Biocompatibility Evaluation of ProRoot MTA Mixed with Human Platelet Lysate on Human Osteoblastic Cells In Vitro Studies
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Objective: The overall goal of this work is to understand the potential role of platelet rich plasma in regenerative endodontic therapy. Materials and methods: Human platelet lysate (HPL) commercially obtained was used as a source of human growth factors present in concentrated platelet enriched plasma. HPL at various concentrations (10-100%) was mixed with ProRoot MTA and in vitro studies were conducted to test the effects of the HPL addition. In these in vitro studies biocompatibility of human osteoblastic cells (HOBs) was evaluated with a spectrophotometric MTT assay that measures cellular mitochondrial activity as well as with a differentiation assay that measures alkaline phosphatase (ALP) activity. Cells were studied either in the presence of the MTA loaded with the HPL or with “conditioned” media that represents media that was first incubated with the loaded MTA without cells and should be reflective of a growth factor enriched medium due to the release of the factors present in HPL that are released from the MTA. Controls in all these experiments were obtained from ProRoot MTA mixed with distilled water in an identical proportion as the HPL loaded MTA samples. In some experiments ProRoot MTA was mixed with bovine serum albumin at various concentrations in order to further quantify the amount of protein that can be released from MTA under the incubation conditions employed in the HPL experiments. Setting times of the various MTA preparations were assessed with a Gillmore apparatus.Statistical analysis: Data were analyzed by ANOVA; p<0.05 was used as a significance level. Results: HPL at concentrations of 10 and 50% in the standard MEM culture medium did result in significant increases (compared to standard MEM medium with no added HPL) in MTT activity of HOB cells. However, when HPL was added to the ProRoot MTA, there were no significant differences in HOB cell activity or ALP activity compared to cells incubated with ProRoot MTA mixed with distilled water. The results were the same whether the cells were incubated directly with the HPL-loaded MTA samples or with “conditioned” media obtained from the HPL-loaded MTA samples. Spectrophotometric measurements of protein released from HPL loaded MTA samples revealed lack of delectable amounts except with samples loaded with 100% HPL after a 7-day incubation period. Similar data were obtained when MTA was loaded with bovine serum albumin (BSA) as a control protein. In these experiments with BSA, it appeared that the amount of protein released with loads (40 and 400 mg/ml) increased over a 14- day period of incubation. The addition of HPL did not affect the setting times of ProRoot MTA although the initial setting time for MTA mixed with 400 mg/ml BSA was the shortest. On the other hand, MTA mixed with 40 mg/m BSA had the longest initial and final sitting time.Conclusion: Gray ProRoot MTA Root Canal Repair Material is biocompatible in direct incubations with human osteoblastic cells. Under the standard growth conditions tested here, addition of HPL did not significantly affect the cellular responses to the MTA. Both time and concentration may affect the protein release from MTA. Further studies should be conducted to evaluate potential effects of MTA mixed with HPL under different cell growth conditions.