Lari Hakkinen

Macromolecular crowding (MMC) supplies synthetic polymeric crowders into cell culture media to better mimic macromolecular concentrations found in tissues. Their use may improve cell culture models for research and provide tools for regenerative therapy. We hypothesized that compared to traditional culture methods (nMMC), MMC can produce improved cultures and cell-derived matrices (CDMs) from cultured human gingival fibroblasts, a cell type associated with fast and scarless wound healing. Our specific aims were to compare fibroblast cultures generated with or without MMC, develop an improved decellularization method to generate CDMs from the cultures, and test if MMC can generate fibroblast cultures under reduced serum concentrations. To this end, fibroblasts were cultured under MMC (medium supplemented with Ficoll 70/400) and nMMC conditions supplemented with 10% or 2% FBS up to 14 days. Accumulation of proteins and specific ECM molecules in cultures and CDMs were studied by Bradford assay and by immunostaining and image analysis. Cell proliferation, morphology, and orientation were studied by image analysis and gene expression was analyzed by RT-qPCR. Results showed that MMC significantly reduced cell growth, but increased accumulation of collagen I and IV, cellular fibronectin, laminin 1, and tenascin C, while suppressing level of SPARC. Out of 62 genes studied, MMC significantly modulated expression of 20 genes compared to nMMC. Interestingly, MMC suppressed myofibroblast markers and promoted deposition of basement membrane molecules by fibroblasts. Decellularization with a novel method using latrunculin B followed by deoxycholate and DNase treatment produced CDMs that showed efficient removal of cellular elements. Molecular composition of cell cultures under MMC was replicated in CDMs, although decellularization caused some loss of ECM molecules. As expected, culture in low serum reduced cell growth and total protein accumulation. However, abundance of the above ECM molecules relative to cell numbers was significantly higher in MMC cultures and CDMs with 2% compared to 10% FBS. Culture in 2% FBS caused gene expression changes resembling a more profibrotic cell phenotype. Overall, the use of MMC and the novel decellularization technique rendered human gingival fibroblast cultures with distinct gene expression and CDMs with enhanced ECM molecule accumulation.

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Scar formation as a result of wound healing in skin is associated with increased deposition of extracellular matrix (ECM) and reduced ECM turnover by fibroblasts. Remarkably, wound healing in the human oral mucosa results in scarless healing, while wound healing in the skin can often result in scarring. Therefore comparing fibroblast phenotype and interactions with their ECM niche in the gingiva and skin may provide novel information about the factors that regulate scar formation. To this end, a novel 3D cell culture model was utilized to yield a cellular microenvironment (niche) that closely mimics the in vivo situation and primary gingival (GFBL) and skin fibroblasts (SFBL) phenotype was characterized. Furthermore, fibroblasts were reseeded on cell-free 3D ECM derived from GFBL and SFBL and the effects of the 3D ECM on cell phenotype were analyzed. Interestingly, SFBL in 3D cultures had greater expression of ECM deposition associated genes, including collagens, matricellular proteins, SLRPs, TGF-β1 and CTGF, intracellular ECM degradation and myofibroblast differentiation and function-associated genes, while GFBL had a greater expression of matrix remodeling associated genes (MMPs). We also found that the 3D cultures showed a significant difference in expression of certain genes (MMPs and myofibroblast function-associated genes) between GFBL and SFBL compared to cells reseeded on the 3D ECM or 2D control substrate. Thus, the 3D culture conditions may differentially regulate expression of a subset of genes in these cells. Interestingly, SFBL had a greater expression of matrix deposition associated genes (collagens, SLRPs, tenascins) irrespective of the culture conditions, suggesting that expression of these genes is inherently distinct between GFBL and SFBL. This was associated with greater autogenous TGF-β expression and SMAD3 phosphorylation in SFBL than GFBL, which may partly explain the innate difference in gene expression. In addition, there was greater ERK1/2 phosphorylation in fibroblasts when seeded on 3D ECM compared to 2D substrate. Greater ERK1/2 phosphorylation may have promoted greater expression of AP-1-dependent MMPs seen in SFBL and GFBL on 3D ECM. In conclusion, reduced expression of matrix deposition associated genes and greater expression of matrix remodeling genes in GFBL may contribute to scarless healing in gingiva.

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Objectives: Wound healing in skin often results in scar formation, whereas wound healing in oral palatal mucosa is fast and rarely results in scarring. Understanding the mechanisms that promote oral scarless wound healing may provide novel approaches to prevent scar formation in skin. The goal of the study was to compare the abundance of the major collagenases MMP-1 and MMP-13 and gelatinases MMP-2 and MMP-9 in normal unwounded oral mucosa and skin and in experimental excisional wounds in skin (healing results to scar formation) and oral mucosa (wounds heal with minimal scar formation) at various time points post-wounding at the protein level. We hypothesized that the abundance of MMPs will be higher in scarless oral mucosal wound healing, compared to skin wound healing.Methods: Experimental wounds were created in oral palatal mucosa and dorsal skin of red Duroc pigs. Wound biopsies were collected before wounding and at various time points after wounding. The abundance of MMPs at the protein level was assessed by Western blotting and zymography. Results: All studied MMPs showed a significantly increased accumulation in the wound tissue already at day three post-wounding. Their abundance remained high until day 28 when MMP-9 and MMP-13 returned to the level of unwounded tissue, while MMP-1 and MMP-2 remained significantly elevated. Oral mucosal wounds showed in general a robust early up-regulation of MMP-1, MMP-2 and MMP-9 as compared to skin wounds already at day 3 after wounding. In contrast, the peak abundance of these MMPs occurred at day 14 in skin wounds. Unwounded oral mucosa showed significantly higher abundance of total MMP-2 and active MMP-9 as compared to unwounded skin. Thus, MMPs needed for early wound healing response are already present in higher abundance in oral mucosa as compared to skin before tissue injury possibly allowing a fast wound healing response. Conclusions: Results suggest that oral mucosal wound healing is associated with fast and robust regulation of MMPs. Rapid controlled processing of wound extracellular matrix may play a key role in scarless palatal wound healing. In addition, MMPs may regulate inflammatory reaction that plays a central role in scar formation.

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