Effect of the ozone on healing of bisphosphonate related wounds using gingival fibroblasts: an in-vitro study

Bisphosphonate related osteonecrosis of the jaws (BRONJ) is a complication seen in patients undergoing long-term bisphosphonate therapy and characterized by non-healing oral mucosa and exposed necrotic jaw bone. Previously, bone tissue studies was commonly performed to explain the pathogenesis of BRONJ, recently soft tissue researches became more important. Treatment of BRONJ is still complicated. Alternative modalities as hyperbaric oxygen and laser have been tired in BRONJ treatment, however; there has not been effective treatment protocol yet. Recently, some case reports have been published stating the effect of ozone therapy in BRONJ treatment have been published. The purpose of this study was to evaluate the effect of the ozone on healing of bisphosphonate related wounds using gingival fibroblasts in-vitro. 6 healthy patients, with age range 18-30 included in this study. Human gingival fibroblasts obtained from gingiva excised above unerupted teeth for orthodontic traction. Sufficient cell number was achieved at passage 6-9. Cultures were treated with only pamidronate, pamidronate + ozone or only ozone and incubated 24, 48 or 72 hours and compared with untreated group. Pamidronate and ozone were applied simultaneously to the cultures incubated 24 hours. The cultures, incubated 48 and 72 hours, treated with ozone 24 hours after pamidronate application. Final concentration of applied pamidronate was regulated as 10-4 M and ozone application was made by OzonytronX medical ozone generator. Total RNA isolation was achieved by 1ml Tripure Isolation Reagent application to cultures at the end of the incubation period and freezed at -200C. COL1A1 and COL1A2 gene expressions were evaluated with RT-PCR. GAPDH was used as housekeeping gene. COL1A1 and COL1A2 expressions were higher in ozone group compared with control group at 48 hours. In pamidronat + ozone group, COL1A1and COL1A2 expressions was increased at 72 hours which means 48 hours after ozone application. After 72 hours of ozone application, there was no increase in COL1A1 and COL1A2 expressions. The results of this study show that; ozone induced the collagen expression, an important phase of wound healing. Maximum effect of ozone on collagen expression was observed at 48 hours after application and collagen expression decreased at 72 hours. In oral mucosal wounds, ozone must be applied at 48 hours intervals to keep increased collagen expression.