Mesenchymal cells as the biological modifiers for stimulation and regulation of cellular and molecular healing and regeneration

Abstract

Biologic modifiers are materials or proteins and factors that have the potential to alter the host tissue so as to stimulate or regulate the wound healing process. Classic examples of biologic modifiers are growth factors. Bioengineered materials are widely utilized due to their biocompatibility and degradability, as well as their moisturizing and antibacterial properties. One field of their application in medicine is to treat wounds by promoting tissue regeneration and improving wound healing. In addition to creating a physical and chemical barrier against primary infection, the mechanical stability of the porous structure of biomaterials provides an extracellular matrix (ECM)-like niche for cells.

Growth factors are polypeptide molecules that control the growth, differentiation, and metabolism of cells during each of the three phases of wound healing. They are present throughout the body in only minute concentrations yet exert a powerful local influence on wound repair. They interact with specific receptors in the cell surface, leading to specific responses determined by the receptor-mediated signal transduction pathways within the target cells. Growth factors (GFs) and cytokines, which are secreted by the cells, are essential parts of the complex process of tissue regeneration and wound healing.

Keywords

Growth factors biomaterials periodontal regeneration

1 Introduction

Periodontal diseases result in destruction of periodontal tissues, including cementum, bone, and periodontal ligament (PDL), with eventual tooth loss if left untreated. Studies targeted at understanding the disease at the cellular and molecular level as well as clinical investigations have resulted in improved therapies for arrest of disease progression. Moreover, beyond arrest of disease progression, substantial evidence exists indicating that regeneration of periodontal tissues is a viable treatment for select situations. There is a need, however, to improve the predictability of regenerative therapies. This need has led to increased efforts, among clinical and basic science researchers, to establish the specific cells, factors, delivery systems, flap design, and host responses required for enhancing the outcome of regenerative therapies.

Although significant advances have been made toward understanding the complexities involved in promoting periodontal regeneration, much remains to be elucidated, including questions regarding placement of growth factors in conjunction with resorbable or non-resorbable membranes directly into the defect. Coating onto the root surface or what cells should these factors be targeted to, and what activities are attractive for promotion and inhibition by these factors.

Biologic modifiers are materials or proteins and factors that have the potential to alter the host tissue so as to stimulate or regulate the wound healing process. Classic examples of biologic modifiers are growth factors. These agents can act through a systemic route (e.g., hormones) or act at the local site (e.g., many polypeptide cytokines and growth factors). Biologic modifiers have the potential to promote regeneration of periodontal tissues (i.e., new bone, new cementum, and new connective tissue attachment) through a variety of cell-tissue interactions, including promoting (1) cell migration, (2) attachment and subsequent spreading of cells at the local site, (3) cell proliferation, (4) cell differentiation, and (5) matrix synthesis [1].

2 Rationale for use in regeneration

The concept that biologic modifiers may serve a role in promoting wound healing is not unique to dentistry. With enhancement in cellular and molecular technologies, great strides have been made in understanding the activities of these modifiers and also in preparing large quantities of recombinant materials.

The interdisciplinary approach to developing new agents and materials for improving tissue function has resulted in substantial progress toward restoring tissues subsequent to disease. In particular, dental procedures rank as one of the most frequent techniques used to enhance tissue deficiencies-” Other areas that rank high are procedures to promote skin healing (e.g., burn patients) and bone procedures. In the development of strategies advancing regeneration of the periodontium, the periodontal field has taken advantage of approaches used for establishing directions to improve regenerative therapies for other tissues. A key factor for enhancing the predictability of regenerative therapies is an understanding of cellular and molecular events required to regenerate periodontal tissues. It is now recognized that an important link, although not exact, to understanding the requirements for regeneration of tissues is to acquire knowledge as to mechanisms involved in development of tissues.

Information gained from studies targeted at understanding the mechanisms and factors controlling development of periodontal structures may prove important for use in regeneration of such tissues, subsequent to disease. For example, data exist suggesting that dental follicle cells (mesenchymal cells surrounding the tooth before root and PDL development) have the capacity to differentiate into osteoblasts, cementoblasts, or PDL cells, when triggered appropriately. Thus, it is possible that factors and proteins identified as required for development of the periodontium may also be operative during periodontal repair and regeneration [1, 2].

Contrasting periodontal development with periodontal regeneration, it is apparent that some common principles exist as well as some concepts that are clearly different between the two processes. Events required for regeneration of periodontal tissues are analogous with those required for normal wound healing and, for the most part, are similar to those events required for development of the periodontium. In contrast to developmental stages, however, in both wound healing and regeneration, the early events include recruitment of marrow cells and clot formation, followed by migration of inflammatory cells and release of cell cytokines and growth factors at the healing sites. During development, it is now recognized that specific growth factors and morphogens trigger differentiation of epithelial and mesenchymal derived cells during tooth formation. The importance of these growth factors (e.g., bone morphogenetic proteins [BMPs]) for regeneration of periodontal tissues as well as the function of endogenous factors present at wound sites is currently being examined in in vivo and in vitro models. Another event considered critical for both development and regeneration of periodontal tissues is attracting appropriate cells to the site of repair or development. Once at the site, the cells must attach to the extracellular matrix of the local environment and become biologically active. That is, such cells must differentiate into osteoblasts, cementoblasts, or PDL cells and lay down the appropriate matrix required for formation of hard and soft connective tissues. To synthesize sufficient matrix, the appropriate cells must be stimulated to proliferate at the local site. Thus, it is reasonable to imagine that many of the molecules involved in triggering development of periodontal tissues may prove to be effective in promoting regeneration of periodontal tissues [2, 3].

3 Basis of action

3.1 Mode of action

The overall scheme of how growth factors act depends on their mode of action. To evoke a biologic effect, a growth factor must be synthesized by an originating cell, travel to its target receptor, interact with the target receptor or binding protein, and activate second messengers or terminal effectors. The mode of action is the way the biologic modifier is meant to interact with its target receptor. Hormones traditionally act in an endocrine manner whereby they are secreted by one cell type and travel in the bloodstream to a distant target cell to exert their actions. Examples of hormones having this type of action are parathyroid hormone, growth hormone, and luteinizing hormone. These factors have the potential for widespread effects because of their circulation in the bloodstream and availability to many different cell types and subsequently are regulated not only by their blood levels, but also by the cells that bear receptors. Local modes of action are more traditionally associated with the term growth factor and involve paracrine, autocrine, juxtacrine, and intracrine modes. Paracrine action involves the production of a factor by one cell, with receptors present on another cell in the local microenvironment. The biologic modifier is secreted from the first cell in a soluble manner and binds to receptors on the target cell to evoke its effects. Examples of this are the growth factors platelet-derived growth factor (PDGF) and transforming growth factor-ß (TGF-ß), which are produced by platelets and act on target cells such as lymphocytes and osteoblasts. Autocrine factors are those that are synthesized by one cell, secreted in a soluble form outside the cell, then bind to surface receptors on the same cell to evoke an effect. Examples of autocrine factors are TGF-∞, which is produced and acts on epithelial cells, and the BMPs, which are produced and act on osteoblastic cells. Less commonly described are juxtacrine effects, which are similar to paracrine effects except that the factor produced by the cell of origin is cell surface bound and requires cell contact by the target cell to evoke a response. An example of juxtacrine mode of action is stem cell factor. Finally, another form of autocrine action is intracrine, whereby a factor is produced by one cell and not secreted but acts intracellularly to facilitate its effects. An example of this mode of action is parathyroid hormone-related protein (PTHrP) in which a portion of the protein has been shown to translocate to the nucleus to inhibit apoptosis. Transcription factors also fit into this category [3 –5].

4 Receptors

For a biologic modifier to exert an effect, its designated receptor must be present in sufficient quantity, orientation, and functional activity to transmit the appropriate stimuli. Growth factor receptors can be divided broadly into two categories: cell surface receptors and intracellular receptors. The most common prototype growth factor receptor is the cell surface receptor, which can be further divided into three main categories: (1) G-protein linked, (2) receptor tyrosine kinases, and (3) serine threonine receptor kinases. Cell surface receptors commonly bind peptide factors that are soluble in water but not easily transported across the lipophilic cell membrane. The intracellular receptors are commonly described for steroids such as vitamin D₃, estrogen, and glucocorticoids. Steroid receptors have been described in both the cytoplasm and the nucleus of target cells. Additionally, intracellular receptors or binding proteins for factors that act in an intracrinc manner are located within the nucleus [6].

Once a cell surface receptor has been bound and activated, a series of second messengers are responsible for taking the next step in evoking a biologic activity. Four main second messengers are outlined. Adenylyl cyclase (AC) is an enzyme activated by G-proteins in the cell membrane in response to activation of G-protein-1 inked receptors such as the parathyroid hormone (PTH)/PTHrP receptor. AC catalyzes the reaction of adenosine triphosphate (ATP) to cyclic adenosine mono-phosphate (cAMP), which activates protein kinase A to cause protein phosphorylation. G-protein-linked receptors also couple to membrane-bound phospholipase C with activation of protein kinase C to evoke protein phosphorylation. The receptor tyrosine kinases and serine threonine kinases are also responsible for phosphorylating their target proteins. Protein phosphorylation is a key component of growth factor activity and is responsible for mediating changes in cell proliferation and differentiation, which are the hallmarks of growth factor activity [1, 6].

5 Cell proliferation

The most fundamental processes of tissue growth and development begin with cell proliferation. Cell growth and division is a prerequisite for regeneration and repair. In cell division, duplication of the cell occurs such that every daughter cell receives an identical copy of genetic material. Cells from different tissues grow and divide at quite different rates. For example, cells of the junctional epithelium typically have a turnover rate of 1 to 6 days, whereas cells of the osteoblast lineage have a turnover rate of 20 to 30 days. Despite this difference, cells undergo a similar pattern of cell cycle events that characterize their process of cell division. There are four main phases of the cell cycle. The S phase is the period in which DNA synthesis occurs. The M phase is the mitotic phase during which the cell is actually dividing. The two G phases represent gap phases G₁ (first gap) and G₂ (second gap) between the S and M phases. If a cell is not actively in the cell cycle (i.e., it is terminally differentiated or at rest), it is considered to be in the G_o phase (exited from the cell cycle). For a cell to reenter the cell cycle from the resting G_o phase and hence initiate cell division, a stimulus designated as a competence factor is required. Competence factors are necessary but not sufficient for the cell to enter into the cell cycle. An example of a competence factor is PDGF. After the cell has been rendered competent to undergo cell division, it requires a progression factor. Progression factors are sufficient once the cells are rendered competent to progress through the cell cycle. An example of a progression factor is insulin like growth factor-1 (IGF-I). Once a cell has progressed to the S phase, it is committed to undergo cell division, although there are growth factors that may act at later stages to delay or block cells in the G₂ phase. Progressing through the cell cycle is an obvious prerequisite for cells to multiply, forming the basis for development and regeneration of tissues. Biologic modifiers are key regulators of this process of cell proliferation via their action at different stages of the cell cycle [6 –8].

6 Cell differentiation

The process of cell differentiation is also a critical component of tissue regeneration. Beyond cell proliferation, differentiation of cells into mature cells bearing the respective phenotypic and functional characteristics composing the tissue type is necessary (e.g., bone, PDL, epithelium). The process of cell differentiation has been outlined for most cell types. For example, cells of mesenchymal origin (fibroblasts, osteoblasts) arise from a common progenitor cell, which is an undifferentiated mesenchymal cell. This cell may progress and differentiate into multiple cell types, including osteoblasts, fibroblasts, adipocytes, or muscle cells with the appropriate signals. Biologic modifiers act in this regard to stimulate or inhibit cell differentiation along the designated pathways. Certain factors may act independently at one or more stages or in concert with other biologic mediators. During development, this process of cell differentiation is exquisitely regulated; hence, the challenge with regeneration is to recreate the appropriate organization of proliferation and differentiation to result in formation of functional tissues.

7 Periodontal wound healing

Periodontal healing and regeneration is complicated because of the large number of cells that participate in the reconstruction: epithelial cells, fibroblasts, osteoblasts, periodontal ligament cells, cementoblasts. We know that regeneration may be limited to the capacity of osteoblasts, and periodontal ligament cells.

Early healing events at the dentogingival interface have been examined using dentin blocks implanted in edentulous alveolar ridges, submerged under gingival flaps in dogs (Wikesjo et al., 1991). In this way, complicating factors such as epithelialization, infection and tensile forces are eliminated [9].

Immediately following wound closure, we see aggregates of red blood cells interspersed in a granular precipitate of plasma proteins. The precipitate adheres to the dentin surface. A few hours later the intercellular matrix is organised with fibrin formation around erythrocyte aggregates. Neutrophils are present and continue to increase. Strands of fibrin become more distinct throughout the clot, binding to the connective tissue as well as to the dentin surface. Degradation of erythrocytes starts. Seven days later we see a cell-rich granulation tissue with fibroblasts and young collagen fibers. Collagenous elements appear parallel to the dentin surface. Resorptive remodeling of the dentin surface may be evident. Within 14 days newly formed collagen fibers may show an arrangement to the dentin. Cementum formation maybe initiated, but is not seen before 3 weeks after wound closure. A functional fiber arrangement followed by cementum formation does not always occur. It was found that 4 months after regenerative therapy a 0.1 to 2.3 mm wide zone of connective tissue adhesion is regularly present between the junctional epithelium and the most coronal extension of newly formed cementum.

Schuppbach and coworkers (1993) studied the healing events under light scanning and electronic microscopy with the concept of guided tissue regeneration in dogs, and they saw that regeneration only occurs in parts of the roots where the original cementum remains [10]. Repair is seen when the layer of peripheral dentin is removed. In the regenerated sites, new collagen fibrils, synthesized by fibroblasts and oriented perpendicular to the root surface, are spliced with spliced ends of Sharpey’s fiber bundles of original cementum. In the repaired sites, where circumpulpal dentin is exposed, intermingling between fibrils and dentinal matrix fibrils occurs. In the regenerated areas (base of the defect) the first event is always the formation of cementoid by a cementoblast monolayer, with subsequent formation of intrinsic fibrils oriented parallel to the root surface. Afterwards the cementoblast monolayer disintegrates and extrinsic fiber bundles become anchored in the new cellular mixed fiber cementum. Linkage between the new cementum and pre-existing tissues always occurs by interfacial intermingling of the fibrils, regardless of whether new attachment occurred at circumpulpal dentin or original cementum.

Graves & Cochran, 1994 studied the periodontal regeneration, endogenous sources of growth factors may exist. These sources can be inflammatory cells (activated leucocytes-macrophages) endothelial cells, osteoblasts, periodontal ligament cells or cementoblasts, factors that are stored in bone and released during bone resorption and factors that have been previously produced and released from binding proteins by proteolysis or decrease in pH [11]. It was suggested that both bone and periodontal ligament tissue compartments supply cells to a periodontal wound containing a surgically created space. Moreover, some of the cells which migrate into the potential space formed by a physical barrier continue to prepare for mitosis as they migrate.

Aukhil & Iglhaut (1988) analyzed the kinetics in the periodontal ligament following experimental regeneration in monkeys and saw a very limited zone of the periodontal ligament apical to the wound that acts as a source for cells migrating into the wound. There appears to be only one peak of activity in the premitotic label uptake by cells in the periodontal ligament, so that the eventual population of the curetted root surfaces by cells in the periodontal ligament may depend on the continued amplifying divisions of these cells [12].

Nojima et al., 1990 studied the periodontal ligament-derived cells have mainly been characterized in vitro. These fibroblast cells have some osteoblast-like characteristics and phenotypes that can differentiate into osteoblasts or cementoblasts. These cells can proliferate, migrate and differentiate [13]. Cell proliferation is a sine qua non in tissue repair. There are three groups of cells according to their mitotic activity. The first group contains the permanent cells. These cells become permanently differentiated after their mitosis (for example, neurons). The second group contains the labile cells (for example, epithelial cells), which can undergo continuous replication even under normal physiological conditions. The third group contains the stable cells that have a decrease or loss of mitosis in adults, but can reenter the cell cycle stimulated by the loss of other cells. Cell differentiation occurs in different stages. Growth factors may play an important role in regulating the cell migration, proliferation and differentiation. Growth factors can influence the cell metabolism in various ways. Certain growth factors act primarily as competency factors, which move cells out of their resting phase into mitosis (G₀ phase), whereas other factors act as progression factors, which stimulate DNA synthesis and completion of the cell cycle (G1 to S phase).

Out of these findings it may be clear that growth factors could play an important role in regeneration of the periodontal tissues to stimulate the migration, proliferation and differentiation of the important cells that have the capacity to regenerate the tissues. Migration and proliferation of the periodontal ligament fibroblasts and synthesis of the matrix components by the cells are needed for the repair of the periodontal ligament in the early phases, while the differentiation of cementoblasts and osteoblasts is needed for the formation of new cementum and new bone in the later phases of the periodontal regeneration. Many of these factors are stored in bone; various growth factors could therefore contribute to the osteoinductive effect of exogenous bone grafts. Maybe regeneration until now has been limited because of the restricted capacity of cells to migrate in the periodontal wound. It is also possible that growth factors should be combined with certain extracellular matrix components (fibronectin and laminin) or with other growth factors (for example, competence growth factors with progression growth factors) to have an optimal chemotactical function.

The healing patterns of guided bone regeneration differ from those of periodontal regeneration. The healing pattern the bone matrix was a rich source for growth factors. Bone regeneration in membrane protected sites followed the pattern of bone development and growth. The first stage corresponded to intramembraneous ossification and resulted in the formation of primary spongiosa. In the second stage, this scaffold was reinforced by parallel fibered and lamellar bone. Simultaneously, a cortical layer and the secondary spongiosa were formed. The third stage was characterized by cancellous and cortical bone remodeling. Bone healing was not completed within the 4-month time frame.

8 Polypeptide growth factors

Graves & Cochran, 1994; in addition to the extracellular matrix, found that there is another class of biological response modifiers, the polypeptide growth factors. Cytokines are a group of multifunctional polypeptides and glycoproteins that are secreted by one or several types of cells and act locally or systemically. Included in this cytokine molecule group are the growth factors. Growth factors are polypeptide hormones that stimulate a wide variety of cellular events, including chemotaxis, proliferation, differentiation and production of extracellular matrix proteins. Growth factors are natural cell products; they cannot diffuse across a cell membrane, and must act by binding high-affinity cell membrane receptors. Their production is tightly regulated in normal cells; unregulated production can occur for example in proliferative disorders. These factors can stimulate the cell that synthesises the molecule (autocrine stimulation) or affect nearby cells (paracrine stimulation) [11]. The term growth factor may be somewhat misleading, since it is known that these compounds can display both stimulatory and inhibitory activities in vitro, even within the same cell type. The resulting cell response may then depend on the presence of other cytokines, the state of cell activation and the degree of cell differentiation.

Freymiller and Aghaloo 2004 found that platelet-rich plasma introduced in oral and maxillofacial surgery as an autologous concentration of platelets, that contained a number of important growth factors such as platelet derived growth factor (PGDF), transforming growth factor-∞, ß, insulin-like growth factor (IGF), epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF). Additionally, PRP also contains proteins (i.e fibrin, fibronectin, vitronectin) known to act as cell adhesion molecules for osteoconduction and as a matrix for bone, connective tissue and epithelial migration. The use of PRP is based on the premise that the large number of platelets in PRP release significant qualities of growth factors that promote chemotaxis of precursor cells, cell mitosis, collagen production, initiating vascular in-growth and inducing cell differentiation [14].

9 Platelet-derived growth factor

Platelet-derived growth factor is a well-characterized protein. There are two different PDGF polypeptides that are 56% homologous and encoded by different genes. It has been found to exist in homo-dimer forms (PDGF-AA, PDGF-BB) as well as in heterodimer form (PDGF-AB). There are two different PDGF receptors: the PDGF- receptor (binds PDGF-AA, PDGF-BB and PDGF-AB) and the PDGF-ß receptor (binds PDGF-BB and PDGF-AB). The capacity of certain cells to respond to these PDGFs depends on the presence of these specific ∞ or ß receptors on the cells (Graves & Cochran, 1990) [11].

It has been demonstrated in rats that fibroblastic cells have both ∞ and ß types of PDGF receptors. PDGF has been isolated from a variety of cells and tissues, including monocytes and macrophages, fibroblasts, endothelial cells and bone matrix. PDGF stimulates cells of mesenchymal origin, such as fibroblasts, glia, smooth muscle and bone cells. PDGF has been identified as a competence growth factor and acts synergistically with progression growth factors, such as the insulin growth factors. PDGF, however, also acts as a paracrine factor by stimulating certain cells to produce their own progression growth factors.

PDGF-BB is the most potent stimulator of mitogenesis, followed by PDGF-AA and -AB. PDGF-BB is twice as potent as PDGF-AA as a chemoattractant for connective tissue cells, and PDGF-AB increases collagen synthesis of periodontal ligament fibroblastic cells [14 –16].

9.1 In vitro studies

Human osteoblasts seem to have a large number of PDGF receptors and respond to PDGF-AA and PDGF-BB. PDGF stimulates mitogenic activity and chemotaxis in osteoblasts. In vitro studies show that PDGF-AA but not PDGF-BB is produced by osteoblastic cells. It was demonstrated that PDGF stimulates the proliferation of cells from the periodontal ligament. The growth rate is dependent on the phenotype of the periodontal ligament cells; osteoblastic phenotypes proliferate higher than fibroblastic phenotypes.

Matsuda et al. (1992) reported also that PDGF has a mitogenic effect on periodontal ligament fibroblastic cells. These cells show a strong chemotactic response to PDGF. PDGF-AB stimulates collagen synthesis and PDGF-BB stimulates proliferation and chemotaxis [18].

PDGF-AA and -BB are major mitogens for human periodontal ligament cells. PDGF stimulates more the proliferation from periodontal ligament cells than from gingival fibroblasts. It was showed that the proliferative responses of PDGF-BB are inhibited by bradykinin in cells of the periodontium. It is important to be aware of the fact that the in vitro effects of growth factors may not always be the same in vivo. Indeed, in vivo there are still a lot of other mediators present that can have inhibitory effects on growth factors in a periodontal lesion. The complex environment in vivo will make it impossible to determine the specific effects of growth factors on various cells involved in the wound healingprocess.

9.2 In vivo studies

Wang et al. (2016) discussed in an animal study in 4 mongrel dogs where fenestration defects were created, the possible stimulating effect of PDGF on proliferation of fibroblasts. In this study there was a control group in which only polytetrafluoroethylene (s-PTFE) barriers were used, a group where only PDGF (as a solution) was used and a group where PDGF was used in combination with an e-PTFE barrier. Their results indicated that PDGF enhanced fibroblast proliferation when compared to the groups without PDGF. The authors did not see significant differences in fibroblast proliferation between the group where PDGF was used alone and the group where PDGF was used in combination with an e-PTFE barrier. This may be explained by the fact that in the group without a membrane, the fibroblast proliferation of cells coming from the gingival connective tissue was also measured [19].

Experimentally created horizontal class III defects were treated with PDGF-BB (solution) and GTR after the roots had been demineralized using citric acid. Antibiotic treatment with Penicillin G benzathine was given. There was also a control group where GTR was used alone. The authors found after 8 weeks that the defects treated with the combination therapy showed 80% new bone and 20% new periodontal ligament. At the sites where only GTR therapy was used, 26% of the repairing area was still empty space and epithelium and 31 % was inflamed tissue. After 11 weeks these GTR-treated areas improved in repair, showing only 4.3% epithelium, 9.4% inflamed tissue, the rest being 12% new connective tissue, 60% bone and 14% periodontal ligament. They concluded that PDGF-BB modulated therapy successfully promotes full periodontal regeneration more rapidly and effectively compared to GTR alone.

10 Fibroblast growth factor

There are 7 forms of fibroblast growth factors (FGF). Two are well described, one is basic (p-FGF), the other acidic (a-FGF).The two fibroblast growth factors are products of different genes but are similar in structure and function. These factors are potent mitogens and chemoattractive for endotheli-al cells and a variety of mesenchymal cells, including fibroblasts, osteoblasts, chondro-cytes, smooth muscle cells and skeletal myoblasts. They are believed to act as competence growth factors and probably need the synergistic action with progression growth factors to maximize DNA synthesis and growth. These factors seem to have important angiogenic effects in vivo. FGF binds tightly to heparan, a major constituent of the extracellular matrix. a-FGF and p-FGF are stored in the bone matrix and may be important factors for the regulation of osteoblastic cells.

10.1 In vitro studies

P-FGF stimulates human endothelial and periodontal ligament cell migration and proliferation, and that the combination with an attachment protein, fibronectin, further enhances periodontal ligament cell chemo-taxis. They reported that p-FGF binds to native dentin and that this binding is increased when the dentin is pre-conditioned with tetracycline HCI or citric acid (exposure of type I collagen). It was showed that p-FGF induces endothelial cell migration and proliferation as well as the formation of capillary-like tubular structures on type I collagen stroma on dentin surfaces [20].

10.2 In vivo studies

Eppley et al. (1991) examined the effects of autografts with or without |3-FGF placed on irradiated bone resection sites in the mandible of animals. In the treated sites the healing and reestablishment of the mandible contour in half the animals was observed, as well as bone formation at the cortical margins adjacent to the growth factor recipient sites. Nontreated sites resulted in sequestration, necrosis and failure to heal [21].

At one side the implants were loaded with lyophilized (3-FGF. The bone ingrowth after 5 months was seen in the whole block, regardless of whether or not the scaffolds were loaded with p-FGF. No further in vivo studies concerning periodontal applications with FGF are found.

10.3 Insulin-like growth factors

The insulin-like growth factors (IGF) are a family of single-chain serum proteins that share 49% homology in sequence with proinsulin. IGF-I and IGF-II are two poly-peptides from this group that have been well described. They are synthesised by multiple tissues, including liver, smooth muscle and placenta, and are carried in plasma as a complex with specific binding proteins. These binding proteins may positively or negatively affect the biological activities of IGF. IGF and their receptors can also be locally produced by osteoblasts. IGF have a similar spectrum of activities to those of insulin. IGF-I acts as a progression factor. IGF have been shown to stimulate bone formation and to have an effect on periodontal ligament cells. It is believed that PDGF and IGF-I have a synergistic effect and that IGF-I alone does not enhance bone repair.

10.4 In vitro studies

IGF-I stimulates the formation of bone matrix in fetal rat calvariae in organ culture. They concluded that IGF-I stimulates bone formation by inducing cellular proliferation and the secretion of extracellular matrix components. Locally, IGF-I and -II are produced by osteoblasts. IGF-I has a mitogenic effect on fibroblasts originating from various connective tissues and cell lines, and that IGF-I can stimulate the DNA synthesis of PDL fibroblasts, likely via binding to high-affinity cell surface receptors. The effect of IGF-I is not enhanced by adding growth hormones, and no effects on the morphology or growth pattern of the fibroblast cells is seen.

Matsuda et al. (1992) demonstrated that IGF-I has mitogenic effects on PDL fibro-blastic cells. They showed that a synergistic effect results from using a combination of PDGF-AB and IGF-I. IGF-I is chemoattractive to periodontal ligament cells [19, 20].

10.5 In vivo studies

The effect of the combination of PDGF and IGF-I in 3 beagle dogs and found a horizontally naturally occurring bone loss in the dogs. An aqueous gel containing PDGF and IGF was applied at the test sites, while a gel without these substances was applied at the control sites. The authors saw a long junctional epithelium and no new bone or cementum formation at the control sites, whereas the sites treated with growth factors showed significant amounts of new bone and cementum formation. The combination of PDGF-B and IGF-I in 13 beagle dogs in which periodontitis occurred naturally. The PDGF-B and IGF-I were applied in a methylcellulose gel at the test site; at the control site only the gel was applied. The authors demonstrated that the short-term application of this combination can significantly enhance the formation of the periodontal attachment apparatus during the early phases of wound healing following surgery.

The implants were installed in extraction sites that healed for nine months. At 7 days they saw that the percentage of bone fill in the peri-implant spaces and the percentage of implant surface in contact with new bone were both significantly increased in the PDGF-B/IGF-I treated sites. At 21 days they saw the same increase. The authors suggested that the PDGF-B/IGF-I combination can stimulate bone regeneration around titanium implants. The effect appears to be prolonged in areas where large bone defects exist, as in the periimplant marrow spaces.

Giannobile et al. (1994) used beagle dogs with natural periodontal disease and non-human primates with ligature-induced periodontitis to evaluate the healing response to periodontal surgery with and without the concurrent use of the combination of PDGF and IGF-I (solution). At 1 month they saw an increase in new attachment formation of 64.1% and 51.4% in the nonhuman primate and the canine, respectively, while the controls showed 34.1% and 8.6% increase in new attachment. Osseous defect fill was seen in 21.6% and 65% of the test sites of the nonhuman primate and canine, respectively, while controls demonstrated 8.5% and 14.5% osseous defect fill in the nonhuman primate and canine, respectively [22].

11 Epidermal growth factor

Epidermal growth factor (EGF) is a single-chain protein. The major sources of EGF are urine and salivary glands, although it also has been isolated from Brunner’s glands and platelets as well as from cerebrospinal and amniotic fluids. EGF stimulates DNA synthesis and cell growth in a large variety of cells, including those of epithelial, endothelial and mesodermal origin. However, EGF stimulates prostaglandin production and induces bone resorption in cultures of neonatal mouse calvaria.

11.1 In vitro studies

Matsuda et al. (1992) saw in vitro that EGF induces moderate mitogenic responses in the fibroblastic cells of the periodontal ligament. EGF only slightly increases the chemotactic effects on these cells, and suppresses collagen synthesis [18]. Although receptors for EGF on periodontal ligament fibroblasts, EGF does not show significant mitogenic and chemotactic effects on PDL fibroblasts in vitro. The up regulation of EGF receptors on PDL fibroblasts is associated with the maintaining of these cells, in an un-differentiated state, while the down regulation of EGF receptors is related to the differentiation of cells into osteoblasts or cementoblasts. EGF may have clinical value in controlling ankylosis.

12 Transforming growth factor

Transforming growth factor (3 is a member of a large family of biologically active protein hormones that are structurally related but differ markedly in their function. TGF-|3 consists of 2 subunits held together by covalent bonds. Five different genes have been identified that encode TGF-ß poly-peptides. An inactive domain of TGF- ß must be removed before TGF- ß is biologically active. It is activated by proteolysis and low pH. Most of the cells express at least one of the TGF- ß genes. It is found in high concentrations in platelets and in bone. TGF- ß appears to be a major regulator of cell replication and differentiation. It can stimulate or inhibit cell growth. It can modulate other growth factors such as PDGF, EGF, and FGF. It inhibits epithelial cell proliferation and stimulates mesenchymal cells. It stimulates fibroblast chemotaxis and proliferation and induces extracellular matrix production. It has stimulatory and inhibitory effects on osteoblast proliferation. In vitro studies [1 , 17].

Matsuda et al. (1992) saw that TGF- ß induces inhibitory effects on mitogenic responses of the periodontal ligament cells. It reveals no chemotactic effect on these cells; however, TGF ß - stimulates the collagen synthesis [18].

12.1 In vivo studies

The effects of topical application of a combination of IGF-II, ß -FGF and TGF-ß 1 in a collagen sponge to treat experimentally created fenestration defects in 4 beagle dogs. At the control sites only collagen sponges as carriers were are placed. Histometric analysis showed no differences in fibroblast and collagen density between control and growth factor defects up to 14 days after surgery. Bone regeneration was significantly greater in control than in growth factor defects. This exemplifies how delicate it is to achieve a clinically efficient combination of growth factors.

12.2 Cementum-derived growth factor

A new growth factor, cementum-derived growth factor (CGF), in the cementum. Although it is similar to PDGF, it has a different molecular weight and electrophoretic mobility after reduction. In addition, monoclonal antibodies to CGF do not recognize PDGF. It is a mitogen distinctly different from PDGF. The cementum still contains other growth factors such as ß -FGF and other weak binding growth factors.

CGF is mitogenic to gingival, periodontal ligament and skin fibroblasts and to bovine and human aortic smooth muscle cells. The presence of CGF and other growth factors in cementum indicates that these substances influence the cells present in cementum and or adjacent periodontal ligament, gingiva and dentin. Interestingly, mitogenic activity appears to be absent, or present only in low concentrations in structures which are in contact with cementum. These observations indicate that cementum has the potential to regulate the metabolism and turnover of surrounding tissues and that it serves as a storage site for these molecules which are liberated from the matrix during inflammation. The CGF and other cementum components could thus play a key role in the formation of connective tissue and restoration of its attachment to tooth root surface previously exposed to disease (Narayanan et al., 1993).

13 Periodontal ligament-derived growth factor

A novel chemotactic factor isolated from human periodontal ligament cells, named PDL-CTX. PDL-CTX induces the direct migration of human PDL cells in vitro and is found to be a more potent chemotactic agent than other known growth factors. Additionally, PDL-CTX has no chemotactic effect on gingival fibroblasts or epithelial cells.

14 Vascular-endothelial growth factor (VEGF)

VEGF has been recently assayed from the GCF of a cross-section of periodontal patients and healthy control subjects. Higher levels of VEGF were found in diseased sites than in healthy sites. In addition to its role in the regulation of angiogenesis VEGF has also been recently suggested to play an important role in the regulation of bone remodeling by attracting endothelial cells and osteoclasts and by stimulating osteoblast differentiation. Its possible contribution to periodontal disease progression was supported by a recent study reporting higher VEGF concentrations in diseased gingiva adjacent to 4– 6-mm pockets. Johnson RB et al 1999 speculated that VEGF may be an important factor in the progression of gingivitis to periodontitis through its role in promoting the expansion of the vascular network observed in inflammation [20 –22].

15 Future perspectives

Growth factors can be provided in large quantities using the recombinant techniques. Further use of these factors in in vivo studies is possible. There is a lot of hope that these growth factors can enhance periodontal regeneration. However, healing of a periodontal lesion is a complex process. Variables such as inflammation, smoking, systemic diseases and others can influence the effect of growth factors. Probably the growth factors need to be combined in a certain sequence for delivery and combinations of growth factors are needed. The concentrations used of the growth factor will be important, as will the delivery system. Thus far, we can conclude from animal; studies that the combined use of growth factors and the guided tissue regeneration technique gives good results and that PDGF and IGF are important growth factors for periodontal regeneration. We do not yet know the impact of certain novel growth: factors on periodontal healing in vivo.

16 Bone morphogenetic proteins

Bone morphogenetic proteins (BMPs) are osteoinductive factors that may have the potential to stimulate bone repair. Unlike the growth factors described above, the main action of BMPs is to stimulate undifferentiated pluripotential cells to differentiate into cartilage or bone-forming cells. In general these factors do not stimulate proliferation and migration of osteoblasts. It appears there are at least 9 BMP-like molecules BMP-1, BMP-2, BMP-3 (osteogenin), BMP 4, etc. Another example is the osteoinductive protein (OP 1). BMPs 2 to 7 are related structurally to TGF- ß, although the latter does not act like BMPs. Osteoinductive proteins were first isolated from demineralized bone matrix. They can be produced by osteoblasts and stored in bone. Since both growth factor and BMPs are found within bone matrix, it is possible that demineralized freeze-dried bone contains small amounts of these proteins. This may explain the bone-stimulating activity of freeze-dried bone allografts. When partially purified BMP from demineralize bone matrix is implanted intraperitoneal into rats in a bone-derived collagenous matrix, it stimulates formation of a nodule cartilage that undergoes remodeling into bone within the surrounding soft connective tissues (i.e. ectopic bone formation) [23 –25].

The primary action of BMPs is to differentiate mesenchymal precursor cells into cartilage and bone-forming cells. BMP-2 induces differentiation of osteoblasts with the capacity to produce bone matrix proteins. BMP-3 (osteogenin) induces cartilage formation preferentially to bone formation. BMPs 5, 6 and 7 augment the capacity of BMP-2 to form bone in vivo, although individually they possess little bone-forming activity. Because of this mode of action, rh BMP-2 heals bony defects in a manner different from the other growth factors which have been used to increase bone mass. TGF- ß, IGF and FGF all affect the already differentiated or committed bone-forming cells present in the bone, causing them to divide and/or increase secretion of the extracellular matrix molecules. By affecting the cells of the bone itself, they have somewhat limited capacity for regeneration. On the other hand, BMP-2 will influence the precursor cells from the marrow environment and the soft tissue surrounding the defect site to infiltrate the defect area and differentiate into cartilage and bone cells. It can thus regenerate an unlimited amount of bone when implanted in soft tissue sites. It is known that BMPs bind to type IV collagen of the extracellular matrix. The biological actions of BMPs are possibly regulated by a complex interaction with extracellular matrix components. Type IV collagen may function as a delivery system by sequestering both initiators and promotors involved in endochondral bone differentiation.

17 BMP in periodontal regeneration

It may be reasonable to expect that BMPs also play a role in periodontal regeneration. Osteogenin (BMP-3) augments new bone and cementum deposition around submerged teeth, it does not significantly enhance new bone or cementum formation around non-submerged teeth. Using demineralized freeze-dried bone or allografts, some better results have been reported. Ankylosis was observed in submerged defects grafted with demineralized osteogenin and demineralized bone. This is not unexpected, since BMP-like molecules do not stimulate PDL formation, but cause bone formation within soft tissues. This can lead to ankylosis in a periodontal wound. Therefore BMPs seem more indicated in promoting bone formation around implants.

Nevertheless, studied the formation of bone and cementum regeneration following periodontal reconstructive surgery using rh BMP-2 in six beagle dogs where they surgically created mandibular supraalveolar defects. During the healing period the defects were submerged. At the test sites the rh BMP-2 was applied with synthetic bio-erodable particles and autologous blood. At the control sites a control vehicle was used. The authors saw cementum regeneration in all test sites and in 15 of the 17 control sites. Small amounts of root resorption were seen in rh BMP-2-treated defects, whereas controls exhibited substantial resorption. Ankylosis was limited and present in both test and control defects. They suggested that wound conditioning with rh BMP-2 has the potential for stimulating periodontal regeneration. The periodontal regeneration here was expressed by cementum regeneration including a fibrous attachment. A possible explanation for the formation of new cementum after the application of BMPs can be found in Melcher’s in vitro study, in which cells isolated from bone were found to synthesize cementum-like and bone-like tissue in vitro. Moreover, periodontal ligament cells express the osteoblastic phenotype and initiate apparently mineralized nodules in vitro. This has raised the possibility that osteoblasts, cementoblasts and their progenitor cells, which are found in the PDL, may have their origin in the endosteal spaces of the alveolar process. This may then have important implications when rh BMP-2 is used in regenerative procedures.

Because of these results we can eventually hypothesize two pathways to reestablish periodontal regeneration. The first is the growth and migration of already differentiated cells into the wound site (alveolar bone cells and PDL cells), which can be influenced by the use of growth factors. The second possible pathway is the regeneration through growth and subsequent differentiation of pluripotent progenitor cells (mesenchymal stem cells). Once the healing sequence is initiated by rh BMP-2, still other cytokines and growth factors are needed to support further differentiation of mesenchymal stem cells into additional periodontal phenotypes. Ripamonti et al. (1994) studied the efficacy of bovine BMPs to induce periodontal regeneration in the baboon. They surgically created large class II furcation defects at the mandibular molars. The healing occurred non-submerged. At one site the BMPs were implanted with a collagenous matrix, while at the control sites only the collagen matrix without BMPs was used. Histological analysis showed that native BMPs in conjunction with a collagenous matrix induce cementum, periodontal ligament and alveolar bone regeneration. Discrete bone and connective tissue regeneration was also observed at the control sites. The mineralized bone and osteoid volumes were significantly greater at the test sites. No significant differences were found between test and control sites with regard to the extent of the remaining furcation exposure [24 –28].

18 Which growth factors promote periodontal regeneration?

Given the preceding overview, the question remains: Which growth factors will promote regeneration of the periodontium? There is as yet no definitive answer to this question (except for the combination of PDGF and IGF-1, which will be discussed below). However, current knowledge suggests advantages and disadvantages to the use of certain growth factors and raises specific questions that need to be answered. Neither EGF nor TGF-a, at least by themselves, appears likely to enhance periodontal regeneration, because both promote bone resorption, are weak enhancers of connective tissue formation, and are strong stimulators of epithelial migration and proliferation. Both a-FGF and b-FGF will likely promote the coronal migration of periodontal ligament fibroblasts. Because coronal migration of periodontal ligament cells is thought to be a prerequisite for new attachment, this would suggest that the FGFs could promote regeneration; however, other studies indicate they may reduce collagen production by fibroblasts while inducing collagenase synthesis. Whether this reduction in collagen is significant enough to affect periodontal ligament formation is not known. Both a-FGF and b-FGF are likely to promote proliferation of alveolar bone cells, but whether this would in turn lead to increased bone matrix formation is unclear since FGFs down regulate transcription of the type I collagen gene. Preliminary data evaluating the effects of a combination of TGF-p, bFGF, and IGF-2 indicated no enhancement of bone formation 4 weeks after application to surgically created defects in dogs (Selvig et al 1994). However, the dose may have been too low to effect a response.

The most potent individual growth factor for stimulating bone matrix apposition in culture is TGF- ß; it thus would likely be a potent stimulus for alveolar bone formation. The fact that TGF- ß is not chemotactic for periodontal ligament fibroblasts suggests that it alone will not promote the coronal migration of these cells, however. The net result of rapid bone growth in the absence of coronal migration of periodontal ligament cells could be ankylosis.

Platelet-derived growth factor is chemotactic for periodontal ligament osteoblasts and fibroblasts and thus will likely promote the coronal migration of periodontal ligament cells. In addition, in vitro data show that PDGF and IGF-1 promote fibroblast and osteoblast proliferation, collagen (especially IGF-1) and noncollagen (especially PDGF) protein synthesis, and bone matrix formation. Nearly all of these activities are increased even further when PDGF and IGF-1 are combined. The following findings suggest that the combination of PDGF and IGF-1 would enhance regeneration of all the components of the periodontium:

PDGF and IGF-1 are chemotactic and mitogenic for periodontal ligament fibroblasts;

PDGF is chemotactic for osteoblasts;

IGF-1 promotes collagenous protein synthesis and PDGF promotes primarily noncollagenous protein synthesis in bone cultures;

the combination of PDGF and IGF-1 promotes greater bone matrix formation than any individual growth factor; and

PDGF and IGF-1 interact synergistically to promote significant collagen formation and healing in soft tissue wounds.

19 Conclusion

The explosion of knowledge and the understanding of the role of growth to factors, their mechanisms of factors and their mechanisms of action and molecular signaling pathways, suggest the potential for many novel therapeutic targets, not only for applying growth factors but also for the agents that target specific parts of the intracellular signaling pathways. There remains an enormous challenge to convert some of the Knowledge from basic studies on cell physiology to therapeutically useful techniques for the future.

Funding

No financial support.

Conflict of interest

No conflicts of interest.

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