Abstract
Aim: Keratin preparations have been shown to have bioactive effects on various cells including osteoblasts and neurons and offer the potential as a biomaterial in tissue regeneration. The aim of this study was to assess the in vitro effect of keratin preparations on the viability and proliferation of cementoblast OCCM-30 and fibroblast L929 cells and on the mineralisation capability of the OCCM-30 cells.
Method: Cementoblast OCCM-30 cells were grown in Dulbecco's modified Eagle's medium (DMEM) with 10% Fetal bovine serum (FBS) and 1% antibiotic-antimycotic. The fibroblast L929 cells were grown in minimum essential media (MEM) with 10% FBS and 1% antibiotic-antimycotic. The cells of L929 and OCCM-30 were assessed for viability in growth media supplemented with 10 mg/ml, 1 mg/ml and 0.1 mg/ml of keratin with a LIVE/DEAD assay™ Kit using confocal laser scanning microscope. Cell proliferation was tested with similar keratin concentrations using an alamarBlue® proliferation assay at 0, 24, 48 and 72 h. The proliferation assay showed that 10 mg/ml was toxic for the cementoblast cell, hence the concentration was ruled out for further testing by mineralisation assays. Alkaline phosphatase assay (SensoLyte® pNPP alkaline phosphatase assay kit) was performed for the OCCM-30 cells with keratin concentrations of 1 mg/ml, 0.1 mg/ml and the control (nil keratin) at 3, 6, and 10 d. Alizarin red assay was performed at 14 d for 1 mg/ml, 0.1 mg/ml and the control group (nil keratin).
Results: The viability assay showed concentrations at 1 mg/ml or greater of keratin proved toxic to the fibroblast L929 cells. However, OCCM-30 cells showed similar viability to the control group with 1 mg/ml of keratin solution. Keratin concentration >1 mg/ml showed a toxic effect on OCCM-30 cells.
The proliferation assay for L929 cells showed 10 mg/ml and 1 mg/ml were toxic compared to 0.1 mg/ml and 0.01 mg/ml keratin groups (P < 0.0001) in that they were unable to proliferate in the presence of 10 mg/ml and 1 mg/ml keratin. Cell proliferation of the control group was significantly higher than 10 mg/ml (P < 0.0001) and 1 mg/ml (P < 0.0001) while cell proliferation in the presence of 0.1 mg/ml and 0.01 mg/ml keratin was not significantly different (P > 0.05) to the control group.
The proliferation assay for cementoblast OCCM-30 cells showed keratin concentration at 10 mg/ml prevented its proliferation and was significantly lower than all other keratin groups tested (P < 0.0001). Keratin concentrations at 1 mg/ml and lower did not hinder the proliferation of the cementoblast OCCM-30 cells with similar proliferation as the control (P > 0.05).
The alkaline phosphatase (ALP) activity of cementoblast OCCM-30 cells exposed to keratin 1 mg/ml showed no signs of ALP activity at any of the time points tested. There was significantly higher OCCM-30 cell ALP activity with the control and 0.1 mg/ml groups compared with the 1 mg/ml group at 6 d and 12 d (P < 0.0001). The alizarin red assay was consistent with the ALP activity of the OCCM-30 cells with significantly lower levels of calcium staining observed between 1 mg/ml of keratin concentration and each of the remaining groups at day 14 (P < 0.0001), but there were no significant differences between 0.1 mg/ml and the control group (P > 0.05).
Conclusion: Keratin preparations were shown to allow normal cell proliferation of fibroblast L929 and cementoblast OCCM-30 cells and normal mineralisation of the cementoblasts. The results show promise that keratin could be developed as an endodontic regeneration scaffold material.