FormalPara Key Summary Points

This article reviews current knowledge about the therapeutic efficacy of bFGF in wound management and scar prevention, which has been accumulated in Japan over the last 2 decades, including Japanese animal model studies, case reports, clinical studies, and randomized controlled trials.

The application of bFGF immediately after debridement and before grafting can improve the healing of chronic ulcers or second-degree burns and prevent pathological scar development.

bFGF signaling promotes both fibroblast proliferation and migration in wound healing by activating the PI3K/Rac1/JNK pathway and may prevent pathological scar formation by inhibiting TGFβ1/SMAD signaling.

Several bFGF delivery systems are being developed, including controlled-release bFGF formulations such as bFGF gelatin sheets, which provide better healing outcomes. However, further research on these alternative formulations may provide optimal clinical advantages.

Introduction

Wound healing normally is a finely orchestrated multi-phase process that involves a variety of cell types. Studies on wound healing have revealed that dysfunction in some growth factors plays a role in non-healing wounds. These findings have directed advanced bioengineering technology to consider the use of exogenously prepared growth factors and cytokines.

Several growth factors have demonstrated potent efficacy for surgical use such as platelet-derived growth factor (PDGF) [1], vascular endothelial growth factor (VEGF) [2], fibroblast growth factor (FGF), epidermal growth factor (EGF), keratinocyte growth factor (KGF), transforming growth factor beta (TGF-β), and granulocyte–macrophage colony-stimulating factor (GM-CSF) [3, 4].

Preparations that contain recombinant growth factors are available for external use in various formulations, for example, solution, spray, ointment, and gel. However, as summarized in Table 1, the use of these preparations is associated with certain limitations regarding safety and drug delivery systems, which require high doses and/or repeated application, resulting in dire side effects including oncogenesis [5,6,7].

Table 1 Specific growth factors and their roles in clinical applications for promoting wound healing
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In this article, we focus on basic fibroblast growth factor (bFGF), a member of the FGF family of growth factors and signaling proteins. In 1988, Kaken Pharmaceutical Co., Ltd., Tokyo, Japan, obtained exclusive licensing rights to recombinant human bFGF (rhbFGF) Trafermin. This drug was originally developed for patients with decubitus ulcers and skin ulcers.

Following several clinical trials and extensive research and development focusing on rhbFGF for tissue regeneration, in 2001, Kaken Pharmaceutical successfully launched Fiblast®, the world’s first rhbFGF preparation marketed in Japan as a topical spray for accelerating healing of burn wounds and diabetic leg ulcers. During the ensuing 8 years, it was observed that Fiblast® spray potently and safely promoted wound healing and reduced scar formation in the clinical setting throughout Japan. Based on these findings, along with additional clinical research and good safety data, the Clinical Practice Guidelines for Burn Injuries published by the Japanese Society for Burn Injuries in 2009 recommended bFGF as treatment for second-degree burns. Since then, bFGF has become widely used as a treatment modality for wound healing and scar reduction in the clinical setting in Japan.

However, the excellent efficacy of bFGF in wound healing remains largely unrecognized internationally. This may reflect the fact that while other countries have also conducted research on the ability of growth factors to improve wound healing, clinical and basic research in Japan has proactively investigated the role of bFGF in wound healing over the last 20 years and are thus at the forefront of clinical experience and research leading this application.

In North America and Europe, significant attention has been paid and massive efforts have been focused on FGFs to generate FGFR inhibitors for application in the field of oncology [8, 9]. In China, recent advances in bioengineering have led to the production of FGF ligands, revealing their role in enhancing wound repair and regeneration of injured tissue [10,11,12,13].

Simultaneously, the clinical application of FGF ligands in the field of oncology as well as in wound healing has been reported in Japan. Nevertheless, the Japanese clinical experience with FGF ligand products, especially bFGF, has demonstrated a remarkable positive impact in accelerating the process of healing and in the management of chronic wounds such as diabetic foot ulcers. Thus, improving the recognition of the potential clinical application of bFGF in wound healing and tissue repair in the West is necessary to overcome the financial burden of wound management, such as diabetic foot ulcers, and to provide sufficiently improved wound healing.

Therefore, in this article we review current knowledge about the therapeutic value of bFGF in wound management and scar prevention, which has been accumulated in Japan over the last 2 decades. In particular, we review animal studies, case reports, clinical studies, and randomized controlled trials (RCTs) that demonstrate the anti-scarring effects of bFGF in Japan. We also exhaustively assess the mechanism by which bFGF exerts these effects.

Methods

Literature Search and Data Selection

Using the search terms “bFGF OR growth factors AND wound healing in Japan” and “bFGF AND scar prevention in Japan,” all original articles related to bFGF treatment in wound and scar management in Japan were identified by a PubMed search. Eligible papers published between 1988 and December 2019 were retrieved and reviewed. In addition, important reference articles from the included articles were reviewed. Three independent researchers performed the literature search to identify all relevant studies.

Inclusion Criteria

This study included articles that (1) reported on the use of topical bFGF in wound healing management and scar prevention; (2) described the effect of bFGF on these wounds. There were no restrictions with regard to the number of patients in the clinical study or the follow-up duration. The systematic review also aimed to include all in vivo studies that employed topical bFGF-treated models of wound healing and scarring along with all in vitro studies that involved bFGF-treated wounds or pathological scar-derived tissues/cells. The language of the article was not restricted to English.

Exclusion Criteria

The following articles were excluded: (1) articles on the use of bFGF for non-cutaneous wound healing; (2) imperfect literature reviews; (3) conference papers that reported data with unresponsive associated authors; (4) repeated publications.

Statistical Analysis

No formal statistical analysis was performed because the extensive methodological heterogeneity of the articles limited this study to a qualitative analysis. This article is based on previously conducted studies and does not contain any studies with human participants or animals performed by any of the authors.

Results

A total of 208 articles were identified from the search, as shown in the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) flowchart for literature attrition (Fig. 1). After screening by title, 82 were relevant to the application of bFGF for dermal wound healing in Japan. Subsequently, 60 were screened by abstract for adherence to the inclusion criteria.

Fig. 1
figure 1

PRISMA flowchart showing literature attrition

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Finally, a total of 27 full-text articles satisfied all the eligibility criteria and were reviewed in their entirety including: 11 laboratory studies, 7 case reports, 4 clinical studies, and 5 RCTs. The included articles are summarized (Tables 2, 3).

Table 2 Laboratory studies on the role of bFGF in cutaneous wound management and scar prevention
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Table 3 Clinical studies on the role of bFGF in cutaneous wound management and scar prevention
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Discussion

Clinical and Experimental Significance of bFGF

Local bFGF Treatment Accelerates Wound Healing

Wound healing normally begins with hemostasis and then progresses through three overlapping phases driven by biomechanical responses, namely, inflammation, proliferation, and maturation. bFGF, along with other growth factors, plays key roles in wound healing as shown by the significantly slower healing of full-thickness skin wounds in bFGF-knockout mice than in wild-type mice [14]. Moreover, many studies show that external application of bFGF promotes wound healing. For example, a study found greatly improved skin hardness with local bFGF injections following lower limb reconstructive surgery with tissue engineering using a tissue scaffold and secondary split-thickness skin graft [15]. Similarly, increased granulation and accelerated epithelialization were observed after treating rat full-thickness acute incisions with bFGF fused to a fibrin-binding peptide (Kringle1) compared with positive controls. The authors also noted that after subcutaneous implantation, a fibrin scaffold suffused with the Kringle1-bFGF fusion protein efficiently induced local angiogenesis, indicating that this approach was also useful for wounds deficient in plasma clot formation [16].

In addition, bFGF treatment of incisional wounds in diabetic rats was found to improve epithelialization, granulation, and wound-breaking strength to levels observed in non-diabetic mice [17].

Several RCTs have been conducted in Japan to examine the efficacy and safety of bFGF in treating chronic skin ulcers, diabetic ulcers, and venous ulcers. An RCT on the utility of Fiblast® spray to improve healing of ulcers or second-degree burns after debridement and split thickness skin grafting revealed that applying bFGF immediately after debridement and before grafting significantly improved scar quality and graft color match [18, 19].

Furthermore, a first-in-human RCT evaluated the utility of an artificial dermis with sustained-release bFGF to improve healing of chronic skin ulcers. The artificial dermis comprising a collagen/gelatin sponge was designed to release bFGF for > 10 days. The patients were randomized to application of either a low-dose (7 mg/cm2 bFGF impregnation) or high-dose (14 mg/cm2 bFGF impregnation) preparation after wound debridement. The degree of wound improvement was assessed 14 days after the sponge was applied. Of the 17 patients divided into the two low- and high-dose groups, 16 demonstrated significant wound improvement, as indicated by the granulation and epithelialization that reduced the wound area by ≥ 50%. No serious adverse effects were reported [20]. Moreover, a placebo-controlled RCT assessed the ability of topical 0.001% (50 mg) or 0.01% (500 mg) bFGF application for up to 8 weeks to improve healing of non-ischemic diabetic ulcers. The low- and high-dose treatments reduced the wound area by > 75% in 72.3% and 82.2% of the patients, respectively. In contrast, this reduction was only seen in 57.5% of the placebo-treated patients. Thus, bFGF treatment accelerated diabetic ulcer healing [21]. In addition, bFGF treatment was found to improve healing of partial thickness skin burns in two RCTs. The first trial was in adult patients; 119 wounds were treated with topical 1 μg/cm2 bFGF, while 122 wounds received standard care. The bFGF-treated wounds closed on average 3 days earlier than the control wounds. The second trial was in pediatric patients who were treated with the same regimen used in the adults; the 15 treated wounds closed on average 3.7 days before the control wounds [22, 23].

Local bFGF Treatment Prevents Pathological Scar Formation

Scarring is influenced by multiple factors, and this accounts for the wide variations of the different clinical types of scars. These range from typical flat white scars, sunken atrophic scars, and aggressively growing fibroproliferative pathological scars, namely, keloids and hypertrophic scars [24].

These pathological scars often develop following trauma and burn injuries and are quite common; in the US alone about 40 million cases of pathological scars are reported every year [25]. Over the past decade, findings from clinical and animal studies have greatly improved our understanding of the pathogenesis of keloids and hypertrophic scars, particularly that their development and progression is driven by chronic inflammation [26]. This knowledge has facilitated the development of therapies that prevent or reduce pathological scarring and act at least in part by blocking or ameliorating wound/scar inflammation. These include silicone tape, compression garment therapy, steroid injections, laser therapy, cryosurgery, radiotherapy, and surgical excision [26,27,28,29,30]. However, because pathological scars remain difficult to treat, there are continued ongoing research efforts to develop new therapies to prevent their formation. In Japan, studies showing that bFGF accelerates wound healing and smooth epithelialization have led to considerations that local application of bFGF could also prevent the formation of pathological scars in trauma and burn injuries. This hypothesis was supported by a report where immediate rinsing with or injection of bFGF into sutured incisional wounds following resection of skin tumors significantly reduced the likelihood of developing hypertrophic scars or scar widening after healing versus untreated control wounds [31]. Also, repeated bFGF treatment started soon after wounding for formed hypertrophic scars in full-thickness excisional wounds in rabbit ears greatly decreased pathological scarring as shown by significant reductions in scar elevation and epidermal thickness indices [32].

Local bFGF Treatment May Reduce Pre-existing Pathological Scars

Some evidence suggests that bFGF treatment could also reduce pre-existing pathological scars. Eto et al. showed that implanting human hypertrophic scars onto nude mice and treating the scars with a controlled-release bFGF formulation decreased the size of the scars. The hydroxyproline content also decreased, and collagen fiber degradation was observed. Thus, bFGF induced collagen degradation in hypertrophic scars. This observation was supported by the fact that bFGF treatment upregulated matrix metalloproteinase-1 expression in fibroblasts from hypertrophic scars, but not in fibroblasts from normal dermis [33]. Also, there is some evidence that bFGF could induce myofibroblast apoptosis. Funato et al. reported that converting palatal fibroblasts into myofibroblasts followed by treatment with bFGF resulted in apoptosis. Interestingly, they showed that compared to normal palatal fibroblasts, myofibroblasts from palatal scars are more sensitive to bFGF, as shown by the greater tyrosine autophosphorylation of their bFGF receptors [34].

Cellular Mechanism and Signaling Pathways of bFGF

Local bFGF Treatment Accelerates Wound Healing by Several Cellular Mechanisms

The mechanisms underlying these wound healing effects of bFGF reflect the multiple properties of this glycoprotein. Specifically, due to its strong mitogenic properties, bFGF accelerates the division and proliferation of cells in the wound bed, namely, endothelial cells, dermal fibroblasts, and keratinocytes. Moreover, its chemoattractant properties influence the migration of these cells during wound healing, thereby promoting neovascularization and epithelialization. In addition, bFGF attracts leukocytes to the wound, thus helping to initiate the inflammatory phase of wound healing. It also strongly stimulates the production and metabolism of major extracellular macromolecules such as collagen, tropoelastin, and hyaluronic acid by fibroblasts, thereby shaping scar tissue maturation [35].

bFGF Signaling Pathways that Participate in Wound Healing

bFGF is known to efficiently promote fibroblast proliferation and migration [36, 37]. The effects of bFGF on signaling pathways that drive these and other important fibroblast functions in normal and abnormal wound healing remain incompletely understood. However, there are several lines of evidence suggesting that FGFs, and in particular bFGF, activate other signaling pathways in fibroblasts, as follows.

In relation to the signaling initiated in fibroblasts by bFGF exclusively, in vitro and in vivo studies show that bFGF accelerates human fibroblast migration by independently activating the PI3K/Rac1/JNK pathway [38]. This is supported by several studies on cultured human dermal fibroblasts, which show that bFGF treatment induces fibroblast migration by significantly increasing Akt, Rac1, and JNK activity [39, 40]. Moreover, Shi et al. showed that reactive oxygen species (ROS) are a key component in this cascade of events [41]. These findings led to proposing the notion that bFGF promotes the migration of human dermal fibroblasts by binding to and activating FGFR, which stimulates Akt. This drives the phosphorylation of Rac1, which activates both JNK and NOX [41]. Both events then lead to the production of ROS, which activates FAK and paxillin, in turn inducing the cytoskeletal rearrangement needed for fibroblast migration (Fig. 2) [41, 42].

Fig. 2
figure 2

Proposed signaling pathway of bFGF in promoting the migration of human dermal fibroblasts by activating PI3K/Akt-Rac1-FAK-JNK signaling, which induces the cytoskeletal rearrangement needed for fibroblast migration

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bFGF Could Prevent Pathological Scarring by Various Mechanisms

Regulating ECM Synthesis and Degradation

bFGF may prevent excessive dermal deposition of collagen, which plays a crucial role in the development and progression of keloids and hypertrophic scars. This deposition is the result of an imbalance between collagen synthesis and degradation that leads to a disorganized and voluminous fiber structure in the dermis [43, 44]. While collagen deposition is a necessary part of normal wound healing, excessive deposition causes pathological scarring [45, 46]. Because bFGF treatment accelerates the appropriate deposition and distribution of collagen in wounds [47,48,49], the application of bFGF to the injured area soon after wounding may hasten the appropriate development of the ECM in the wound, and this effect of local bFGF may help to prevent the onset of the chronic inflammation that drives pathological scarring.

Inducing Apoptosis of Granulation-Tissue Fibroblasts

bFGF treatment was reported to reduce collagen density, decrease expression of fibronectin, tissue inhibitor of metalloproteinase-1, collagen I, and collagen III, and increase expression of matrix metalloproteinase-1 in ear wounds. bFGF also increased apoptosis of fibroblasts in granulation tissue [32]. This is consistent with a report where bFGF treatment induced apoptosis in granulation-tissue fibroblasts. In contrast, bFGF had no effect on fibroblasts from uninjured dermis, suggesting that bFGF might inhibit scar formation by promoting programmed cell death of fibroblasts in granulation tissue throughout the remodeling stage of wound healing [50].

Interfering with Myofibroblast Formation

bFGF may also prevent pathological scarring by interfering with the formation of myofibroblasts, which is considered the most important mechanism underlying the development of keloids and hypertrophic scars. Myofibroblasts are activated collagen-hypersecreting cells that differentiate terminally from the normally quiescent dermis-derived cells (particularly dermal fibroblasts) into activated collagen-hypersecreting myofibroblasts. This event occurs by a process called endothelial/epithelial to mesenchymal transition (EMT). Formation of myofibroblasts is the most important mechanism underlying the development of keloids and hypertrophic scars [36]. Indeed, Tiede et al. showed that multipotent human dermis-derived progenitor cells tend to differentiate into myofibroblasts in the presence of serum and that co-culture with bFGF inhibits this mesodermal differentiation; bFGF-cocultured cells expressed much lower levels of the myofibroblast marker alpha-smooth muscle actin (α-SMA) than control cells [36]. It is likely that this effect of bFGF is also mediated by the ability of this cytokine to inhibit the TGFβ1/SMAD-dependent pathway, and the study of Shi et al. in hypertrophic rabbit ear showed that topical application of bFGF downregulated α-SMA expression as well as TGFβ1/SMAD signaling [32]. This is believed to be initiated through the expression of miR-20a, which in turn represses the expression of the TGFβ receptor complex (ALK5 and TGFBR2) and SARA, thus inhibiting Smad2 and Smad3 activation, which plays an important role in the development of fibroproliferative disorders (Fig. 3) [51].

Fig. 3
figure 3

bFGF downstream mediators Ras and PI3K activate miR-20a expression, which in turn inhibits the expression of the TGFβ receptor complex (ALK5 and TGFBR2) and SARA, thus blocking the Smad2 and Smad3 activation, which plays an important role in the development of fibroproliferative disorders

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Finally, bFGF may impede pathological scarring by promoting fibroblast proliferation [37, 52]. It may also act by enhancing fibroblast migration. Kanazawa et al. showed that bFGF promoted the migration of fibroblasts when the proliferation of these cells was blocked by mitomycin C treatment [53].

Effect of bFGF on Non-fibroblast Cell Types

The findings described above show that bFGF promotes fibroblast migration and perhaps proliferation, differentiation, and survival of these cells also. It should be noted that the name fibroblast growth factor (FGF) reflects the fact that these molecules were initially known for these effects on fibroblasts. However, it was later shown that FGFs were identical to endothelial cell growth factors [54, 55]. Thus, FGFs do not act specifically on fibroblasts. Moreover, several animal studies showed that bFGF affects not only fibroblast and endothelial cell functions [56, 57], but also influences keratinocyte proliferation and migration [58, 59], nerve regeneration [60], and vascular smooth muscle cell proliferation [61].

Methods of bFGF Delivery for Improving Wound Healing

In Japan, hrbFGF preparation Fiblast® spray has been used as a topical spray to improve wound healing by directly spraying it onto the wound area. This is then followed by applying a standard dressing. Its efficacy in this formulation is demonstrated in a study by Sugamata et al. where the healing period was shortened and good reconstruction of the fingertip shapes was achieved in amputated human fingertips reconstructed with artificial dermis and then treated with Fiblast® spray [62].

Currently, Fiblast® spray is routinely used throughout Japan for negative pressure wound therapy. This combination therapy became available after the Japanese national health insurance system coverage of the use of the V.A.C. ATS® Negative Pressure Wound Therapy System (Kinetic Concepts, Inc., Tokyo, Japan). This widespread use of the Fiblast® spray is emblematic of its considerable effectiveness in wound healing.

Controlled Release of bFGF Is Associated with Increased Efficacy

Free bFGF is water soluble and is rapidly degraded in vivo. Thus, it has a short half-life [63]. Therefore, various formulations have been developed to prevent this degradation. Xiang et al. showed that bFGF-encapsulated liposomes (bFGF-lip) improved healing of deep second-degree burns in rats relative to treatment with blank liposomes [64]. Similarly, another study by the Department of Plastic and Reconstructive Surgery in the Graduate School of Medicine of Kyoto University showed that artificial dermis with incorporated biodegradable gelatin bFGF-containing microspheres when implanted into full-thickness skin defects in guinea pigs accelerated fibroblast proliferation and wound angiogenesis [65]. Further experiments by this group showed that implantation of murine full-thickness skin wounds with bFGF-impregnated collagen/gel sponges greatly accelerated the formation of dermis-like tissue [66]. This group also found that similar treatment of debrided decubitus ulcers in diabetic mice accelerated wound closure, epithelialization, and angiogenesis [67]. Thus, this group began an investigator-initiated first-in-human RCT in 2010 to examine the effects of this approach in patients with persistent ulcers. The findings were reported in 2013 as described previously. Briefly, an artificial dermis consisting of a collagen/gelatin sponge with sustained release of bFGF for > 10 days significantly improved chronic ulcer healing [20].

Another group examined the effect of implanting a controlled-release bFGF-impregnated gelatin hydrogel in mouse incisions. This innovative approach significantly increased neovascularization when subcutaneously implanted in the back of the mice [68, 69]. This early study showed that a gelatin scaffold could release a single growth factor or drug in a controlled fashion, thereby increasing the efficacy of the active agent in wound healing.

Fiblast® Spray vs. bFGF-impregnated Gelatin Sheet

In contrast with studies reporting the effectiveness of bFGF in accelerating wound healing, a study has shown no significant difference between bFGF-chitosan film compared with chitosan film only when applied to surgically created full-thickness wounds in diabetic mice [70]. Additionally, a pilot, randomized, double-blind study compared the topical application of bFGF with placebo in 17 diabetic patients with neurotrophic foot ulcers and reported similar outcomes of bFGF and placebo for healing chronic diabetic ulcer of the foot. Thus, it was hypothesized that using a single growth factor such as bFGF alone might be insufficient for healing of such severe wounds [71].

The effectiveness of the bFGF-impregnated gelatin sheet was compared with conventional spray administered in a murine model for wound healing. Compared with the bFGF spray group, the bFGF-gelatin group demonstrated significantly better wound healing outcomes including faster wound closure, greater area of mature collagen at the early stage of wound healing, and higher vascular density. This suggests that bFGF-impregnated gelatin sheet yields a better acceleration effect on wound healing compared with bFGF spray [72].

There is now considerable evidence showing that bFGF accelerates healing of a wide range of wounds, including pressure ulcers, second degree burns, leg ulcers, and diabetic ulcers, in addition to prevention of scar formation. A large body of research also indicates that bFGF effectively accelerates the process of wound healing and can prevent the development of pathological scars as illustrated in Fig. 4. This is as a result of stimulating the proliferation, differentiation, and migration of fibroblasts and endothelial cells and keratinocytes in the skin, in addition to regulating ECM production and metabolism as previously discussed. These potent effects on key cellular players in wound healing account for the widespread routine use of the commercially available rhbFGF preparation Fiblast® spray to promote wound healing and prevent fibroproliferative scarring in the clinical setting throughout Japan.

Fig. 4
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Illustrative overview of the biological actions of bFGF application in chronic ulcer and burn injury involved in accelerating wound healing and preventing pathological scar development

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This study has some limitations. First, the review was restricted to the Japanese literature and as such may not reflect research findings abroad. Second, the list of keywords used for retrieving articles in the PubMed database search was incomplete and thus may not represent the full range of such studies. Third, the majority of the studies were animal studies and so are not representative of clinical applicability. Hence, further translational research is needed.

Conclusion

In the 2 decades since Fiblast® spray was launched in Japan, several other bFGF delivery systems have been developed, including controlled-release bFGF formulations such as bFGF gelatin sheets. Further research on these alternative formulations is needed to clarify additional clinical advantages, including greater convenience and lower treatment costs, while simultaneously efficiently improving wound healing and reducing the risk of post-surgical scar formation.