Methods: Donor-matched oral mucosal and dermal fibroblasts were obtained. TGFb1-stimulation was used to induce fibroblast-myofibroblast differentiation. Myofibroblast transition was assessed by mRNA (QPCR) and protein expression of the myofibroblast marker a-smooth muscle actin (a-sma) as well as actin cytoskeleton reorganisation (Immunocytochemistry). QPCR was used to assess expression of HA Synthase (HAS) enzymes. HA synthesis was analysed using metabolic radio-labelling and column chromatography. Pericellular HA was assessed using the particle exclusion assay. Inhibition of HA synthesis was performed by 4-methyl umbelliferone depletion of the UDP-glucuronic acid pool.
Results: The two fibroblast populations demonstrated intrinsic differences in their response to TGFb1. Dermal fibroblasts demonstrated increased a-sma mRNA and protein expression and prominent cytoskeletal re-organisation in response to TGFb1-stimulation. In contrast, oral mucosal fibroblasts demonstrated resistance to TGFb1-mediated differentiation as shown by reduced a-sma expression and intermediate cytoskeletal re-organisation. In dermal fibroblasts differentiation was associated with induction of HA Synthase (HAS1 and HAS2) transcription, an 8-fold increase in intra- and extracellular HA synthesis and increased pericellular HA coat assembly. In comparison resistance to differentiation in oral mucosal fibroblasts was associated with failure of HAS1 and HAS2 transcription, failure of induction of HA synthesis and failure of pericellular coat assembly. Furthermore, inhibiting HA synthesis significantly attenuated TGFb1-mediated dermal fibroblast differentiation.
Conclusion: Oral fibroblasts are resistant to myofibroblastic differentiation and HA is involved in regulating TGFb1-driven differentiation. This represents an important target for future research into the investigation of scaring versus scar-free healing.