Cytotoxicity, inflammation, biomineralization, and immunoexpression of IL-1β and TNF-α promoted by a new bioceramic cement

Abstract Aim To evaluate the cytotoxicity, biocompatibility and mineralization capacity of BIO-C PULPO, and MTA. Methodology L929 fibroblasts were cultured and MTT assay was used to determine the material cytotoxicity on 6, 24, and 48 h. A total of 30 male rats (Wistar) aged between 4 and 6 months, weighing between 250 and 300 g were used. Polyethylene tubes containing BIO-C PULPO, MTA, and empty tubes were implanted into dorsal connective tissue. After the experimental periods (7, 15, 30, 60, and 90 days) the tubes were histologically analyzed using hematoxylin-eosin (H&E), immunolabeling of IL-1β and TNF-α, and von Kossa staining, or without staining for polarized light analysis. The average number of inflammatory cells was quantified; the mineralization assessment was determined by the area marked in μm2 and semiquantitative immunolabeling analyses of IL-1β and TNF-α were performed. Then, data underwent statistical analysis with a 5% significance level. Results It was observed that BIO-C PULPO and MTA presented cytocompatibility at 6, 24, and 48 similar or higher than control for all evaluated period. On periods 7 and 15 days, BIO-C PULPO was the material with the highest number of inflammatory cells (p<0.05). On periods 30, 60, and 90 days, BIO-C PULPO and MTA presented similar inflammatory reactions (p>0.05). No statistical differences were found between Control, BIO-C PULPO, and MTA for immunolabeling of IL-1β and TNF-α in the different periods of analysis (p<0.05). Positive von Kossa staining and birefringent structures under polarized light were observed in all analyzed periods in contact with both materials, but larger mineralization area was found with BIO-C PULPO on day 90 (p<0.05). Conclusion BIO-C PULPO was biocompatible and induced mineralization similar to MTA.

摘要 目的：评估BIO-C PULPO与MTA（Mineral Trioxide Aggregate）的细胞毒性、生物相容性及矿化能力。
方法：培养L929成纤维细胞（L929 fibroblasts），采用MTT比色法（MTT assay）在6、24、48小时检测材料的细胞毒性。本研究共纳入30只4~6月龄、体重250~300g的雄性Wistar大鼠（Wistar rat），将装载BIO-C PULPO、MTA的聚乙烯小管及空白聚乙烯小管植入大鼠背部结缔组织。分别于7、15、30、60、90天取材，采用苏木精-伊红（hematoxylin-eosin，H&E）染色、IL-1β与TNF-α免疫标记、冯库萨（von Kossa）染色进行组织学分析，部分样本不染色以用于偏振光观察。对炎症细胞平均数量进行定量统计；以μm²为单位量化矿化区域面积，并完成IL-1β与TNF-α的半定量免疫标记分析。所有数据采用显著性水平为5%的统计学方法进行分析。
结果：研究观察到，在6、24、48小时的所有评估时间点内，BIO-C PULPO与MTA的细胞相容性均不低于对照组。在第7天和第15天时，BIO-C PULPO组的炎症细胞数量最多（p<0.05）。在第30、60、90天时，BIO-C PULPO组与MTA组的炎症反应无统计学差异（p>0.05）。不同分析时间点下，对照组、BIO-C PULPO组与MTA组的IL-1β及TNF-α免疫标记结果均无统计学差异（p<0.05）。在接触两种材料的所有样本中，各分析时间点均观察到冯库萨染色阳性结果及偏振光下的双折射结构，但在第90天时，BIO-C PULPO组的矿化区域面积更大（p<0.05）。
结论：BIO-C PULPO具备良好生物相容性，且可诱导与MTA相当的矿化作用。