The effects of extracellular citric acid acidosis on the viability, cellular adhesion capacity and protein synthesis of cultured human gingival fibroblasts

Root surface demineralization is widely used as an adjunct to periodontal treatment. To clarify the influence of citric acid root conditioning on periodontal wound healing, the effects of citric acid and associated extracellular acidosis on the viability (MTT assay), attachment and protein synthesis ([H-3]-proline incorporation into trichloroacetic acid-precipitated proteins) of human gingival fibroblasts (GF) were investigated. A concentration of 47.6 mmol/L of citric acid (pH 2.3) in water led to total cell death within three minutes of incubation. Media containing 23.8 mmol/L and 47.6 mmol/L of citric acid exerted strong cytotoxicity (47 to 90 per cent of cell death) and inhibited protein synthesis (IC50=0.28 per cent) of GF within three hours of incubation. Incubation of cells in a medium containing 11.9 mmol/L of citric acid also suppressed the attachment and spreading of fibroblasts on culture plates and Type I collagen, with 58 per cent and 22 per cent of inhibition, respectively. Culture medium supplemented with 11.9, 23.8 and 47.6 mmol/L of citric acid also led to extracellular acidosis by decreasing the pH value from 7.5 to 6.3, 5.2 and 3.8, respectively. In addition, it was confirmed that the toxic effect of media containing citric acid was due to their acidity rather than the citrate content. Most of the citric acid-induced cell death could be prevented by adjusting the pH value of the culture medium to pH 7.5. Sodium citrate, at a concentration of 47.6 mmol/L, also exerted little cytotoxicity. The results suggested that toxicity of citric acid in specific stages of the healing process must be considered prior to its clinical application. Careful management of citric acid in order to avoid contact with tissue or the development of other demineralizing agents is important in enhancing periodontal wound healing.