Effects of different transforming growth factor beta (TGF-β) isomers on wound closure of bone cell monolayers

Abstract

This study aimed at determining the role of the transforming growth factor-beta (TGF-β) isomers and their combinations in bone cell behaviour using MG63 cells. The work examined how TGF-β1, 2 and 3 and their solvent and carrier (HCl and BSA, respectively) effected cell morphology, cell proliferation and integrin expression. This study also aimed at examining how the TGF-βs and their solvent and carrier influenced wound closure in an in vitro wound closure model and how TGF-βs influence extracellular matrix (ECM) secretion and integrin expression. The wound healing response in terms of healing rate to the TGF-βs and their solvent/carrier was investigated in 300 μm ± 10-30 μm SD wide model wounds induced in fully confluent monolayers of MG63 bone cells. The effect of different TGF-β isomers and their combinations on proliferation rate and cell length of human bone cells were also assessed. Immunostaining was used to determine if TGF-βs modifies integrin expression and ECM secretion by the bone cells. Imaging with WSPR allowed observation of the focal contacts without the need for immunostaining. The wound healing results indicated that TGF-β3 has a significant effect on the wound healing process and its healing rate was found to be higher than the control (p < 0.001), TGF-β1 (p < 0.001), TGF-β2 (p < 0.001), BSA/HCl (p < 0.001) and HCl (p < 0.001) in ascending order. It was also found that TGF-β1 and TGF-β2 treatment significantly improved wound closure rate in comparison to the controls (p < 0.001). All TGF-β combinations induced a faster healing rate than the control (p < 0.001). It was expected that the healing rate following treatment with TGF-β combinations would be greater than those healing rates following treatments with TGF-β isomers alone, but this was not the case. The results also suggest that cell morphological changes were observed significantly more in cells treated with TGF-β(2 + 3) and TGF-β(1 + 3) (p < 0.001). Any cell treated with TGF-β1, TGF-β(1 + 2) and TGF-β(1 + 2 + 3) showed significantly less elongation compared to the control and other TGF-β isomers. In terms of proliferation rate, TGF-β3 and TGF-β(2 + 3) increased cell numbers more than TGF-β1, TGF-β2 and other combinations. TGF-β1 and its combinations did not show significant proliferation and attachment compared to the control. Immunostaining indicated that treatment with TGF-β3 significantly enhanced the secretion of collagen type I, fibronectin and integrins α3 and β1. The WSPR experiments also indicated that TGF-βs influenced the distribution of focal contacts. In conclusion, combining TGF-β3 with any other TGF-β isomer resulted in a faster model wound closure rate (p < 0.001), while treatment with TGF-β1 in any TGF-β combination reduced the healing rate (p < 0.001). It can therefore be concluded that the presence of TGF-β1 has an inhibitory effect on bone wound healing while TGF-β3 had the opposite effect and increased the rate of wound closure in a 2 dimensional cell culture environment.

Keywords: Bone cell engineering; Transforming growth factor beta (TGF-β); Widefield surface plasmon microscopy (WSPR); Wound healing.