聚乳酸降解终产物对成骨样细胞增殖和成骨表型影响的体外实验研究

IN VITRO EXPERIMENTAL STUDY ON INFLUENCES OF FINAL DEGRADATION PRODUCTS OF POLYACTIC ACID ON PROLIFERATION AND OSTEOBLASTIC PHENOTYPE OF OSTEOBLAST-LIKE CELLS

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.