Resident fibroblasts in tissues are responsible for the formation of the extracellular matrix (ECM) and play a crucial role in tissue repair. Human gingival fibroblasts (hGF) and human periodontal ligament fibroblasts (hPDL) are key participants in the repair of oral tissues. hGF is the most abundant cell type in gingival connective tissue, secreting various ECM components and cytokines to promote tissue repair without scarring. hPDL is essential for the maintenance and repair of the periodontal ligament, demonstrating capabilities in proliferation and matrix secretion.

Growth factor technology is pivotal in changing the local tissue microenvironment during periodontal repair. Studies have shown that TGF-β1 treatment results in different protein synthesis and proliferation rates between hGF and hPDL. Understanding the specific phenotypic responses of hGFs and hPDLs to various growth factors is vital for the development of targeted therapies aimed at improving oral wound healing and tissue regeneration.

Recently, Douglas W. Hamilton et al. published an article titled 'FGF and TGF-β growth factor isoform modulation of human gingival and periodontal ligament fibroblast wound healing phenotype' in Matrix Biology. This research compared the reactions of hPDL and hGF to TGF-β1, TGF-β3, FGF-2, and FGF-9, revealing that TGF-β3 combined with FGF-2 can regulate ECM synthesis, reduce fibrosis, and enhance cell migration, thus providing a novel approach to wound healing therapy.

Materials and Methods

hPDL was collected from the lower two-thirds of extracted teeth and hGF from explant cultures of five patients. Both were cultured in high-glucose DMEM with 10% fetal bovine serum and 1% Antibiotic-Antimyotic. Cells from passage 2-7 were used and growth factors were added at set concentrations. RNA was isolated with TRIzol and QIAGEN kit for RT-qPCR. Cells were fixed, permeabilized, blocked, and incubated with antibodies for staining. After cell lysis, Western blot was carried out. For cell migration, scratch assay was used with specific culture conditions and imaging. Collagen gel contraction assay was done following the preparation and treatment process and then weighing. Statistical analysis was performed using specific methods and data were from 3-5 patients' cells.

Results

To study cell migration affected by growth factors, scratch wound assays were done. FGF-2-treated hPDL cells closed wounds faster than others (Fig. 1A). Quantification showed that hPDL with FGF-2 closed the scratch area quicker at 24h (Fig. 2B). For hGF cells, FGF-2 also led to faster closure than other conditions, verified by quantification at 36h and 48h (Fig. 1C).

Fig. 1 FGF-2 enhanced migration.

The presence of myofibroblasts is often marked by α-SMA expression and its integration into stress fibers. In hPDL and hGF cells, TGF-β1 and TGF-β3 treatment significantly boosted α-SMA expression (Fig. 2A'C). Immunocytochemistry (ICC) further verified this increase and showed fiber incorporation (Fig. 2B'D). For hGF cells, it showed more fiber incorporation in TGF-β1 treatment than TGF-β3 treatment.

Fig. 2 TGF-β1 and TGF-β3 upregulated α-SMA, with distinct effects on fiber incorporation.

Tissue regeneration depends on extracellular matrix production. ICC showed more fibronectin in TGF-β1/TGF-β3-treated hPDL and hGF (Figs. 3A-B). At day 7, FGF-2-treated cells had less dense fibronectin matrix than TGF-β3-treated ones (Figs. 3C-D), with similar fiber thickness.

In hPDL, TGF-β1/TGF-β3 upregulated fibronectin, collagen I, and periostin mRNA (Fig. 3E). In hGF, FGF-2 cut fibronectin and collagen I mRNA (Fig. 3F), periostin unchanged. TGF-β1 hiked fibronectin protein in hPDL (Fig. 3G), FGF-2 lowered it in hGF (Fig. 3H).

Fig. 3 TGF-β promoted matrix production.

The response of hGFs and hPDL cells to combination treatments of TGF-β3+FGF-2 was further investigated. Cells exhibited higher wound closure rates compared to TGF-β3 treatment alone in hPDL at 24h (Fig. 4A-B). In hGF cells, the combined treatment did not increase cell migration compared to untreated controls (Fig. 4C).

Fig. 4 TGF-β3+FGF-2 co-treatment does not impede cell migration.

To assess fibroblast activation, α-SMA was used to detect myofibroblast transition. In hPDL, TGF-β3 upped α-SMA more than control, FGF-2, and TGF-β3 + FGF-2 groups (Fig. 5A), confirmed by ICC (Fig. 5B). For hGF, TGF-β3-treated cells had higher α-SMA than FGF-2-treated ones (Fig. 5C); the combined treatment had α-SMA levels between control and FGF-2-only, verified by ICC (Fig. 5D). Collagen gel assays for contraction showed TGF-β3-treated hPDL/hGF and TGF-β3 + FGF-2-treated hPDL contracted more than relevant controls (Fig. 5E-F).

Fig. 5 Absence of myofibroblast differentiation with TGF-β3+FGF-2 co-treatment.

Next, the impact of TGF-β3 + FGF-2 treatment on ECM production was examined. In hPDL, it didn't change fibronectin protein levels (Fig. 6A), though ICC showed TGF-β3 addition led to extracellular fibril formation (Fig. 6B). In hGF, the combined treatment hiked fibronectin on day 1 and day 2 (Fig. 6C). Fibronectin was intracellular at days 1-2, with fibril formation by day 7 in all cases. Day 7 analysis found no effect on fiber traits (Fig. 6E-F).

Fig. 6 Enhanced Fibronectin Production Induced by TGF-β3 and Combined TGF-β3 + FGF2 Treatment.

Next, the role of signal transduction pathways in response to combined growth factor treatment was explored. In hPDL cells, compared to the control, the combined treatment significantly boosted p-SMAD3 and p-ERK1/2 levels (Fig. 7A-C). The same was true for hGF cells; their p-SMAD3 and p-ERK1/2 levels also rose with the combined treatment (Fig. 7D-F). ICC results revealed p-SMAD3's nuclear localization (Fig. 7G). Adding the FGF-2 inhibitor BGJ398 to the culture attenuated p-ERK1/2 levels (Fig. 7H-I).

Fig. 7 Both TGF-β3 and FGF-2 pathways are activated simultaneously.

Conclusion

In conclusion, this study firstly fully explored how TGF-β1, TGF-β3, FGF-2, FGF-9 and TGF-β3+FGF-2 combination affected human gingival and periodontal ligament fibroblasts. FGF-2 boosted migration while FGF-9 had no effect. TGF-β1 and TGF-β3 raised α-SMA, and only TGF-β1 induced stress fiber incorporation. The TGF-β3 and FGF-2 combination showed beneficial effects. The study shows growth factors' unique and complementary roles, promising for periodontal soft tissue regeneration.