Chemcially Induced Growth Factor Release from Fractured Mandible Cultures

Endogenous growth factors bound in bone matrix may be released from sites of fracture and signal optimal reparative processes at these sites leading to bone regeneration. Our previous studies have demonstrated that growth factors can be released from the bone following chemical treatment with EDTA, citric acid and calcium hydroxide but further studies are required to investigate cellular responses to released growth factors. Objectives: To investigate cellular response to chemical agents used to released endogenous growth factors in a dynamic ex-vivo fractured rodent mandible culture system. Methods: 2mm transverse mandible slices from 28 day old Wistar rats, were prepared and fractured using a sterile scalpel prior to treatment with 10ml 17% EDTA, 0.02M Ca(OH)_2, 10% citric acid and PBS for 5 and 15 min. Slices were cultured in semi-solid agar based medium as Trowel-type cultures for 7 days. Following culture, tissues were fixed and demineralised prior to histological examination. Cellular characteristics within the fracture site in response to released growth factors were assessed by immunohistochemistry for PCNA, bone sialoprotein (BSP) and osteopontin (OPN). Condition media from cultures was concentrated and TGF-β1, BMP2 and VEGF levels in media quantified by ELISA. SEM / Immuno-gold staining for the above growth factors was performed on treated surfaces and gold particles quantified. Results: Fractured mandible slices treated with Ca(OH)₂ exhibited a significant increase in cell numbers and immuno-positivity to PCNA, OPN, and BSP within the fracture site, compared with EDTA, citric acid and PBS. Fractured mandible slices treated with Ca(OH)₂ exhibited a significant increase in VEGF, TGF-β1 and BMP2, compared with EDTA, citric acid and PBS in both immuno-gold particle expression on fractured treated surfaces and within conditioned media, with significantly greater VEGF release when compared with other growth factors. Conclusion: Ca(OH)₂ may release growth factors from bone surfaces which influences cellular reparative processes.