intermediate seeding density can yield the best proliferation,differentiation and extracellular matrix formation.The best seeding density of BMSCs on CPC may vary between 5 mol/mL and 25 mol/mL.