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Growth factors are polypeptide hormones or biological factors. They mediate many cellular processes necessary for the various stages of growth, induction of phenotypic expression, and even the metamorphosis of totipotent germ cells into pluripotent stem cells.1, 2 A totipotent germ cell is a single cell that can recapitulate or repeat development, demonstrating the capacity to develop and differentiate into a complete organism.2, 3 A pluripotent stem cell is “capable of differentiating into several different final differentiated types.”3 When germ cells are cultured in certain polypeptide growth factors, they can be stimulated to transform into pluripotent stem cells.2 Growth factors are also known to influence the growth rate of certain cancers.4
Many tissues are mediated by growth factors: epithelium and endothelium; myocardium and neural tissue; cartilage, bone, and cementum; the more specialized types of connective tissue and blood: and the less specialized connective tissue replete with extracellular matrix, such as collagen and proteoglycans that surround other more highly developed tissues and organs.1
Polypeptide growth and differentiation factors (GDFs) are classified as biological mediators that have demonstrated a significant role in stimulating and regulating the wound healing process. The growth factors associated with repair and regeneration coordinate important cellular processes such as:1
The sequential steps that are critical for periodontal regeneration depend on the processes of osteogenesis, cementogenesis, and formation of connective tissue. Various in vitro and in vivo studies revealed that specific growth factors modify reputed elements of periodontal wound healing, thus achieving significant periodontal regeneration in animals.1
In 1976, Melcher 5 proposed the initial hypothesis on the potential of periodontal regeneration. Since then the rapidly growing sector of regenerative research has striven to identify modes of treatment that support regeneration of lost periodontal tissues. Currently utilized regenerative techniques incorporate guided tissue regeneration (GTR) alone or with bioabsorbable or nonresorbable membranes, allografts and bone autographs, and agents such as citric acid or tetracycline used to condition roots and enhance the attachment of periodontal tissues.1
The past decade has witnessed a concentrated effort by cell and molecular biologists to determine how polypeptide growth and differentiation factors (GDFs) effect the repair and regeneration of tissues. These molecules exist in nature and demonstrate pleiotrophic or multiple effects on wound repair in almost all tissues, including the periodontium. Growth and differentiation factors exert effects, both in vivo and in vitro, on soft tissue components of the periodontium such as the periodontal ligament (PDL) and gingival connective tissue as well as the hard tissue structures such as alveolar bone and cementum.1
As natural biological mediators, polypeptide growth factors modulate significant cellular events in tissue repair:1
The following nine growth and differentiation factors (GDFS) are found in bone, cementum, and healing wound tissues.1
After an injury occurs, healing proceeds in a succession of “well orchestrated cell-cell and cell-macromolecular interactions”.1 In the process of normal wound healing, various growth factors act in conjunction to form a complex arrangement of molecules that regulate cellular activity within and bordering the wound.1
When an acute injury of tissue extends into the level of subepithelial tissues, the interruption of wound vasculature results in the formation of fibrin and an aggregation of platelets that form a cellular plug. Platelets at the wound margins become activated and produce various growth factors such as PDGF, TGF-β1, an epidermal growth factor (EGF)-like protein, and platelet-derived endothelial cell growth factor (PD-ECGF).1 Additionally plasma exudate produces insulin-like growth factors, an important source of these regulatory molecules.1
Within a few hours after the injury occurs, cells bordering the site of injury produce growth factors such as IGF-I, PDGF, TGF-?? and TGF-?1. Subsequent to tissue injury, neutrophils accumulate followed by migration of macrophages within the next several days into the area. Macrophages provide for the debridement of damaged tissue and also serve as another source of growth factors such as PDGF, TGF-α, and TGF-β1.1
Substantial amounts of growth factors such as IGF-I, -II, TGF-?1, and PDGF are found in bone and cementum and they may be released after injury. Differentiation or osteoinductive factors, such as bone morphogenetic proteins, are thought to be expressed in mature bone, during repair of a fracture, and during embryological development.1
When a gene is expressed, it promotes the full use of information it contains via transcription and translation, resulting in the production of a protein and the subsequent appearance of the physical characteristics coded for by that gene.3 Messenger RNA transcripts of various bone morphogenetic proteins have been detected in a variety of growing tissues such as developing craniofacial structures, tooth buds, odontoblastic layers, and palatal shelves.1
After injury of bone and soft tissue, and perhaps during the process periodontal disease, the expression of various growth and differentiation factors may regulate repair and possibly the regenerative process. In the treatment of chronic adult-onset periodontitis, the objective of the administration of growth and differentiation factors is the enhancement of the normal wound healing response, which if given alone may be inadequate to promulgate complete regeneration of all attachment structures.1
In the wound healing process multiple growth factors appear to interact to promote repair of injured tissues. In acute skin injury various different growth factors facilitate wound repair and influence processes such as re-epithelialization, angiogenesis, and synthesis of extracellular matrix.1 Growth and Differentiation Factors in Wound Healing of Periodontal Soft Tissues Growth and Differentiation Factors in Wound Healing of Periodontal Soft Tissues Wound healing studies conducted on animals provide evidence that soft tissue wound repair is enhanced by epidermal growth factor (EGF), transforming growth factors-α (TGF-β), platelet derived growth factor (PDGF), transforming growth factors-β1 (TGF-β1), and acidic and basic fibroblast growth factors, (a- and bFGF). Combinations of different growth and differentiation factors yield greater repair than can be achieved by individual factors alone.1
Growth and Differentiation Factors in Wound Healing of Periodontal Soft Tissues Gingival epithelial cells, gingival fibroblasts, and periodontal ligament fibroblasts are the major cells involved in the soft tissue repair mechanisms that achieve new attachment in periodontal wound healing. The periodontal ligament is a soft tissue structure comprised of a neurovascular supply, extracellular matrix made of primarily of type I collagen and noncollagenous protein, and various cell types such as fibroblasts, macrophages, polymorpholeukocytes, etc.Growth and Differentiation Factors in Wound Healing of Periodontal Soft Tissues
For periodontal regeneration to occur, the coronal aspect of the periodontal ligament must be re-established along with the related cementum and supporting alveolar bone. Factors that facilitate the proliferation and migration of periodontal ligament fibroblasts and the biosynthesis of collagen are believed to be crucial mediators in promoting the formation of new periodontal ligament fibers.1
Among the most extensively studied growth factors that affect the activity of periodontal ligament fibroblasts are:Growth and Differentiation Factors in Wound Healing of Periodontal Soft Tissues
In vitro and in vivo, PDGF has been depicted as being the most thoroughly described growth factor associated with the periodontium. There are different forms of PDGF called isoforms and all of them have been shown to have a PDL fibroblast proliferative activity in vitro.1 An isoform is a protein having the same function and similar or identical sequence, but it is the product of a different gene and is usually specific to a particular tissue.3
Since platelet-derived growth factor is chemotactic for fibroblasts in the periodontal ligament, it induces collagen and total protein synthesis. Platelet-derived growth factor stimulates gingival fibroblasts to synthesize hyaluronate, which is necessary for the formation of large groups of proteoglycans that supply the framework on which the extracellular matrix can develop.1
Lipopolysaccharide is a major constituent of the cell walls of gram-negative bacteria3, i.e., periodontal pathogenic bacteria; it is associated with loss of alveolar bone in periodontal disease. Lipopolysaccharide inhibits the proliferation of gingival fibroblasts; and platelet-derived growth factor decreases this inhibitory effect.1
Platelet-derived growth factor also increases the proliferation of fibroblasts under teflon membranes (polytetrafluoroethylene membranes [ePTFE]) in fenestration defects in dogs. Apparently, platelet-derived growth factor supports the healing in the periodontal soft tissue wound in a variety of ways.1
Transforming growth factor-beta 1 strongly induces the production of extracellular matrix in periodontal ligament fibroblasts as well as many other cell types. Although transforming growth factor-beta 1 does not seem to be involved with the migration of periodontal ligament fibroblasts, it has a mild influence on the passage of periodontal ligament fibroblast cells through the cycle of cell division with resultant daughter cells. Transforming growth factor-beta 1, alone or in combination with platelet-derived growth factor-BB, induces periodontal ligament fibroblasts to proliferate at greater levels than gingival fibroblasts.1 The PDL must be able to proliferate at a faster rate than gingival epithelium in order to achieve true new attachment.
Transforming growth factor-beta 1 increases the number of platelet-derived growth factor-b receptors, but at the same time decreases the number of PDGF-a subunits.1 A receptor is a molecular structure located within or on the surface of a cell; it selectively binds a specific substance followed by a specific physiologic effect, for example, cell surface receptors for hormones.3 TGF-β1 hinders the reproduction of epithelial cells. Considering all of these facts together, TGF-β1 may participate in periodontal wound healing.1
Basic fibroblast growth factor (bFGF) is a strong chemotactic agent (movement directed by a gradient)3 and mitogenic (cell division or transformation)3 agent for periodontal ligament fibroblasts.1 Dentin blocks coated with basic fibroblast growth factor induce the migration and multiplication of human endothelial cells. Exposure of type I collagen on dentinal surfaces increases the binding of basic fibroblast growth factor.1
Epidermal growth factor seems to exert only a minor effect on the promotion of mitogenesis, chemotaxis, or matrix synthesis in periodontal ligament fibroblasts. During differentiation, EGF and EGF receptors (EGF-R) are localized on periodontal ligament fibroblasts in rats. Epidermal growth factor receptors may stabilize the periodontal ligament fibroblast phenotype or cellular physical characteristics.1
Stimulation of epidermal growth factor receptors and periodontal ligament fibroblasts is related to sustaining the cells in an undifferentiated state (with decreased alkaline phosphatase activity). Inhibition of epidermal growth factor receptors is associated with the differentiation of cells into osteoblasts or cementoblasts. In vivo testing is needed on the effects of epidermal growth factor on periodontal wound healing.1
Insulin-like growth factor-1 is chemotactic for cells that come from the periodontal ligament and demonstrates significant effects on the mitogenesis of periodontal ligament fibroblasts and protein synthesis in vitro. Insulin-like growth factor-I receptors are present on the surface of periodontal ligament fibroblasts. However, the effect of insulin-like growth factor-II on the metabolism of gingival and periodontal ligament fibroblasts is uncertain.1
Cementum-derived growth factor is a more recently characterized growth factor and is thought to be found only in cementum. It is mitogenic for gingival and periodontal ligament fibroblasts. Cementum-derived growth factor helps progenitor cells located in structures adjacent to the dentin matrix to differentiate in to cementoblasts. It may also encourage their growth and migration.1
Questions remain about the effects of cementum-derived growth factor on the synthesis of collagen by periodontal ligament fibroblasts. However, cementum-derived growth factor is responsible for more collagen synthesis in human lung fibroblasts than PDGF-AB or –BB. In vivo experimentation would be aided by producing larger quantities of cementum-derived growth factor via molecular cloning and expression.1
Much remains to be learned about the effect of bone morphogenetic proteins on periodontal ligament fibroblast metabolism. Yet, research has yielded information that at concentrations up to 200 ng/ml, bone morphogenetic protein-7 is not mitogenic for periodontal ligament fibroblasts.1
Bone morphogenetic protein-7 does alter the periodontal ligament fibroblast phenotype by stimulating alkaline phosphatase activity in a dose- and time-dependent manner. Neither bone morphogenetic proteins-2 or 12 have been able to elicit a mitogenic response from cultured periodontal ligament fibroblasts according to more recent studies.1
Growth factors are found in bone matrix and are thought to link the bone formation to bone resorption.3 Bone matrix is the bone tissue located in between bone cells; it consists of collagen fibers and ground substance, into which the inorganic salts (phosphate, carbonate, and some fluoride) are deposited in the form of an apatite.3 Hydroxyapatite is the calcium phosphate mineral that is a component of bone and dentin.3
Many in vitro and in vivo model systems have been used to determine how polypeptide growth factors affect bone. Less data seems to be available on how growth factors affect alveolar bone metabolism.1 Various growth factors have been located in cementum matrix, but the specific effects that they exert on cementogenesis is less well understood.1
Platelet-derived growth factor induces synthesis of DNA, collagen and non-collagen proteins, as well as chemotaxis in bone organ cultures. Platelet-derived growth factor inhibits alkaline phosphatase activity and osteocalcin in osteoblast-like cell cultures. Demineralized bone matrix-induced cartilage and bone formation are stimulated by platelet-derived growth factor in vivo. Bone repair is stimulated when platelet-derived growth factor-BB is combined with a collagen vehicle and then applied to surgically-created tibial osteotomy defects.1
Studies on stimulation or inhibition of osteoblast proliferation suggest that transforming growth factor-b 1 is markedly dependent upon the source of bone cells, the dose applied, and the local environment. In addition to bone matrix deposition and chemotaxis, transforming growth factor-beta 1 enhances the biosynthesis of type I collagen, fibronectin, and osteonectin.1 (see definitions)
Transforming growth factor-beta 1 decreases the destruction of connective tissue matrix by inhibiting the synthesis of metalloproteinases and plasminogen activator, which increases synthesis of tissue inhibitor of metalloproteinase (TIMP) and plasminogen activator inhibitor (PAI). While transforming growth factor-beta 1 seems to decrease formation of osteoclast-like cells, it may use a prostaglandin-mediated mechanism to increase bone resorption.1 Repeated injections of transforming growth factor-beta 1 leads to cartilage formation in long bones, and ultimately ossifies to bone through endochondral bone formation. During the normal healing process in human fractures, the body expresses genes for transforming growth factor-beta 1. Transforming growth factor-beta 1 is abundant in bone and exhibits multiple effects on the formation of bone matrix. As a result, it is thought to act as a bone coupling factor that links bone resorption to bone formation.1
Both forms of fibroblast growth factors (acidic and basic) are present in bone matrix; they enhance synthesis of DNA and replication of cells in vitro. Not only do fibroblast growth factors fail to directly stimulate mature osteoblasts, but they inhibit the activity of alkaline phosphatase.1
In an immortal cell line, basic fibroblast growth factors were reported to decrease mRNA levels of osteocalcin and type I collagen. However, researchers reported a significant finding that fibroblast growth factors were potent stimulators of angiogenesis, a process vital to the vascular invasion of bone.1
Although insulin-like growth factor-II is the most plentiful growth factor found in bone matrix, insulin-like growth factors-I and II both exist in abundant amounts in bone. Osteoblasts produce insulin-like growth factor-I, which stimulates cellular proliferation, differentiation, and type I collagen biosynthesis in order to form bone.1
Since high levels of insulin-like growth factor-I are synthesized and secreted by osteoblasts in bone, insulin-like growth factor-I might control the formation of bone in an autocrine manner.1 Autocrine secretion refers to the secretion of a substance, such as a growth factor, that stimulates the secretory cell itself.3 It provides for independence from control of the rate of cellular division rather than of the size of an individual cell.3 Insulin-like growth factor-I has also been reported to increase the numbers of osteoclastic multinucleated cells.1
When insulin-like growth factor-I was applied to the root surfaces of rat teeth, it promoted cementogenesis within 8 days after the teeth were reimplanted. However, only small increases in new cementum and bone formation were reported when insulin-like growth factor-I was applied to naturally occurring periodontitis lesion in dogs.1
When tested in vitro, insulin-like growth factor-II demonstrates effects similar to insulin-like growth factor-I. High levels of insulin-like growth factor-II can bind to the insulin-like growth factor-I receptor. Yet, results from studies imply that insulin-like growth factor-II is less potent than insulin-like growth factor-I in stimulating bone formation.1
However, both insulin-like growth factor-I and insulin-like growth factor-II are equally potent in stimulating osteoblast chemotaxis in vitro. Researchers have also evaluated insulin-like growth factors in various combinations with other growth factors both in vitro and in vivo.1
When insulin-like growth factor-I was combined with transforming growth factor-b 1 or with platelet-derived growth factor, either combination led to more bone matrix apposition in calvarial organ culture than with individual exposure to any of these 3 growth factors. This synergistic increase was also observed when osteoblast mitogenesis increased in bone culture cells after insulin-like growth factor-I was combined with other growth factors such as basic fibroblast growth factor, platelet-derived growth factor, or transforming growth factor-b 1.1
Cultured adult human osteoblasts responded with maximal proliferation to a mixture of insulin-like growth factor-I, platelet-derived growth factor, transforming growth factor-b 1, and epidermal growth factor. Insulin-like growth factor-II in combination with transforming growth factor-b 1 acted in synergy to increase new callus formation in tibial osteotomy defects beyond what could have been expected from the effects of the individual growth factors.1
Therefore, results from various studies indicate that insulin-like growth factor-I or insulin-like growth factor-II, in combination with other growth factors, may enhance the wound healing process in bone.1
Many cells including osteoblasts, activated lymphocytes, and keratinocytes produce parathyroid hormone-related protein which is a polypeptide growth factor. It is related to parathyroid hormone, produces bone resorption, and has a strong anabolic effect on bone. Studies have identified receptors for parathyroid hormone-related protein on PDL cells and evidence of expression of parathyroid hormone-related protein on cells resembling cementoblasts in the developing tooth.1
Structurally similar, bone morphogenetic proteins are members of the transforming growth factor-b 1 superfamily. Individually, bone morphogenetic proteins 2-12 stimulate new endochondral bone formation. While bone morphogenetic proteins are capable of inducing new bone formation, other growth factors such as TGF-b 1 are unable to achieve this.1
Undifferentiated bone cell precursors are stimulated by bone morphogenetic proteins to proliferate and migrate. Yet, bone morphogenetic proteins seem to exert minimal to no effect on mature osteoprogenitor cells. Therefore the primary function of bone morphogenetic proteins is to transform undifferentiated pluripotential cells into committed or differentiated cartilage and bone-producing cells.1
Bone morphogenetic proteins are found in significant amounts in bone and they are secreted by osteoblasts as well as various other cell types. Different levels of bone morphogenetic proteins such as BMP-2, -4, and -7 are found in dental bone allograft material. Commercially prepared proteins retained in allograft material demonstrate the ability to influence cell behavior in vivo.1
Molecules similar to bone morphogenetic proteins are found in dentin and stimulate reparative dentin formation in vivo. Inhibition of bone cell differentiation and failure of fractures to heal may both be associated with a deficiency of bone morphogenetic proteins. Recombinant bone morphogenetic proteins support bone formation in segmental long bone osteotomies and in calvarial defects.1 The term ‘recombinant’ refers to a cell or individual that has a new combination of genes not found together in either parent.3
To summarize, bone morphogenetic proteins exert a large number of effects on bone. They stimulate cell division in undifferentiated mesenchymal cells and osteoblast precursors. They stimulate bone cells to increase their alkaline phosphatase activity. They elicit an accumulation of mesenchymal cells and monocytes. They adhere to extracellular matrix type IV collagen.1
Not all growth factors have received equal attention in research studies, with some being studied more extensively than others. Various early in vivo studies of the impact of growth factors on periodontal regeneration concentrated on platelet-derived growth factor combined with insulin-like growth factor-I (PDGF + IGF-I). This combination of growth factors stimulated the formation of new bone, cementum and periodontal ligament in natural periodontal disease lesions in dogs and lesions induced by ligatures in non-human primates. In addition PDGF + IGF-I stimulates bone formation around press-fit and immediate extraction socket dental implants.1
Investigators also examined the periodontal regenerative potential of platelet-derived growth factor in combination with dexamethasone when experimental periodontitis was induced in non-human primates. It promoted new bone growth and periodontal attachment 4 weeks after administration of platelet-derived growth factor + dexamethasone.1
Subsequent studies with dogs evaluated platelet-derived growth factor used in conjunction with guided tissue regeneration therapy. Measurements at 5, 8, and 11 weeks revealed significant increases in the growth of new bone and periodontal attachment after platelet-derived growth factor + guided tissue regeneration therapy were used to treat Class III furcation defects in the dogs.1
In 1995 investigators reported on the first human clinical trial that evaluated the safety and efficacy of platelet-derived growth factor combined with insulin-like growth factor-I. The study consisted of 38 patients presenting with moderate-severe periodontal disease. They were treated with three regimens: the test subjects were treated with platelet-derived growth factor combined with insulin-like growth factor-I in a methylcellulose vehicle; the 2 control groups were treated with the vehicle only or with surgery only.1
The test group treated with platelet-derived growth factor combined with insulin-like growth factor-I experienced a 43.2% fill in the bony defect. The combined control groups had an 18.5% fill in the bony defects. The differences in the amount of bone defect fill in the different groups was statistically significant.1
Earlier studies concentrated their efforts on evaluating the effects of platelet-derived growth factor alone or combined with other growth factors on periodontal regeneration. Far fewer studies evaluated the effects of how other growth factors might promote periodontal repair.1
Transforming growth factor-b 1, basic fibroblast growth factor, and insulin-like growth factor-II were applied to surgically created fenestration defects in dogs to evaluate their osseous regenerative potential. At 4 weeks these 3 growth factors had still not produced any bone formation in the osseous defects. The investigators suggested that the nanogram levels of these growth factors might not have been appropriate to stimulate bone formation.1
In 1991, the first human study was conducted using bone morphogenetic protein-3 to induce periodontal regeneration. Bone morphogenetic protein-3, also known as osteogenin, was combined with demineralized bone allograft and applied in a submerged tooth model.1
In the group treated with bone morphogenetic protein-3 + bone grafting, new bone and cementum formation were reported around the periodontally involved submerged teeth. The groups treated with bone morphogenetic protein-3 + collagen vehicle experienced no increased bone or cementum formation.1
However, the group treated with bone morphogenetic protein-3 + bone grafting did not experience significantly better results than the group treated with bone grafts alone. An undesirable result of pinpoint ankylosis was reported in submerged teeth treated with bone morphogenetic protein-3 + bone grafting.1
When bone morphogenetic protein-2 in a vehicle of synthetic bioabsorbable particles was applied to surgically created Class III furcation lesions in dogs, it stimulated significant periodontal regeneration in the formation of new bone and cementum. The authors reported that almost 95% of the missing bone in the surgically created Class III furcation lesions was regenerated 8 weeks following the application of bone morphogenetic protein-2.1
However, the sites treated with bone morphogenetic protein-2 in the vehicle of synthetic bioabsorbable particles demonstrated a nearly 4-fold increase in ankylosis compared to the sites treated with the vehicle alone. The authors suggested that the proximity of the bone to the cemento-enamel junction may have been a contributing factor in the development of ankylosis in the submerged defect model. It was recommended that bone morphogenetic proteins need to be evaluated in further studies in non-submerged models.1
They also demonstrate promise in stimulating wound healing around dental implants. Bone morphogenetic protein-7 (osteogenic protein-1 or OP-1) was tested in a pilot study in non-human primates. When it was applied around immediate extraction socket implants, histological evaluation at 3 weeks revealed increased bone growth.1
Bovine bone morphogenetic protein was applied around cylindrical uncoated endosseous implants in a dog model. Histomorphometric measurements 4 weeks after implantation revealed an increase in the rate of osseointegration around cylindrical uncoated endosseous implants.
Scanning electron microscopy was used for 12 weeks to evaluate how tissue reacted to titanium implants coated with bovine bone morphogenetic protein in the same dog model. A copious amount of lamellar bone formation around bBMP-coated implants was evident by 8 weeks after treatment. The new bone was located adjacent to the implant threads and repeatedly entered into the implant holes.1
In earlier studies, the application of growth and differentiation factors provided differing degrees of success in stimulating wound healing in the periodontal and peri-implant areas. These studies did establish the need to further evaluate the biologic mechanisms that may be responsible for the promotion of tissue regeneration by growth and differentiation factors.1
Researchers noticed a prolonged enhancement of new bone and attachment apparatus for several weeks to months following the application of growth and differentiation factors. Temporal changes in gene expression by cells within and adjacent to the wound site may be responsible for this regeneration.1
The delivery systems in the earlier studies permitted the periodontal tissues to be exposed to the growth and differentiation factors for a brief time of only a few hours. Longer exposure and improved delivery systems may enhance regeneration in future studies. Ultimately, these studies seek evidence to conclusively support the addition of growth and differentiation factor therapy to the protocol for reconstructive periodontal treatment.1