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Article

Fibroblast Growth Factor-2 and Enamel Matrix Derivative Enhance Proliferation, Migration, and Wound Healing in Gingival Epithelial and Fibroblast Cells

by
Nanako Tsuchimochi
1,
Naoki Maruo
1,
Kimiko Ohgi
1,
Hiroaki Yamato
1,
Masanobu Nakagami
1,
Aya Fujioka
1 and
Yasunori Yoshinaga
1,2,*
1
Section of Periodontology, Department of Odontology, Fukuoka Dental College, Fukuoka 814-0175, Japan
2
Oral Medicine Research Center, Fukuoka Dental College, Fukuoka 814-0175, Japan
*
Author to whom correspondence should be addressed.
Medicina 2026, 62(2), 244; https://doi.org/10.3390/medicina62020244
Submission received: 17 December 2025 / Revised: 8 January 2026 / Accepted: 20 January 2026 / Published: 23 January 2026
(This article belongs to the Section Dentistry and Oral Health)
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Abstract

Background and Objectives: Soft-tissue healing, particularly rapid epithelialization, is a critical determinant of successful periodontal regenerative therapy. Fibroblast growth factor-2 (FGF-2) and enamel matrix derivative (EMD) are regenerative biomaterials used clinically. However, their comparative effects on gingival epithelial and fibroblast cell behavior remain unclear. The objective of this study was to examine the effects of FGF-2 on the proliferation, migration, and wound closure dynamics of human gingival epithelial-like cells (Ca9-22) and human gingival fibroblasts (HGF-1) and to compare its effects with those of EMD. Materials and Methods: Ca9-22 and HGF-1 cells were stimulated with FGF-2 (10 µg/mL) or EMD (100 µg/mL) or left unstimulated (control). Wound closure was assessed via scratch assay, migratory capacity via Transwell assay, and proliferation via automated cell counting at pre-defined time points. Results: In Ca9-22 cells, both FGF-2 and EMD significantly accelerated wound closure in a time- and concentration-dependent manner and markedly enhanced cell migration and proliferation compared to controls. EMD consistently induced a stronger migratory response. In HGF-1 cells, FGF-2 significantly advanced wound closure by day 5, whereas EMD induced a non-significant favorable trend. Both treatments significantly increased cell proliferation and migration of HGF-1 cells, with EMD yielding the highest migratory cell count. Conclusions: FGF-2 promotes gingival soft-tissue healing by enhancing epithelial-like cell and fibroblast migration and proliferation, supporting rapid epithelialization. EMD produced comparable wound-healing effects, indicating that the activation of both epithelial and mesenchymal cells is a central mechanism shared by distinct regenerative agents.

1. Introduction

Advances in periodontal regenerative therapy have increasingly enabled the preservation of teeth that would previously have required extraction [1,2]. Among the determinants of successful regeneration, soft-tissue wound healing is essential, and epithelialization plays a particularly critical role in wound closure and infection control [3]. However, tissues affected by periodontitis often exhibit delayed or incomplete healing, highlighting the need for strategies that promote stable soft-tissue repair.
Clinical studies have demonstrated that fibroblast growth factor 2 (FGF-2) formulations yield excellent outcomes in periodontal hard-tissue regeneration, including significant probing depth reduction, clinical attachment gain, and substantial radiographic bone fill [4,5]. These effects are attributed to FGF-2-mediated activation of periodontal ligament-derived cells and osteoblastic precursors that promote regeneration of cementum and alveolar bone [6]. In addition, FGF-2 accelerates wound closure in skin models by stimulating angiogenesis and enhancing epithelial and fibroblast proliferation and migration [7,8]. A previous study reported that locally applied FGF-2 markedly promoted gingival soft-tissue healing after periodontal surgery in rats, mainly through the acceleration of epithelialization [9]. These findings suggest that FGF-2 contributes not only to hard-tissue regeneration but also to early soft-tissue healing; however, the cellular mechanisms underlying its effects on gingival epithelialization remain unclear.
Soft-tissue healing is a tightly regulated cascade consisting of inflammatory, proliferative, and remodeling phases. Successful healing depends on the rapid reestablishment of a protective epithelial barrier, making re-epithelialization the central event in the proliferative phase. Activated basal epithelial cells migrate across the wound surface to restore tissue continuity in close cooperation with the underlying fibroblasts [10]. During proliferation, fibroblasts synthesize extracellular matrix (ECM) components and form granulation tissue that provides a scaffold for epithelial migration [11]. In addition to matrix production, fibroblasts secrete cytokines and growth factors that modulate epithelial cell activity, thereby indirectly influencing proliferation and migration [12,13]. Thus, evaluating both the direct and indirect effects of FGF-2 on epithelial cells and fibroblasts is crucial for understanding its role in promoting soft-tissue healing.
Enamel matrix derivative (EMD) is a porcine-derived enamel matrix protein extract rich in amelogenins, which play a key role in cementogenesis. Basic and clinical research has established that EMD promotes periodontal regeneration by stimulating periodontal ligament cells, cementoblasts, and osteoblasts while modulating inflammation [14]. More recently, EMD has been shown to exert favorable effects on soft-tissue repair, including enhanced angiogenesis, fibroblast migration, collagen synthesis, and accelerated epithelialization [15,16]. These properties suggest that EMD contributes to stable soft-tissue healing following regenerative therapy. However, direct comparisons between EMD and the more recently adopted regenerative agent FGF-2, particularly regarding soft-tissue wound healing and epithelialization, remain limited.
This study sought to examine how FGF-2 formulations influence soft-tissue healing following periodontal surgery, with particular emphasis on epithelialization and intercellular interactions. Using human gingival epithelial-like cells (Ca9-22) and fibroblasts (HGF-1), we evaluated the influence of FGF-2 on three essential cellular functions: wound closure ability, proliferation, and migration. Furthermore, we conducted a comparative analysis with EMD, an established regenerative agent. Through these assessments, this study sought to provide insights into the cellular mechanisms by which FGF-2 enhances soft-tissue healing and contributes foundational knowledge toward optimizing postoperative soft-tissue outcomes in periodontal regenerative therapy.

2. Materials and Methods

2.1. Cell Culture

Ca9-22 and HGF-1 cells were used in this study. Ca9-22 cells were obtained from the RIKEN BioResource Center (Tsukuba, Japan), and HGF-1 cells were obtained from the ATCC (Manassas, VA, USA). Ca9-22 cells were cultured in high-glucose Dulbecco’s modified Eagle medium (Fujifilm, Osaka, Japan), and HGF-1 cells were cultured in Dulbecco’s modified Eagle medium (ATCC), each supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin. Cells were cultured at 37 °C under a humidified 5% CO2 atmosphere.
Cells were grown as monolayers, and the medium was changed every 2–3 days. At 70–80% confluence, the cells were passaged by washing with PBS and treated with 0.5% trypsin-EDTA (no phenol red; Thermo Fisher Scientific, Waltham, MA, USA). After neutralization with complete medium and centrifugation (1500× g for 3 min), the cells were resuspended and seeded at densities appropriate for each experiment (typically 0.05–1.0 × 105 cells/mL).
The concentrations of FGF-2 (10 μg/mL) and EMD (100 μg/mL) used in this study were selected based on previous in vitro studies and clinical relevance [17,18,19]. Specifically, EMD was applied at 100 μg/mL, corresponding to approximately a 300-fold dilution of the commercially available formulation (Emdogain®; Straumann, Basel, Switzerland), a concentration that has been widely used and shown to elicit biological responses in gingival epithelial cells and fibroblasts. To allow a meaningful comparison between the two regenerative agents, the concentration of FGF-2 was similarly adjusted to reflect an approximately 300-fold dilution of the original clinical formulation, resulting in a final concentration of 10 μg/mL.

2.2. Scratch Wound-Healing Assay

Ca9-22 and HGF-1 cells were seeded in 12-well plates (Corning, NY, USA) and grown to near confluence. To minimize the influence of cell proliferation, the cells were preincubated for 24 h in serum-free (0% FBS) medium supplemented with FGF-2 (10 μg/mL) or EMD (100 μg/mL). A linear scratch was made across the monolayer using a 200 µL pipette tip, and the wells were washed with PBS to remove detached cells. The cells were then incubated in serum-free medium with or without FGF-2 (10 µg/mL) or EMD (100 µg/mL).
Phase-contrast images were captured at specific intervals. Ca9-22 cells were evaluated at 0, 6, and 12 h, whereas HGF-1 cells were evaluated at 0 and 5 days. Wound areas were quantified using ImageJ version 1.54 (National Institutes of Health, Bethesda, MD, USA), and closure rates were calculated relative to the initial wound area. Each condition was tested in at least triplicate wells, and all experiments were independently repeated at least three times.

2.3. Transwell Migration Assay

Migration assays were performed with 24-well Transwell inserts containing 8 µm pore membranes (Corning). A total of 5.0 × 104 cells in 500 µL medium, with or without stimulants (FGF-2, 10 µg/mL; EMD, 100 µg/mL), were placed in the upper chamber. The lower chamber contained 750 µL of medium with 10% FBS as a chemoattractant.
Following 24, 48, and 72 h of incubation at 37 °C, any cells that had not migrated and remained on the upper membrane surface were wiped away using a cotton swab. Migrated cells on the lower surface were fixed with 100% methanol and stained with 0.5% crystal violet. The stained cells were evaluated by capturing images from three randomly selected fields under an inverted microscope, and the percentage of the area occupied by migrated cells was quantified using ImageJ software.

2.4. Cell Proliferation Assay

Cells were plated in 35 mm culture dishes at a density of 1 × 104 cells per dish and incubated for 24 h to allow adhesion. After this period, the medium was replaced with fresh medium containing FGF-2 (10 µg/mL), EMD (100 µg/mL), or vehicle as a control. After 24–72 h, the cells were harvested using 0.5% trypsin-EDTA (no phenol red; Thermo Fisher Scientific), centrifuged (1500× g for 3 min), washed with PBS, and mixed 1:1 with Trypan Blue. Total cell numbers were measured by the Countess 3 Automated Cell Counter (Thermo Fisher Scientific). Each condition was analyzed in three to five technical replicates, and the experiments were repeated at least three times independently.

2.5. Statistical Analysis

All experiments were performed using at least three independent biological replicates, each conducted with three to five technical replicates per condition. For the scratch wound-healing and Transwell migration assays, quantitative analyses were performed using mean values obtained from three independent experiments. Cell proliferation assays were conducted with three to five technical replicates per experiment and repeated independently at least three times.
Statistical testing was performed with IBM SPSS Statistics 29.0 (IBM Corp., Armonk, NY, USA). Prior to performing one-way ANOVA, data distributions were assessed for normality using the Shapiro–Wilk test. As the data satisfied normality assumptions, one-way ANOVA followed by Tukey’s post hoc test was applied. Statistical significance was defined as p < 0.05. The results are presented as mean values with corresponding standard deviations.

3. Results

3.1. Effects of FGF-2 and EMD on Cell Migration in Ca9-22 Cells

In Ca9-22 cells, the control group gradually migrated into the scratched area over time. FGF-2 treatment induced a clear concentration- and time-dependent increase in the number of migrating cells, and EMD produced a similar dose- and time-dependent enhancement (Figure 1A). When wound closure was calculated by defining the 0 h wound area as 100%, the 10 μg/mL FGF-2 group and all EMD concentrations showed significantly greater closure at 6 h than the control group. A similar pattern was observed at 12 h, with FGF-2 (10 μg/mL) and all EMD groups exhibiting significantly increased wound closure (Figure 1B).

3.2. Effects of FGF-2 and EMD on Cell Proliferation in Ca9-22 Cells

Cell proliferation was evaluated by counting the Ca9-22 cells after stimulation with FGF-2 or EMD. At 48 h, both treatments significantly increased the cell numbers compared to the control. At 72 h, FGF-2 showed no significant difference, although a mild increase was observed (Figure 2A).

3.3. Effects of FGF-2 and EMD on the Migratory Capacity of Ca9-22 Cells

In the Transwell assay, the control Ca9-22 cells showed a gradual increase in migration over time. Both FGF-2 and EMD enhanced cell migration, with a more pronounced effect in the EMD group (Figure 2B). Quantitative analysis revealed significantly greater migration in both FGF-2- and EMD-treated cells at all time points. Moreover, EMD consistently induced significantly higher migration than FGF-2 (Figure 2C). Taken together, both FGF-2 and EMD consistently enhanced wound closure, migration, and proliferation in Ca9-22 cells across the different assays, with EMD generally exhibiting a stronger migratory effect than FGF-2.

3.4. Effects of FGF-2 and EMD on Cell Migration in HGF-1 Cells

In HGF-1 cells, the control group showed gradual migration by day 5, whereas FGF-2- and EMD-treated cells displayed more extensive movement into the scratched area (Figure 3A). Wound-closure analysis showed that FGF-2 significantly increased closure on day 5 compared with the control. The EMD demonstrated a similar trend, although it was not significant (Figure 3B).

3.5. Effects of FGF-2 and EMD on Cell Proliferation in HGF-1 Cells

Proliferation assays revealed that FGF-2 significantly increased the number of HGF-1 cells at 48 and 72 h. EMD significantly enhanced proliferation at all measured time points and produced significantly greater increases than FGF-2 at 48 and 72 h (Figure 4A).

3.6. Effects of FGF-2 and EMD on the Migratory Capacity of HGF-1 Cells

In the Transwell assay, the control HGF-1 cells showed a time-dependent increase in migration. Both FGF-2 and EMD markedly enhanced cell migration, with EMD showing the strongest effect (Figure 4B). Quantitative analysis confirmed that both agents significantly increased migration at all time points compared with the control, and EMD induced significantly greater migration than FGF-2 (Figure 4C). Overall, these results indicate that both FGF-2 and EMD promote migratory and proliferative responses in HGF-1 cells, although the magnitude and statistical significance of their effects differed depending on the assay and time point.

4. Discussion

In this study, FGF-2 was applied to Ca9-22 gingival epithelial-like cells and HGF-1 human gingival fibroblasts to evaluate its effects on wound closure using a scratch assay. The results indicate that FGF-2 promotes gingival soft-tissue healing by enhancing epithelialization and connective tissue repair, accompanied by increased cell migration and proliferation.
Rather than reiterating the quantitative results presented in the Results section, the following discussion focuses on the biological interpretation and potential mechanisms underlying the observed cellular responses.
Although the present study did not directly investigate intracellular signaling pathways at the molecular level, the observed functional responses are consistent with signaling mechanisms previously reported in the literature. Accordingly, references to ERK/MAPK, PI3K/Akt, JNK, and TGF-β/Smad signaling pathways in this section should be interpreted as literature-based mechanistic considerations rather than direct experimental evidence from the present study.
Both FGF-2 and EMD activated migratory and proliferative responses in gingival epithelial-like cells and fibroblasts. Previous studies have shown that FGF-2 activates the ERK/MAPK and PI3K/Akt pathways, thereby enhancing cell motility [20,21]. Furthermore, activation of ERK1/2 and JNK signaling has been shown to promote fibroblast proliferation [22]. Taken together with the present functional data, these reports suggest that the FGF-2-mediated enhancement of migration and proliferation observed in this study is likely regulated by these known intracellular signaling pathways.
With respect to EMD, previous studies have shown that it induces rapid phosphorylation of MAPK family members and nuclear translocation of Smad2 in oral epithelial-like cells, resulting in altered adhesion and improved migratory behavior [23]. In fibroblasts, EMD elicits biphasic ERK1/2 phosphorylation, and inhibition of ERK signaling abolishes its proliferative effect, indicating that ERK activation is essential for its mitogenic response [24]. Furthermore, studies using palatal fibroblasts have shown that EMD-induced gene expression changes depend on TGF-βRI kinase activity, suggesting that TGF-β-like signaling via Smad2/3 contributes to enhanced ECM production and cytokine expression [25]. These findings support the possibility that similar mechanisms underlie the EMD-mediated enhancement of migration and proliferation in gingival epithelial-like and fibroblast cells in this study.
The finding that FGF-2 enhanced wound closure in both epithelial-like and fibroblast cells has implications for periodontal regenerative therapy. Previous in vivo studies have demonstrated that FGF-2 promotes re-epithelialization following periodontal surgery in rats [9]. Early epithelial closure is essential for protecting the wound surface, reducing infection risk, and providing mechanical stability, all of which contribute to successful regeneration [26,27]. Rapid epithelialization also minimizes bacterial infiltration and wound dehiscence, thereby supporting stable healing around regenerative materials [28,29]. A clinical study reported that local application of FGF-2 increases regenerated tissue volume [4], and the present in vitro results support the cellular mechanisms underlying this clinical outcome. The ability of FGF-2 to simultaneously promote epithelial and connective tissue healing may therefore contribute to wound stability, infection control, and maintenance of the regenerative space following periodontal surgery.
Gingival epithelialization is driven by coordinated epithelial migration and proliferation, supported by fibroblast activity. The activation of epithelial-like cells and fibroblasts observed in this study suggests several mechanisms by which early epithelialization may be accelerated. Enhanced epithelial migration directly promotes re-epithelialization, whereas fibroblast activation contributes to wound-bed maturation through ECM production and remodeling. Fibroblasts also secrete paracrine mediators such as TGF-β, HGF, and FGF-7, which stimulate epithelial proliferation and migration [10], creating a microenvironment conducive to epithelialization. Additionally, epithelial–mesenchymal interactions play a crucial role. For example, fibroblast-derived FGF-7 promotes epithelialization [30], whereas epithelial cells induce fibroblast activation via IL-1 and EGF [31]. Together, these reciprocal interactions likely contribute to the substantial improvement in wound closure induced by FGF-2 and EMD.
This study has several limitations. First, an important limitation is the use of Ca9-22 cells, which are derived from an oral squamous cell carcinoma and therefore do not fully represent normal human gingival epithelial cells. Tumor-derived epithelial cells may exhibit altered proliferative or migratory behavior compared with primary gingival epithelial cells. Nevertheless, Ca9-22 cells retain key epithelial characteristics and have been widely used as a reproducible in vitro model to investigate gingival epithelial migration and wound-healing-related responses in periodontal research [32,33]. Accordingly, the present findings should be interpreted with caution, and future studies using primary human gingival epithelial cells will be necessary to further validate the translational relevance of these results.
Second, this study focused on functional cellular outcomes and did not directly assess intracellular signaling pathways at the molecular level. Although the observed responses are consistent with established ERK/MAPK, PI3K/Akt, FAK, and TGF-β/SMAD signaling mechanisms reported in previous studies, molecular analyses will be required in future investigations to directly confirm the involvement of these pathways and to further strengthen the translational relevance of the present findings.
Although a previous study reported that EMD suppresses epithelial proliferation [17], the present study demonstrated a stimulatory effect. This discrepancy may be partly explained by differences in the types of epithelial cells used, as well as the biphasic, dose-dependent nature of EMD activity, in which lower concentrations are inhibitory and higher concentrations exert stimulatory effects [34,35]. Differences in culture conditions, including serum concentration, extracellular matrix substrates, and incubation periods, may also account for variations among studies. These factors suggest that the stimulatory effect observed in the present study reflects both cell-type-specific sensitivity and the known dose-dependent characteristics of EMD activity.
EMD was used at a concentration of 100 μg/mL, corresponding to a 300-fold dilution of the commercial product Emdogain®, a concentration widely used in previous in vitro studies [17,18,19]. To ensure comparability, the FGF-2 concentration was similarly determined to reflect a 300-fold dilution of the original formulation, resulting in a final concentration of 10 μg/mL.

5. Conclusions

In summary, this study demonstrated that FGF-2 and EMD enhanced wound healing in gingival epithelial-like and fibroblast cells, likely through improved migration and proliferation. These findings suggest that both biomaterials may serve as effective agents to promote soft-tissue healing in periodontal regenerative therapy.

Author Contributions

Conceptualization, Y.Y. and N.T.; methodology, N.T. and H.Y.; software, Y.Y.; validation, N.M. and K.O.; formal analysis, Y.Y.; investigation, N.T., M.N. and A.F.; resources, Y.Y. and K.O.; data curation, N.T. and Y.Y.; writing—original draft preparation, Y.Y. and N.T.; writing—review and editing, K.O., N.M. and H.Y.; visualization, Y.Y. and N.T.; supervision, Y.Y. and K.O.; project administration, Y.Y.; funding acquisition, Y.Y. and K.O. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by JSPS KAKENHI Grants Numbers JP25K21791, JP24K12958, JP23H00442 and JP21K09907, and by the Japan Agency for Medical Research and Development (AMED) under Grant Number JP24ek0410120.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data supporting the present study can be obtained upon reasonable request.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
FGF-2Fibroblast growth factor-2
EMDenamel matrix derivative
Ca9-22human gingival epithelial-like cells
HGF-1human gingival fibroblasts
ECMextracellular matrix
FBSfetal bovine serum
ANOVAanalysis of variance
ERKextracellular signal-regulated kinase
MAPKmitogen-activated protein kinase
PI3K/Aktphosphatidylinositol 3-kinase/protein kinase B
JNKJun N-terminal kinase
Smadsuppressor of mothers against decapentaplegic
TGFtransforming growth factor

References

  1. Cortellini, P.; Stalpers, G.; Mollo, A.; Tonetti, M.S. Periodontal regeneration versus extraction and dental implant or prosthetic replacement of teeth severely compromised by attachment loss to the apex: A randomized controlled clinical trial reporting 10-year outcomes, survival analysis and mean cumulative cost of recurrence. J. Clin. Periodontol. 2020, 47, 768–776. [Google Scholar] [PubMed]
  2. Ng, E.; Tay, J.R.H. Periodontal regeneration of vital poor prognosis teeth with attachment loss involving the root apex: Two cases with up to 5 years follow-up. Dent. J. 2024, 12, 170. [Google Scholar] [CrossRef] [PubMed]
  3. Cho, Y.D.; Kim, K.H.; Lee, Y.M.; Ku, Y.; Seol, Y.J. Periodontal wound healing and tissue regeneration: A narrative review. Pharmaceuticals 2021, 14, 456. [Google Scholar] [CrossRef] [PubMed]
  4. Kitamura, M.; Akamatsu, M.; Machigashira, M.; Hara, Y.; Sakagami, R.; Hirofuji, T.; Hamachi, T.; Maeda, K.; Yokota, M.; Kido, J.; et al. FGF-2 stimulates periodontal regeneration: Results of a multi-center randomized clinical trial. J. Dent. Res. 2011, 90, 35–40. [Google Scholar] [CrossRef]
  5. Harada, R.; Yamashita, K.; Imamura, K.; Inagaki, S.; Saito, A. Periodontal regenerative therapy with recombinant human fibroblast growth factor-2 and autogenous bone graft in generalized aggressive periodontitis: A 1-year case report. Bull. Tokyo Dent. Coll. 2025, 66, 123–132. [Google Scholar] [CrossRef]
  6. Nagayasu-Tanaka, T.; Anzai, J.; Takaki, S.; Shiraishi, N.; Terashima, A.; Asano, T.; Nozaki, T.; Kitamura, M.; Murakami, S. Action mechanism of fibroblast growth factor-2 (FGF-2) in the promotion of periodontal regeneration in beagle dogs. PLoS ONE 2015, 10, e0131870. [Google Scholar] [CrossRef]
  7. Tsuboi, R.; Rifkin, D.B. Recombinant basic fibroblast growth factor stimulates wound healing in healing-impaired db/db mice. J. Exp. Med. 1990, 172, 245–251. [Google Scholar] [CrossRef]
  8. Yun, Y.R.; Won, J.E.; Jeon, E.; Lee, S.; Kang, W.; Jo, H.; Jang, J.H.; Shin, U.S.; Kim, H.W. Fibroblast growth factors: Biology, function, and application for tissue regeneration. J. Tissue Eng. 2010, 2010, 218142. [Google Scholar] [CrossRef]
  9. Yoshinaga, Y.; Maruo, N.; Ohgi, K.; Yamato, H.; Tsuchimochi, N.; Nakagami, M.; Sakagami, R. Recombinant human fibroblast growth factor-2 promotes surgical-wound healing in the rat gingiva. J. Dent. Sci. 2025, 20, 2407–2415. [Google Scholar] [CrossRef]
  10. Werner, S.; Grose, R. Regulation of wound healing by growth factors and cytokines. Physiol. Rev. 2003, 83, 835–870. [Google Scholar] [CrossRef]
  11. Tracy, L.E.; Minasian, R.A.; Caterson, E.J. Extracellular matrix and dermal fibroblast function in the healing wound. Adv. Wound Care 2016, 5, 119–136. [Google Scholar] [CrossRef] [PubMed]
  12. Ahangar, P.; Mills, S.J.; Smith, L.E.; Gronthos, S.; Cowin, A.J. Human gingival fibroblast secretome accelerates wound healing through anti-inflammatory and pro-angiogenic mechanisms. NPJ Regen. Med. 2020, 5, 24. [Google Scholar] [CrossRef]
  13. Jettanacheawchankit, S.; Sasithanasate, S.; Sangvanich, P.; Banlunara, W.; Thunyakitpisal, P. Acemannan stimulates gingival fibroblast proliferation; expressions of keratinocyte growth factor-1, vascular endothelial growth factor, and type I collagen; and wound healing. J. Pharmacol. Sci. 2009, 109, 525–531. [Google Scholar] [CrossRef] [PubMed]
  14. Sculean, A.; Gruber, R.; Bosshardt, D.D. Soft tissue wound healing around teeth and dental implants. J. Clin. Periodontol. 2014, 41, S6–S22. [Google Scholar] [CrossRef] [PubMed]
  15. Yuan, K.; Chen, C.L.; Lin, M.T. Enamel matrix derivative exhibits angiogenic effect in vitro and in a murine model. J. Clin. Periodontol. 2003, 30, 732–738. [Google Scholar] [CrossRef]
  16. Sakoda, K.; Nakajima, Y.; Noguchi, K. Enamel matrix derivative induces production of vascular endothelial cell growth factor in human gingival fibroblasts. Eur. J. Oral Sci. 2012, 120, 513–519. [Google Scholar] [CrossRef]
  17. Kawase, T.; Okuda, K.; Yoshie, H.; Burns, D.M. Cytostatic action of enamel matrix derivative (EMDOGAIN®) on human oral squamous cell carcinoma-derived SCC25 epithelial cells. J. Periodontal Res. 2000, 35, 291–300. [Google Scholar] [CrossRef]
  18. Kawase, T.; Okuda, K.; Momose, M.; Kato, Y.; Yoshie, H.; Burns, D.M. Enamel matrix derivative (EMDOGAIN) rapidly stimulates phosphorylation of the MAP kinase family and nuclear accumulation of smad2 in both oral epithelial and fibroblastic human cells. J. Periodontal Res. 2001, 36, 367–376. [Google Scholar] [CrossRef]
  19. Schlueter, S.R.; Carnes, D.L.; Cochran, D.L. In vitro effects of enamel matrix derivative on microvascular cells. J. Periodontol. 2007, 78, 141–151. [Google Scholar] [CrossRef]
  20. Lau, M.T.; So, W.K.; Leung, P.C.K. Fibroblast growth factor-2 induces E-cadherin downregulation via PI3K/Akt/mTOR and MAPK/ERK signaling in ovarian cancer cells. PLoS ONE 2013, 8, e59083. [Google Scholar] [CrossRef]
  21. Hardy, K.M.; Yatskievych, T.A.; Konieczka, J.H.; Bobbs, A.S.; Antin, P.B. FGF signalling through RAS/MAPK and PI3K pathways regulates cell movement and gene expression in the chicken primitive streak without affecting E-cadherin expression. BMC Dev. Biol. 2011, 11, 20. [Google Scholar] [CrossRef]
  22. Makino, T.; Jinnin, M.; Muchemwa, F.C.; Fukushima, S.; Kogushi-Nishi, H.; Moriya, C.; Igata, T.; Fujisawa, A.; Johno, T.; Ihn, H. Basic fibroblast growth factor stimulates the proliferation of human dermal fibroblasts via the ERK1/2 and JNK pathways. Br. J. Dermatol. 2010, 162, 717–723. [Google Scholar] [CrossRef] [PubMed]
  23. Xiang, C.; Zhang, L.; Tao, E. Research progress of enamel matrix derivative on periodontal tissue regeneration: A narrative review. Front. Dent. Med. 2025, 6, 1611402. [Google Scholar] [CrossRef] [PubMed]
  24. Zeldich, E.; Koren, R.; Nemcovsky, C.; Weinreb, M. Enamel matrix derivative stimulates human gingival fibroblast proliferation via ERK. J. Dent. Res. 2007, 86, 41–46. [Google Scholar] [CrossRef] [PubMed]
  25. Stähli, A.; Bosshardt, D.; Sculean, A.; Gruber, R. Emdogain-regulated gene expression in palatal fibroblasts requires TGF-βRI kinase signaling. PLoS ONE 2014, 9, e105672. [Google Scholar] [CrossRef]
  26. Pastar, I.; Stojadinovic, O.; Yin, N.C.; Ramirez, H.; Nusbaum, A.G.; Sawaya, A.; Patel, S.B.; Khalid, L.; Isseroff, R.R.; Tomic-Canic, M. Epithelialization in wound healing: A comprehensive review. Adv. Wound Care 2014, 3, 445–464. [Google Scholar] [CrossRef]
  27. Sculean, A.; Chapple, I.L.C.; Giannobile, W.V. Wound models for periodontal and bone regeneration: The role of biologic research. Periodontol. 2000 2015, 68, 7–20. [Google Scholar] [CrossRef]
  28. Rojas, M.A.; Marini, L.; Pilloni, A.; Sahrmann, P. Early wound healing outcomes after regenerative periodontal surgery with enamel matrix derivatives or guided tissue regeneration: A systematic review. BMC Oral Health 2019, 19, 76. [Google Scholar] [CrossRef]
  29. Kaner, D.; Soudan, M.; Zhao, H.; Gassmann, G.; Schoenhaueser, A.; Friedmann, A. Early healing events after periodontal surgery: Observations on soft tissue healing, microcirculation, and wound fluid cytokine levels. Int. J. Mol. Sci. 2017, 18, 283. [Google Scholar] [CrossRef]
  30. Werner, S.; Krieg, T.; Smola, H. Keratinocyte–fibroblast interactions in wound healing. J. Investig. Dermatol. 2007, 127, 998–1008. [Google Scholar] [CrossRef]
  31. Aden, N.; Nuttall, A.; Shiwen, X.; de Winter, P.; Leask, A.; Black, C.M.; Denton, C.P.; Abraham, D.J.; Stratton, R.J. Epithelial cells promote fibroblast activation via IL-1α in systemic sclerosis. J. Investig. Dermatol. 2010, 130, 2191–2200. [Google Scholar] [CrossRef]
  32. Imamura, K.; Kokubu, E.; Kita, D.; Ota, K.; Ishihara, K.; Saito, A. Cigarette smoke condensate modulates migration of human gingival epithelial cells and their interactions with Porphyromonas gingivalis. J. Periodontal Res. 2015, 50, 411–421. [Google Scholar] [CrossRef]
  33. Mu, M.; Inoue, H.; Mao, D.; Sougawa, N.; Goda, S. β1 Integrin/FAK signaling regulates interleukin-8 production in human gingival epithelial Ca9-22 cells. J. Oral Biosci. 2025, 67, 100615. [Google Scholar] [CrossRef]
  34. Takahashi, A.; Morita, T.; Murata, K.; Minowa, E.; Jahan, A.; Saito, M.; Tanimura, A. Effects of full-length human amelogenin on the differentiation of dental epithelial cells and osteoblastic cells. Arch. Oral Biol. 2019, 107, 104479. [Google Scholar] [CrossRef]
  35. Alberti, A.; Francetti, L.; Taschieri, S.; Corbella, S. The applications of enamel matrix derivative in implant dentistry: A narrative review. Materials 2021, 14, 3045. [Google Scholar] [CrossRef]
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Figure 1. Scratch wound-healing assay evaluating the effects of FGF-2 and EMD on Ca9-22 cell migration. (A) Representative images of Ca9-22 cells subjected to a scratch wound-healing assay and treated with FGF-2 (1, 5, 10 μg/mL) or EMD (10, 50, 100 μg/mL). Images were captured at 0, 6, and 12 h after scratching. (B) Quantitative analysis of wound closure rates calculated by defining the wound area at 0 h as 100%. Values are expressed as mean ± SD. Significant differences were evaluated using one-way ANOVA followed by Tukey’s post hoc test. A p-value < 0.05 was considered significant. * p < 0.05, ** p < 0.01, *** p < 0.001. Ca9-22, human gingival epithelial-like cells; EMD, enamel matrix derivative; FGF-2, fibroblast growth factor-2.
Figure 1. Scratch wound-healing assay evaluating the effects of FGF-2 and EMD on Ca9-22 cell migration. (A) Representative images of Ca9-22 cells subjected to a scratch wound-healing assay and treated with FGF-2 (1, 5, 10 μg/mL) or EMD (10, 50, 100 μg/mL). Images were captured at 0, 6, and 12 h after scratching. (B) Quantitative analysis of wound closure rates calculated by defining the wound area at 0 h as 100%. Values are expressed as mean ± SD. Significant differences were evaluated using one-way ANOVA followed by Tukey’s post hoc test. A p-value < 0.05 was considered significant. * p < 0.05, ** p < 0.01, *** p < 0.001. Ca9-22, human gingival epithelial-like cells; EMD, enamel matrix derivative; FGF-2, fibroblast growth factor-2.
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Figure 2. Influence of FGF-2 and EMD on Ca9-22 cell proliferation and migration activity. (A) Ca9-22 cells were stimulated with FGF-2 (10 μg/mL), EMD (100 μg/mL), or left unstimulated (control). The cell numbers were quantified at 24, 48, and 72 h using an automated cell counter. (B) Representative images of migrated Ca9-22 cells stained after the Transwell migration assay. Migration was evaluated at 24, 48, and 72 h. (C) Quantitative analysis of the number of migrated Ca9-22 cells. Values are expressed as mean ± SD. Significant differences were evaluated using one-way ANOVA followed by Tukey’s post hoc test. A p-value < 0.05 was considered significant. * p < 0.05, ** p < 0.01, *** p < 0.001. Ca9-22, human gingival epithelial-like cells; EMD, enamel matrix derivative; FGF-2, fibroblast growth factor-2.
Figure 2. Influence of FGF-2 and EMD on Ca9-22 cell proliferation and migration activity. (A) Ca9-22 cells were stimulated with FGF-2 (10 μg/mL), EMD (100 μg/mL), or left unstimulated (control). The cell numbers were quantified at 24, 48, and 72 h using an automated cell counter. (B) Representative images of migrated Ca9-22 cells stained after the Transwell migration assay. Migration was evaluated at 24, 48, and 72 h. (C) Quantitative analysis of the number of migrated Ca9-22 cells. Values are expressed as mean ± SD. Significant differences were evaluated using one-way ANOVA followed by Tukey’s post hoc test. A p-value < 0.05 was considered significant. * p < 0.05, ** p < 0.01, *** p < 0.001. Ca9-22, human gingival epithelial-like cells; EMD, enamel matrix derivative; FGF-2, fibroblast growth factor-2.
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Figure 3. Scratch wound-healing assay evaluating the effects of FGF-2 and EMD on HGF-1 cell migration. (A) Representative images of HGF-1 cells subjected to a scratch assay and treated with FGF-2 (10 μg/mL) or EMD (10 μg/mL). Images were obtained at 0 h and day 5. (B) Quantitative analysis of wound closure rates calculated by defining the wound area at 0 h as 100%. Values are expressed as mean ± SD. Significant differences were evaluated using one-way ANOVA followed by Tukey’s post hoc test. A p-value < 0.05 was considered significant. ** p < 0.01, *** p < 0.001. EMD, enamel matrix derivative; FGF-2, fibroblast growth factor-2; HGF-1, human gingival fibroblasts.
Figure 3. Scratch wound-healing assay evaluating the effects of FGF-2 and EMD on HGF-1 cell migration. (A) Representative images of HGF-1 cells subjected to a scratch assay and treated with FGF-2 (10 μg/mL) or EMD (10 μg/mL). Images were obtained at 0 h and day 5. (B) Quantitative analysis of wound closure rates calculated by defining the wound area at 0 h as 100%. Values are expressed as mean ± SD. Significant differences were evaluated using one-way ANOVA followed by Tukey’s post hoc test. A p-value < 0.05 was considered significant. ** p < 0.01, *** p < 0.001. EMD, enamel matrix derivative; FGF-2, fibroblast growth factor-2; HGF-1, human gingival fibroblasts.
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Figure 4. Influence of FGF-2 and EMD on HGF-1 cell proliferation and migration activity. (A) HGF-1 cells were treated with FGF-2 (10 μg/mL), EMD (100 μg/mL), or left unstimulated (control). The cell numbers were measured at 24, 48, and 72 h using an automated cell counter. (B) Representative images of migrated HGF-1 cells stained after the Transwell migration assay. Migration was evaluated at 24, 48, and 72 h. (C) Quantitative analysis of HGF-1 cell migration at each time point. Values are expressed as mean ± SD. Significant differences were evaluated using one-way ANOVA followed by Tukey’s post hoc test. A p-value < 0.05 was considered significant. * p < 0.05, ** p < 0.01, *** p < 0.001. EMD, enamel matrix derivative; FGF-2, fibroblast growth factor-2; HGF-1, human gingival fibroblasts.
Figure 4. Influence of FGF-2 and EMD on HGF-1 cell proliferation and migration activity. (A) HGF-1 cells were treated with FGF-2 (10 μg/mL), EMD (100 μg/mL), or left unstimulated (control). The cell numbers were measured at 24, 48, and 72 h using an automated cell counter. (B) Representative images of migrated HGF-1 cells stained after the Transwell migration assay. Migration was evaluated at 24, 48, and 72 h. (C) Quantitative analysis of HGF-1 cell migration at each time point. Values are expressed as mean ± SD. Significant differences were evaluated using one-way ANOVA followed by Tukey’s post hoc test. A p-value < 0.05 was considered significant. * p < 0.05, ** p < 0.01, *** p < 0.001. EMD, enamel matrix derivative; FGF-2, fibroblast growth factor-2; HGF-1, human gingival fibroblasts.
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MDPI and ACS Style

Tsuchimochi, N.; Maruo, N.; Ohgi, K.; Yamato, H.; Nakagami, M.; Fujioka, A.; Yoshinaga, Y. Fibroblast Growth Factor-2 and Enamel Matrix Derivative Enhance Proliferation, Migration, and Wound Healing in Gingival Epithelial and Fibroblast Cells. Medicina 2026, 62, 244. https://doi.org/10.3390/medicina62020244

AMA Style

Tsuchimochi N, Maruo N, Ohgi K, Yamato H, Nakagami M, Fujioka A, Yoshinaga Y. Fibroblast Growth Factor-2 and Enamel Matrix Derivative Enhance Proliferation, Migration, and Wound Healing in Gingival Epithelial and Fibroblast Cells. Medicina. 2026; 62(2):244. https://doi.org/10.3390/medicina62020244

Chicago/Turabian Style

Tsuchimochi, Nanako, Naoki Maruo, Kimiko Ohgi, Hiroaki Yamato, Masanobu Nakagami, Aya Fujioka, and Yasunori Yoshinaga. 2026. "Fibroblast Growth Factor-2 and Enamel Matrix Derivative Enhance Proliferation, Migration, and Wound Healing in Gingival Epithelial and Fibroblast Cells" Medicina 62, no. 2: 244. https://doi.org/10.3390/medicina62020244

APA Style

Tsuchimochi, N., Maruo, N., Ohgi, K., Yamato, H., Nakagami, M., Fujioka, A., & Yoshinaga, Y. (2026). Fibroblast Growth Factor-2 and Enamel Matrix Derivative Enhance Proliferation, Migration, and Wound Healing in Gingival Epithelial and Fibroblast Cells. Medicina, 62(2), 244. https://doi.org/10.3390/medicina62020244

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