An insight into the possibilities of fibroblast growth factor in periodontal regeneration.

Periodontitis is caused by bacterial biofilms and is modulated by a variety of risk factors. The periodontal ligament comprises heterogeneous cell populations which are lost in the disease process. A variety of regenerative therapies, such as bone grafts, guided tissue regeneration treatment, application of enamel matrix derivative, have been introduced, with some success in periodontal tissue regeneration. Topical application of recombinant cytokines is now one of the most effective methods to stimulate stem cells. Researchers are now exploring the potential applications and uses of fibroblast growth factor in periodontal regeneration.

INTRODUCTION

Periodontitis is caused by bacterial biofilms and is modulated by a variety of risk factors. No conventional periodontal therapy can regenerate the lost periodontal tissue to a statistically or clinically significant degree. A variety of regenerative therapies, such as bone grafts, guided tissue regeneration treatment, application of enamel matrix derivative, have been introduced, with some success in periodontal tissue regeneration. Numerous issues including technique sensitivity, limitation of indications, as well as the predictability and longevity of outcomes still persist in each method chosen for periodontal tissue regeneration.

The periodontal ligament comprises heterogeneous cell populations. Researchers have predicted the existence of progenitor cells that can differentiate into cementoblasts or osteoblasts.[12] One important question is how to efficiently enhance and ⁄ or induce the biological potential of such cells to regenerate destroyed periodontal tissue. In the 1990s, the concept of tissue engineering emerged with three key elements: Signaling molecules, scaffolds, and stem cells. A physiologically efficient method for stimulating cells is the use of cytokines or growth factors. Researchers have attempted to accelerate the regeneration of periodontal tissue by using topical application of human recombinant cytokines to stimulate the undifferentiated mesenchymal cells into osteoblasts and cementoblasts. Clinical application of cytokines has been enabled by the availability of recombinant products. To date, only cocktails of growth factors have been evaluated for their efficacy in inducing periodontal tissue regeneration in multicenter clinical trials, the platelet-derived growth factor (PDGF) being the most prominent among them. Apart from PDGF, researchers are now exploring the potential applications and uses of fibroblast growth factor (FGF).

FIBROBLAST GROWTH FACTOR

FGF was discovered in 1974 as a protein in cow pituitary glands that strongly induced proliferation of fibroblasts.[3] In 1984, two proteins with different basic and acidic isoelectric points were identified as acidic FGF (aFGF, FGF-1) and basic FGF (bFGF, FGF-2).[45] The proteins have been classified into seven subfamilies: FGF-1 (FGF-1/2), FGF-4 (FGF-4/5/6), FGF-7 (FGF-3/7/10/22), FGF-8 (FGF-8/17/18), FGF-9 (FGF-9/16/20), FGF-11 (FGF-11/12/13/14), and FGF-19 (FGF-19/21/23), based on structural similarities, with each subfamily consisting of two to four types of FGF. Nearly all FGFs transmit signals via receptor-type tyrosine kinase. When FGF binds to a receptor, tyrosine kinase is activated. Following activation, various tyrosine residues on the receptor are phosphorylated, and signal transmission is triggered by the binding of effector proteins to these sites.[6]

FGF signaling also plays an important role in tissue repair and regeneration. FGF-2 induces particularly strong angiogenic activity and proliferative capacity in undifferentiated mesenchymal cells. It is widely expressed in various tissues from fetal stages through to adulthood because it binds to most of the FGF receptors (FGFR)[7] and showers its effects in various cell types. In 2001, FGF-2 was first applied clinically in Japan as a decubitus ulcer medication (Fiblast spray; Kaken Pharmaceutical, Tokyo, Japan). The efficacy of a human recombinant basic FGF (FGF-2) for periodontal tissue regeneration has also been evaluated. At present, a drug containing FGF-2 for periodontal tissue regeneration is not commercially available. However, a series of in vitro and preclinical studies have indicated the efficacy of FGF-2 in periodontal tissue regeneration.[8] In the field of regenerative medicine, the most advanced research among FGFs is being performed on FGF-2.

Medical uses of FGF-2

FGF-2 facilitates reactions that are necessary for revascularization, migration, and proliferation of endothelial cells,[9] and regenerates capillary blood vessels in vivo[10] in the healing of intractable ulcers. Fibroblasts not only produce collagen, but also develop into myofibroblasts and induce so-called wound constriction. It has been reported that when anti–FGF-2 antibodies are administered to normal animals that show normal wound healing, both granulation and wound healing are inhibited.[11]

FGF-2 facilitates fracture healing by promoting the proliferation of marrow-derived mesenchymal cells and inducing their differentiation into osteoblasts. It is thought that FGF-2 promotes the healing of fractures by stimulating both the growth and biochemical functions of mesenchymal stem cells.[12] Pitaru et al.[13] found that after FGF-2 stimulation, cell proliferation occurred and increased amounts of hydroxyproline and proteins were present in the initial stage. This was followed by alkaline phosphatase (ALP) activity, and increased osteocalcin and calcification in later stages of healing of fractures.

Based on the fact that FGF-2 has a powerful angiogenic action, clinical trials are being performed on intermittent claudication in peripheral arterial disease and atherosclerotic peripheral vascular disease.[14] Clinical studies are also being performed on peripheral occlusive arterial disease with FGF-2 gene transfer, and the powerful angiogenesis action of FGF-2 is thought to be one of the most promising treatment factors in therapeutic angiogenesis.

In vitro analyses of effects of FGF-2 in periodontal bioregeneration

FGF-2 acts on various cell types. It promotes proliferation of fibroblasts and osteoblasts, and enhances angiogenesis. These activities are directly associated with periodontal tissue regeneration. However, periodontal ligament cells are the key players during periodontal tissue regeneration. To reveal the molecular and cellular mechanisms by which FGF-2 induces periodontal tissue regeneration, various in vitro experiments were conducted in which the effects of FGF-2 on periodontal ligament cells were examined. Experiments showed that periodontal ligament cells express FGFR1 and FGFR2 mRNA.[15] In contrast, gingival epithelial cells express mRNA of FGFR1, 2, 3, and 4.[15] The responsiveness to FGF-2 is higher in undifferentiated periodontal ligament cells than in mature periodontal ligament cells.

Proliferation and differentiation

It has been demonstrated that FGF-2 enhances the proliferative responses of periodontal ligament cells in a dose-dependent manner. FGF-2 weakly induces the proliferation of gingival epithelial cells in a dose-dependent manner. Also, epithelial downgrowth at FGF-2-treated sites was significantly inhibited compared to control sites. However, co-stimulation with fetal calf serum inhibited FGF-2–induced proliferation of gingival epithelial cells, but synergistically enhanced FGF-2–induced periodontal ligament cell proliferation.[15] This suggests that the biological effects on periodontal ligament cells may be synergistically increased in vivo, as serum components are present, and that FGF-2 acts differently on periodontal ligament cells and gingival epithelial cells in vivo in terms of the proliferative response.

Migration

Cell migration is essential for a wide range of biological events, including embryogenesis, tissue development, wound healing, and tissue regeneration. FGF-2 activated significant migration of periodontal ligament cells. In addition, FGF-2 activated the growth and migration of human dental pulp cells.[16]

Extracellular matrix production

The extracellular matrix provides structural support and anchorage for cells, and regulates a wide range of cellular functions and behaviors. It sequesters growth factors and acts as a local reservoir for them. To create suitable environments for subsequent tissue regeneration, orchestrated regulation of various extracellular matrix products is essential.

Collagen, the main component of the periodontal ligament, plays an important role in supporting the tissues and cells. It also functions as the main bone matrix component. Interestingly, it was demonstrated that FGF-2 downregulates the expression of type I collagen mRNA and production of total collagen.[17] This inhibitory effect is reversible, and is apparently correlated with the temporary inhibition of cytodifferentiation of periodontal ligament cells into hard-tissue forming cells, such as osteoblasts and cementoblasts.

Osteopontin is a protein found in the bone-related matrix. It is detected within the cementum surface and periodontal ligament cells, and is implicated in cementogenesis and homeostasis of periodontal tissues. FGF-2 upregulated the expression of osteopontin in periodontal ligament cells at both mRNA and protein levels and enhanced the concentration of osteopontin in the culture supernatant,[18] but decreased the transcription of genes encoding almost all the bone-related proteins, including osteocalcin, osteonectin, and bone sialoprotein. So, FGF-2 induces expression of osteopontin, which plays a role different from other bone-related proteins during the process of periodontal tissue regeneration by FGF-2.

The extracellular matrix is composed of collagens and non-collagenous proteins such as proteoglycans. Heparan sulfate proteoglycans mediate a variety of physiological responses in development, cell growth, cell migration, and wound healing. Treatment of periodontal ligament cells with FGF-2 for 72 h resulted in a pronounced increase in heparin sulfate levels in the culture supernatant in a dose-dependent manner.[19] FGF-2 had no effect on transcription of genes encoding enzymes associated with heparan sulfate biosynthesis. Interestingly, FGF-2 enhanced the levels of syndecan 4 in culture supernatants from FGF-2–stimulated periodontal ligament cells. These results suggest that the FGF-2–activated increase in the levels of heparan sulfate in conditioned medium may be due to shedding of syndecan 4 from the periodontal ligament cell surface. Taken together, this indicates that FGF-2 may differentially regulate the expression of heparan sulfate proteoglycans in a heparan sulfate proteoglycan subtype-dependent manner. The shed heparan sulfate may be involved in the migration of periodontal ligament cells and enhancement of local wound healing, resulting in periodontal tissue regeneration. In contrast, the production of chondroitin sulfate was barely influenced by FGF-2.[20]

Hyaluronan is a non-sulfated glycosaminoglycan. It plays important roles in homeostasis, cell migration, and inflammatory ⁄ wound healing responses as one of the chief components of the extracellular matrix. FGF-2 significantly increases hyaluronan production in periodontal ligament cells in a dose-dependent manner.[20] FGF-2 promotes the production of hyaluronan with high molecular mass by upregulating hyaluronan synthase expression, resulting in increased biosynthesis. Thus, local production of hyaluronan by FGF-2 stimulation plays important roles in cell migration and the early stages of wound healing, resulting in enhanced periodontal tissue regeneration.

In vivo analyses of effects of FGF-2 on periodontal regeneration

In order to evaluate the activity of topical application of FGF-2 in inducing significant periodontal tissue regeneration, a series of preclinical animal studies was performed. Female beagle dogs and male Macaca fascicularis were used.[2122] Furcation defects were created and filled with gelatinous carrier alone or 0.3% FGF-2 plus gelatinous carrier, and then surgical closure was performed. Periodontal tissue regeneration at the test sites was examined 6 weeks after application of FGF-2 to the defects. In M. fascicularis, the first and second molars (M1 and M2) were utilized. Furcation defects were filled with gelatinous carrier alone, or either 0.1 or 0.4% FGF-2 plus gelatinous carrier, and periodontal regeneration at the test sites was evaluated 8 weeks after FGF-2 application. Local application of FGF-2 significantly enhanced periodontal regeneration in both the beagle dog and non-human primate models compared to control sites. Histological observation showed new cementum with Sharpey's fibers, new functionally oriented periodontal ligament fibers, and new alveolar bone. These data suggest that topical application of FGF-2 is efficacious in the regeneration of human periodontal tissue that has been destroyed by periodontitis. More importantly, there was no evidence of epithelial downgrowth, ankylosis, or root resorption at the FGF-2 sites in any of the in vivo experiments. Furthermore, no severe inflammation or swelling was observed at any of the sites examined throughout the experimental period.

Possible mode of action of FGF-2 to induce periodontal tissue regeneration

A series of in vitro analyses revealed that FGF-2 regulates proliferation, differentiation, migration, and extracellular matrix production of periodontal ligament cells. However, calcified nodule formation and alkaline phosphatase activity in periodontal ligament cells were inhibited in the presence of FGF-2. Furthermore, the suppressive effects of FGF-2 on differentiation are reversible; when FGF-2 is eliminated, the suppressive effects of FGF-2 also dissipate, thus initiating periodontal ligament differentiation. Tracer experiments using radiolabeled FGF-2 showed that topically applied FGF-2 disappeared from the sites in approximately 1 week. This suggests that the effects of topically applied FGF-2 will disappear within 1 week. The effects of FGF-2 on various cellular functions in periodontal ligament cells decreased gradually over the course of periodontal ligament cell culture. Taken together, these data suggest that FGF-2 acts effectively on immature periodontal ligament cells at the early stages of wound healing. Based on these findings, it can be inferred that during the early stages of periodontal tissue regeneration, FGF-2 increases the number of periodontal ligament cells while suppressing differentiation into hard tissue-forming cells such as osteoblasts and cementoblasts. During the subsequent healing processes, probably 1 week after application, when FGF-2 activity disappears at the administration site, periodontal ligament cells begin to differentiate, inducing marked periodontal tissue regeneration. In addition, FGF-2 does not simply induce angiogenesis, an action that is indispensable in the regeneration of tissue, but also increases the production of osteopontin, heparin sulfate, and macromolecular hyaluronan from periodontal ligament cells.[181920] Thus, FGF-2 creates a local environment suitable for regeneration of periodontal tissue through these activities.

Future outlook for FGF-2 therapy

Numerous studies have been performed in order to examine the safety, efficacy, and mechanism of FGF-2–induced periodontal tissue regeneration. With regard to the clinical application of FGF-2, a large trial must be performed in order to confirm the efficacy and safety of FGF-2–based drugs. Preclinical studies should also be performed in order to expand the indications of FGF-2 therapy in the dental field.

For ideal tissue regeneration, it is very important to fully apply the concept of “tissue engineering.” Topical application of FGF-2 significantly induces periodontal tissue regeneration, including osteogenesis and cementogenesis in animal models. It is also noteworthy that no gingival epithelial downgrowth was observed at FGF-2–treated sites without barrier membranes. This suggests that FGF-2 is efficacious for periodontal regeneration of intraosseous bone defects. However, in order to treat severe bony defects or horizontal bone resorption with FGF-2, it is essential to introduce the concept of a “scaffold” into the FGF-2 carrier. An FGF-2 carrier that could provide a moldable and osteoconductive scaffold for undifferentiated cell types would dramatically increase its applications.

Many clinicians are seeking effective treatment procedures that enable resolution of bone volume deficits where implants are placed. Cytokines or growth factors have attracted particular attention in this field. Topical application of FGF-2 may be helpful in inducing bone augmentation and ⁄ or promoting osseointegration of implants. Studies to investigate its efficacy are now in progress.

CONCLUSIONS

The ultimate goal of periodontal therapy is to achieve complete regeneration of the periodontal tissue destroyed by periodontal diseases. Since the 1990s, the knowledge associated with stem cells has advanced and it is now certain that undifferentiated stem cells exist within the periodontal ligament, which allows periodontal regeneration to be stimulated in clinical settings. Also, cytokine therapy may provide a possible solution for the dilemma involved in periodontal tissue regeneration. However, few cytokines have been approved for use in the dental field. This will open further avenues that will provide better understanding of cytokine therapy for periodontal regeneration.