摘要
目的探讨聚乳酸(polylactic acid,PLA)降解终产物乳酸对成骨样细胞增殖和成骨表型的影响,为骨组织工程支架材料的设计和降解调控提供理论依据。方法实验分为A、B、C 3组,A、B组分别采用含4、8、16、22、27 mmol/L右旋乳酸(L-lactic acid,L-LA)和外消旋乳酸(D,L-lactic acid,D,L-LA)的培养液培养Ros17/2.8成骨样细胞,C组以不含乳酸的培养液(p H7.4)培养细胞。培养1、3、5 d,采用MTT法检测细胞相对增殖率(relative growth ratio,PGR)和细胞毒性评级。另取含4 mmol/L L-LA(A组)及4 mmol/L D,L-LA(B组)的培养液培养Ros17/2.8成骨样细胞,以不含乳酸的培养液(p H7.4)培养细胞作为空白对照组(C组)。培养1、5 d时检测相对ALP活性(relative ALP ratio,RAR),21 d时采用von Kossa染色法检测细胞矿化结节生成并行定量分析。结果乳酸浓度为4 mmol/L时,培养5 d内A、B组RGR均值均>80%,细胞毒性评级为0级或Ⅰ级,无明显细胞毒性;浓度增大至8 mmol/L和16 mmol/L时,A、B组表现出毒性作用的时间分别为培养5 d和3 d;浓度≥22 mmol/L时,A、B组乳酸培养液在培养1 d时即对细胞增殖产生明显抑制。相同浓度下,各时间点A组RGR均高于B组,除4 mmol/L浓度培养1 d和3 d外,差异均有统计学意义(P<0.05)。培养1 d,A、B组RAR分别为144.1%±3.2%和115.2%±9.8%,5 d时分别为129.6%±9.8%和78.2%±6.9%,A组均显著高于B组(P<0.05)。von Kossa染色示,培养21 d,A组黑色团状物明显多于B、C组,定量分析分别为91.2%±8.2%、50.3%±7.9%和54.2%±8.6%,A组显著高于B、C组(P<0.05);B、C组间差异无统计学意义(P>0.05)。结论 PLA降解产物的浓度和组成对成骨样细胞增殖和成骨表型均有显著影响。
Objective To investigate the influences of lactic acid(LA),the final degradation product of polylactic acid(PLA) on the proliferation and osteoblastic phenotype of osteoblast-like cells so as to provide theoretical basis for bone tissue engineering.Methods Ros 17/2.8 osteoblast-like cells were harvested and divided into 3 groups.In groups A and B,the cells were cultured with the medium containing 4,8,16,22,and 27 mmol/L L-LA and D,L-LA,respectively.In group C,the cells were cultured with normal medium(pH7.4).The cell proliferation was determined with MTT method after 1,3,and 5 days.The relative growth ratio(RGR) was calculated,and the cytotoxicity was evaluated according to national standard of China.In addition,the alkaline phosphatase(ALP) activity of cells cultured with medium containing 4 mmol/L L-LA(group A),4 mmol/L D,L-LA(group B),and normal medium(group C) after 1 and 5 days were detected with ALP kits,and the relative ALP ratio(RAR) was calculated;after 21 days,the calcium nodules were tested with von Kossa staining method,and were quantitatively analyzed.Results When LA concentration was 4 mmol/L,the mean RGR of both groups A and B were all above 80%,and the cytotoxic grades were grade 0 or 1,which meant non-cytotoxicity.When LA concentration was 8 mmol/L and 16 mmol/L,groups A and B showed cytotoxicity after 5 days and 3 days,respectively.When LA concentration was above 22 mmol/L,cell proliferations of groups A and B were inhibited evidently after 1-day culture.At each LA concentration,RGR of group A was significantly higher than that of group B at the same culture time(P〈0.05) except those at 4 mmol/L after 1-day and 3-day culture.After 1 day,the RAR of group A was significantly higher than that of group B on 1 day(144.1%± 3.2%vs.115.2%±9.8%,P〈0.05) and on 5 days(129.6%± 9.8%vs.78.2%± 6.9%,P〈0.05).The results of von Kossa staining showed that the black gobbets in group A were obviously more than those of groups B and C.The staining area of group A(91.2%±8.2%) was significantly higher than that of groups B(50.3%± 7.9%) and C(54.2%± 8.6%)(P〈0.05).Conclusion The concentration and composition of LA have significant effects on the cell proliferation and osteoblastic phenotype of osteoblast-like cells.
出处
《中国修复重建外科杂志》
CAS
CSCD
北大核心
2014年第12期1525-1529,共5页
Chinese Journal of Reparative and Reconstructive Surgery
基金
国家自然科学基金资助项目(51273125
30800223)~~
关键词
骨组织工程
支架材料
聚乳酸
乳酸
成骨样细胞
细胞增殖
成骨表型
Bone tissue engineering
Scaffold material
Polylactic acid
Lactic acid
Osteoblast-like cells
Cell proliferation
Osteoblastic phenotype