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In 2001, Giannobile et al.14 published a study on the use of platelet-derived growth factor for gene delivery to promote periodontal tissue engineering. The targeted delivery of growth-promoting molecules to the tooth root surface presents a difficult challenge to scientists who are attempting to reconstruct periodontal structures. Fortunately, polypeptide growth factors such as platelet-derived growth factor (PDGF) have demonstrated the capacity to stimulate both cementogenesis and osteogenesis.14
Recent developments in gene therapy now enable scientists to deliver recombinant proteins to tissues over extended time periods. Researchers constructed recombinant adenoviral vectors encoding for the PDGF-A gene to enable delivery of platelet-derived growth factor transgenes to cells. The success of tissue engineering is highly dependent upon the purification and production of signaling molecules on a large scale, and the methodology for delivery of these factors to their targets. At the present, the extremely short half-life of the factors interferes with their delivery to periodontal wounds.14
It is thought that proteolytic breakdown, receptor-mediated endocytosis, and the solubility of the delivery vehicle are probably responsible for the limited duration during which topically-administered growth factors remain in the periodontal defect. Since existing protein delivery systems are capable of producing only a transient action of the administered growth factor, utilization of DNA delivery systems may provide an alternative method for directing proteins to periodontal wounds.14
Gene therapy has been used to treat various diseases associated with tissue deficiencies, such as skin and bone injuries. As such, an active area of research has developed around the use of gene therapy to promote repair and regeneration. In the case of wound repair, the transient expression of a presumed transgene may prove beneficial in restoration of the tissue defect.14 A transgene is defined as DNA that has been integrated into the germ line of a transgenic organism.3 Transgenic organisms are those organisms that have integrated foreign DNA into their germ line as a result of the experimental introduction of DNA, most often through the use of recombinant DNA techniques.3
The use of DNA-lipid complexes and adenovirus are examples of gene delivery methods for the short-term expression of genes. Normally, the regulation of wound repair takes place in a controlled manner over a defined period of time. As a result, the use of gene therapeutics in chronic wounds, such as those found in periodontal disease, may stimulate both an elevated and sustained production of growth factors, therefore promoting tissue regeneration.14
Depending on the tissue transduced and route of administration, recombinant adenoviral expression has been observed to persist for up to 35 days. It is the ability of the adenovirus to remain extrachromosomal (i.e., episomal) as a nonintegrating virus that enables the expression pattern to be transient. The decrease over time of the total level of recombinant virus in vivo depends on the lifespan of the cells infected and the how effectively the host immunologic mechanisms attenuate viral expression.14
Researchers evaluated the feasibility of platelet-derived growth factor (PDGF) for gene delivery because of its history of consistently stimulating periodontal tissue regeneration. Platelet-derived growth factor produces its biologic effects when it binds to the cell-surface receptors (PDGF- alphaR and -betaR) because they possess intrinsic tyrosine kinase activity which stimulates the cell to undergo mitosis, chemotaxis or matrix synthesis.14
Giannobile et al.14 inserted the PDGF gene into adenovirus, which is a transiently expressing viral vector, in order to target quiescent and replicating cells. The use of a longer term delivery method of PDGF enables researchers to utilize the extended expression pattern of PDGF receptors, which is advantageous following tissue injury. Various research groups have utilized plasmid DNA to deliver growth factor genes to healing skin, bone, and periodontal wounds.14
Another method thought to be promising in the promotion of tissue repair is the use of biodegradable polymers to deliver PDGF DNA. Also yielding impressive results is the use of adenovirus to deliver bone morphogenetic protein (BMP) genes to orthotopic (in the normal or usual position3) wounds by ex vivo (in vitro) approaches.14 Others have developed unique methods of transducing wounds by the technique of in vivo microseeding which Giannobile et al.14 have modified for use in periodontal wounds.
Root lining cells that possess phenotypic characteristics similar to cementoblasts have recently been characterized. This has enabled researchers to evaluate the delivery of PDGF transgenes to cementoblasts.14 “A transgene is DNA that has been integrated into the germ line of transgenic organisms.”3 A transgenic organism is an “organism that has integrated foreign DNA into its germ line as a result of the experimental introduction of DNA. Recombinant DNA techniques are commonly used to produce a transgenic organism”.3
In this investigation they evaluated the capacity of a recombinant adenovirus encoding PDGF-A to transduce and regulate the activity of cementoblasts. The study of growth factor gene therapy may produce a greater understanding of cementogenesis and periodontal tissue regeneration and thus enhance future therapeutic techniques.14
According to Giannobile et al.14 their test results “demonstrate for the first time the use of growth factor gene delivery to root lining cells”. Their study provided evidence “that PDGF-A gene transfer is stable in vitro and that genes known to be modulated by PDGF are induced by Ad2/PDGF-A, and in a prolonged fashion”14. In addition, their data indicates that the gene transfer to a cell type reputed to facilitate periodontal regeneration, such as the cementoblast, can be successfully transduced at high levels of greater than 90% of the cells and for extended time periods in vitro.14
Giannobile et al.14 pointed out that the gene expression profile would probably be diminished in vivo by the rising cytotoxic T-lymphocyte response to the adenoviral protein release by Ad2/PDGF-A. However, the viral loads, the immune response, and the fate of the transduced cells in vivo are hazards involved in the use of this technique in periodontal wounds, and they necessitate careful evaluation.14
One important goal of the ongoing studies in ex vivo and in vivo gene transfer to the periodontium is the careful examination of the degree to which the above effects modulate periodontal wound healing. For example, PDGF has multiple or pleiotropic effects on cells that originate in the periodontium including osteoblasts, periodontal ligament cells, and cementoblasts. When PDGF is applied topically, it is known to accelerate tissue repair in many wound model systems such as skin, bone, and periodontium.14
Encouraging results using recombinant growth factors have been documented in many clinical studies. Yet, there are limitations associated with topical protein delivery, for example, short half-life, protease inactivation, and poor bioavailability from the available delivery vehicles. Thus, Giannobile et al.14 developed a method that optimized growth factor delivery and consequently maximized therapeutic efficacy. It is thought that the use of a genetic approach to tissue engineering might yield a method to deliver growth factors directly from the target cells, thus eliminating many of the disadvantages of protein delivery.14
The findings of this study confirmed the following:14
In addition, this study confirmed that the PDGF-AA protein delivered by gene transfer modulates cementoblasts in a manner compatible with its response by protein delivery, that is, by stimulating the immediate-early gene c-myc. This is significant because “the proto-oncogene c-myc is an important member of the early responsive nuclear oncogene family that is induced typically within minutes of growth factor stimulation”.14
In the situation of gene transfer, there is a delay in the induction of c-myc attributed to the steps needed for the virus to enter into the cell, followed by transcription and subsequent translation of PDGF protein to induce c-myc mRNA. Surprisingly, the biological effects occur relatively rapidly in view of the steps mandatory for viral infection.14
PDGF-A gene transfer also results in the induction of osteopontin mRNA, further demonstrating the consistent effects of viral versus protein delivery of PDGF-AA. They also documented the strong induction of OPN gene expression alone and in combination with ascorbic acid, which is a significant co-factor in the production of extracellular matrix. Osteopontin has been identified in many connective tissues; it promotes adhesion of both osteoblasts and osteoclasts, and regulates mineral crystal growth.14
It has been proposed that prolonged delivery of PDGF may have a variety of different effects on the mineralization process. Earlier studies revealed that long-term exposure of osteoblastic cells to PDGF stimulates proliferation, but it decreases expression of the osteoblast phenotype and subsequently inhibits mineralization in vitro. However, it is to be expected that PDGF gene delivery inhibits ALP activity, because by definition, rapidly proliferating cells are not expressing the differentiated cell phenotype. Consequently, regulation of the spatial and temporal levels of PDGF gene expression in vivo is most probably going to impact the composition of newly regenerating periodontal tissues.14
Giannobile et al.14 concluded that this study provides evidence of the successful gene transfer of PDGF-A to cementoblasts in vitro. The successful transduction of cementoblasts by PDGF-A stimulated mitogenesis, proliferation and expression of PDGF-inducible genes. Further studies are recommended to evaluate the long-term effects of PDGF gene transfer on cementogenesis in vitro and in vivo, as well as the role of PDGF gene therapy in the modulation of periodontal tissue regeneration in vivo.14
Currently there is a scarcity of information about the therapeutic value of non-surgical periodontal therapy in conjunction with subantimicrobial dose doxycycline (SDD) when treating severe, generalized periodontitis.13 In 2005, Gürkan et al.13 published the results from a 6-month study during which they evaluated how adjunctive subantimicrobial dose doxycycline (SDD) affected the clinical periodontal parameters and gingival crevicular fluid (GCF) levels of transforming growth factor-beta1 (TGF-β1) in patients with generalized chronic severe periodontitis.
The host produces enzymes, cytokines and other mediators derived from both resident and inflammatory cells. In concert, they amplify immune and inflammatory reactions that the body initiates in response to microbial factors. The result is the subsequent degradation of connective tissues in the presence of periodontal diseases. A considerable amount of evidence indicates that matrix-metalloproteinases are the primary mediators of the breakdown of connective tissues that is so characteristic of chronic inflammatory diseases such as periodontitis.13
Transforming growth factor-beta1 is a cytokine generally recognized for its potential to effect repair and regeneration. In addition, it plays an important role in regulating matrix-metalloproteinases. Transforming growth factor-beta1 inhibits the release of procollagenase and suppresses the production of collagenase by fibroblasts and macrophages. It also down-regulates transcription of matrix-metalloproteinase genes.13
TGF-b amplifies the expression of matrix-metalloproteinase inhibitors, for example, tissue inhibitors of metalloproteinases (TIMPs) and plasminogen activator inhibitor. Not only is transforming growth factor-beta1 a key mediator in the regulation of matrix-metalloproteinases, but also in the limitation of inflammation and its resolution.13
Transforming growth factor-beta1 is a multifunctional cytokine, producing both pro-inflammatory and anti-inflammatory effects. Thus, it is thought to possibly play a role in both the early inflammatory and the chronic destructive aspects of periodontal disease. The anti-inflammatory properties of transforming growth factor-beta1 are known to suppress both cell-mediated and humoral immune responses.13
The effects of transforming growth factor-beta1 on cell proliferation and the differentiation process indicates that this cytokine may have a significant role in wound healing, tissue remodeling and regeneration. Moreover, transforming growth factor-beta1 stimulates many cell types, thereby increasing synthesis of extracellular matrix molecules.13
Transforming growth factor-beta1demonstrates significant influence as it modulates collagen matrix in both physiological and pathological conditions such as periodontitis. This cytokine is thought to contribute to the maintenance of tissue integrity, as evidenced by its expression and production at healthy sites as well as at periodontally diseased sites. Thus these properties demonstrated by transforming growth factor-beta1 are indicative of its obvious function in the pathogenesis of periodontal diseases and inflammatory wound healing.13
As treatment modalities, scaling and root planing are effective in curtailing or preventing the progression of periodontal diseases, but they do not directly address the host response component of periodontal disease. In the search for novel therapeutic approaches, a trend has emerged in the direction of both microbial elimination and pharmacologic regulation of the exaggerated host response.13
Promising results were obtained as researchers evaluated several pharmacological agents such as tetracyclines (TCs), non-steroidal anti-inflammatory drugs and bisphosphonates for their potential to beneficially modulate the host response. Comprehensive research has revealed that this antibiotic family of tetracyclines and tetracycline analogues possess non-antimicrobial properties capable of modulating the host-response by a variety of mechanisms.13
Evidence from current research indicates that tetracyclines inhibit collagenolytic activity by inhibiting matrix-metalloproteinases. When tetracyclines suppress events that are mediated by matrix-metalloproteinases, there is a decrease in the breakdown of connective tissue and the resorption of bone. Tetracyclines have been shown to sustain their inhibitory effect on matrix-metalloproteinases even at lower doses than those required for antimicrobial efficacy. In 1999, investigators reported that the Food and Drug Administration had approved a specially formulated subantimicrobial dose doxycycline (SDD) as an adjunct to non-surgical periodontal therapy.13
From 1996-2004, various double-blind, placebo-controlled studies conducted on patients with chronic periodontitis have reported a significant reduction in probing depth and an increase in clinical attachment level in patients treated with adjunctive subantimicrobial dose doxycycline therapy compared to those treated with an adjunctive placebo. However, for patients with a severe and generalized form of chronic periodontitis, data on the clinical effect of non-surgical therapy supplemented with subantimicrobial dose doxycycline is limited.13
Studies published in 1998 and 2000 that evaluated the host modulatory effects of tetracyclines reported that they utilized a variety of mechanisms including the enhancement of collagen production in order to inhibit connective tissue breakdown. The fundamental role of the cytokine transforming growth factor-beta1 in regulating matrix-metalloproteinases and collagen turnover, in addition to matrix deposition, implies that tetracyclines might demonstrate potential effects on this cytokine.13
Research has established a relationship between the baseline pocket depth at an individual site and the effectiveness of non-surgical therapy; deeper pockets demonstrate a greater potential for a reduction in pocket depth and a gain in clinical attachment level. Scaling and root planing therapy promotes healing from inflammation and interrupts the progression of periodontal disease, consequently achieving a gain in clinical attachment and a reduction in probing depth.13
In this study both therapies SRP + placebo therapy and SRP + SDD achieved and maintained significant improvements in clinical parameters all through the study. The study group received SDD capsules containing doxycycline hyclate equivalent to 20 mg of doxycycline, twice daily for 3 months. The placebo (control) group received placebo capsules containing inactive filler, cornstarch, twice daily for 3 months.13 Gürkan et al.13 reported that subantimicrobial dose doxycycline appears to be an adjunct to non-surgical periodontal therapy, and provides a safe therapeutic approach for the long term management of chronic periodontitis.
Transforming growth factor-beta1 (TGF-ß1) plays an essential role in mediating the resolution of gingival inflammation and in initiating wound healing. It stimulates proliferation of fibroblasts, increases production of extracellular matrix molecules and inhibitors of MMPs, and inhibits synthesis of matrix-metalloproteinase.13
Transforming growth factor-beta1 has been shown to increase the matrix synthesis of gingival fibroblasts by 1.7-fold at a concentration of 1 ng/ml. This TGF- ß1 cytokine can also stimulate the production of the formative fibroblast phenotype, which may then synthesize connective tissue matrix. Previously, some investigators proposed the hypothesis that, during inflammation and wound healing, an alteration occurs in the matrix composition; and that this alteration is regulated in part by the altered phenotype of matrix producing cells, and in part by the altered response demonstrated by these cells to transforming growth factor-beta1.13
Other investigators proposed that transforming growth factor-beta1 enjoys a pivotal role in regulating collagen metabolism under normal physiologic conditions and also under pathologic conditions like periodontitis. Others have suggested that transforming growth factor-beta1 might help to create equilibrium between factors associated with destruction and repair by inhibiting destruction of the collagen matrix during phases of disease remission and healing in periodontal disease.13
When the microbial load is sustained as it is in chronic periodontitis, a state of equilibrium results. After the microbial factors are eliminated, the inflammatory process is resolved and the repair of tissues follows, to be completed in time.13 In the study by Gürkan et al.13 gingival crevicular fluid transforming growth factor-beta1 increased in both concentration and total amount after therapy with adjunctive subantimicrobial dose doxycycline and placebo therapy at 3 months.
The rapid and effective restoration of the periodontium probably accounts for the increased cytokine levels after the microbial factors were eliminated. Compared to baseline levels, the concentration of gingival crevicular fluid transforming growth factor-beta1 was higher in both groups at 6 months, although the difference is not statistically significant. Constant wound healing and cellular activity in periodontal tissues might account for this finding.13
At the end of medication period the total amount and concentration of GCF TGF-β1 in the adjunctive subantimicrobial dose doxycycline group was significantly higher than that of placebo group. It is well known that tetracyclines stimulate collage production in connective tissue. There is ample documentation that TGF-β1 can stimulate the production of a variety of connective tissue matrix constituents including collagen.13 Gürkan et al.13 suggest the possibility that TGF-ß1 may partly contribute to the regulation of increased collagen synthesis following the application of a tetracycline.
An in vitro study in 2001 found that doxycycline was able to increase to mRNA levels of TGF-ß receptors in osteoarhtritic cartilage cultures. A number of other in vitro studies provided evidence that the application of transforming growth factor-beta1 decreased synthesis of several MMPs including MMP-1, -2, -8, -9 and -13. These findings suggest that subantimicrobial dose doxycycline may promote connective tissue healing and collagenous matrix production by increasing transforming growth factor-beta1, which modulates collagenase activity.13
It has been suggested that transforming growth factor-beta1 might alternate between pro-inflammatory or anti-inflammatory roles associated with the nature of the host response during periodontal diseases. In the early phases of inflammation, transforming growth factor-beta1 is thought to play a pro-inflammatory role before it later switches to anti-inflammatory role. It is not yet known at what stages of periodontal disease and healing that transforming growth factor-beta1 demonstrates a pro or anti-inflammatory role.13
Since cytokine levels increased instead of decreasing after the inflammation resolved, it has been suggested that the anti-inflammatory and reparatory properties of transforming growth factor-beta1 might influence its pro-inflammatory properties in chronic periodontitis. Since bone loss does not occur in gingivitis, this might also be a reflection of the remodeling of alveolar bone by transforming growth factor-beta1. It remains to be determined whether or not the resolution of gingival inflammation is responsible for the decrease in the levels of transforming growth factor-beta1 during the early stages of healing after non-surgical treatment for chronic periodontitis.13
The results of this study suggest that, in patients with severe, generalized chronic periodontitis, subantimicrobial dose doxycycline therapy improves the clinical parameters and increases the levels of gingival crevicular fluid transforming growth factor-beta1. It is possible that the beneficial effects of adjunctive subantimicrobial dose doxycycline might be sustained after the treatment ceases.13 Gurken et al.13 concluded that in patients with severe, generalized chronic periodontitis, SDD combined with non-surgical therapy results in improved clinical parameters of periodontal disease and increased levels of GCF TGF-ß1 levels, as well as a decreased prevalence of residual pocket depths.
However, the effectiveness of subantimicrobial dose doxycycline at the biochemical level is thought to continue for the duration of its use. Improvements at the biochemical level probably occur prior to the detection of improvements at the clinical level. Thus they recommended a cyclical regimen of SDD with 3-month intervals to achieve improvement in biochemical markers.13
Gürkan et al.13 reported that, to best of their knowledge, their study was the first to investigate the effect of scaling and root planing, and scaling and root planing + subantimicrobial dose doxycycline on the levels of gingival crevicular fluid transforming growth factor-beta1 in patients with chronic periodontitis. They suggested that the increased levels of GCF TGF-ß1 subsequent to SDD therapy might indicate the presence of “a novel pleiotrophic mechanism” by which tetracyclines can inhibit the breakdown of connective tissue.13
The characteristics of doxycycline that enable it to increase levels of transforming growth factor-beta1 may yield another mechanism by which tetracyclines can inhibit the breakdown of connective tissue. Additional studies are needed to determine the pathway utilized by doxycycline to increase transforming growth factor-beta1 levels and subsequently, its mechanism of action.13
One of the primary goals of oral therapy is to facilitate the repair of tooth-supporting structures that have been destroyed by periodontal chronic inflammatory disease. Materials, science, and biology are critical components of tissue engineering necessary for the repair of organs and tissues. Guiding tissue regeneration with cell occlusive membranes and bone grafting techniques have yielded only limited success in periodontal tissue engineering. Periodontitis is the most common bone disease affecting humans. It is only in the last decade that scientists began to use signaling molecules such as growth factors in their quest to restore tooth support that had been destroyed by periodontal disease.15
Biofilms, or bacterial plaque, accumulate on the surface of the tooth root. It is the subsequent inflammatory response of the gingival tissues to the bacteria that characterizes periodontal disease. Following this inflammatory response is the destruction of periodontal structures including alveolar bone, tooth root cementum and periodontal ligament. If periodontal disease is left untreated tooth loss is the result.15
In conventional periodontal therapy, the clinician utilizes mechanical removal of the bacterial accumulation in an attempt to eradicate the infection, often followed by surgical intervention and/or anti-infective therapy. The purpose of the mechanical removal of plaque is to change the local microenvironment, decrease the bacterial load, and ultimately stabilize the disease process. Unfortunately, this method rarely succeeds in restoring the tooth supporting structures that have been destroyed by periodontal disease.15
There are several key factors required to enhance healing in the attempt to achieve the most effective regeneration of periodontal structures:15
Consequently, clinicians have utilized a variety of surgical procedures:15
Due to recent advances in molecular cloning, an infinite amount of recombinant growth factors have become available for use in tissue engineering as an optional avenue to treatment for periodontal regeneration. Recombinant GFs such as platelet derived growth factors (PDGFs), insulin like growth factors (IGFs), fibroblast growth factors (FGFs) and bone morphogenetic proteins (BMPs) are known to promote skin and bone wound healing. They have been employed in periodontal pre-clinical and clinical trials.15
Tissue engineering has enlisted a variety of delivery vehicles to transport GFs to the wound site in order to maximize their bioavailability. The dosage, release pattern (pulsatile, constant or time programmed), kinetics of release and duration of delivery can be optimized depending on the device and application of the growth factor or cytokine.15
Anusaksathien et al.15 emphasized that “Ideally, the delivery vehicles that form the scaffold should possess the following properties:
Therefore the focus is on the biological properties of each factor and their applications in the endeavor to regenerate periodontal and bone tissues in concert with the theory of tissue engineering.15
Anusaksathien et al.15 described polypeptide growth factors as “a class of naturally occurring biological mediators that regulate the proliferation, migration and/or extracellular matrix synthesis of a variety of cell types including those derived from the periodontium”. Primarily these polypeptide growth factors are produced in the form of pro-peptides, inactive biologically, and stored in the cytoplasm.15
It is the C-terminal mature forms that are the cleaved products of the pro-peptides; they are secreted extracellularly. The C-terminal mature forms then bind to their cognate receptor. They form a receptor ligand complex that transduces signals to the nucleus leading to a climate of cellular events.15
After the growth factors are released, they can affect target cells via several means:15
Various growth factors have been evaluated extensively for their ability to stimulate wound repair as well as periodontal tissue regeneration:15
The alpha granules of platelets comprise a significant source of platelet-derived growth factor. Many studies have confirmed that platelet-derived growth factor is manufactured by numerous cell types, including fibroblasts, myocytes, neurons, endothelial cells and bone marrow hematopoetic cells.15
Platelet-derived growth factor has 4 isoforms: PDGF –A, -B, -C and -D. An isoform is a protein that has “the same function and similar (or identical sequence), but is the product of a different gene and is usually tissue specific. The term isoform carries a stronger implication than the term homologous”.3 The mature sections of the A and B chains are approximately 100 amino acid residues in long; they share approximately 60% of the amino acid identities.15
Proteolysis, cleavage of proteins by proteases, activates PDGF-C and D. When platelet-derived growth factor-C and D are overexpressed in transgenic mice, this induces proliferation of fibroblasts. Although not much is known about the interaction of PDGF-C and PDGF-D with the other PDGF-A and –B chains, each chain is known to be regulated independently at both the transcriptional and translational levels.15
PDGFs induce various biological responses in an array of cell and tissue types. For example, platelet-derived growth factors regulate migration, proliferation, and inhibition of apoptosis, and/or matrix synthesis of endothelial, smooth muscle, fibroblast, and bone cells. The PDGF receptors are transmembrane peptides that contain various tyrosine kinase domains. For example, PI-3 kinase is reported to mediate a variety of cellular events, including actin reorganization, chemotaxis, cell growth, and anti-apoptosis.15
Extensive in vitro studies have been conducted on the biological effects of PDGF on cells derived from periodontium. Platelet-derived growth factors also demonstrated an increased migratory and proliferative influence on fibroblasts derived from gingival tissue. In addition PDGFs optimized proliferative activity on cementoblasts, preosteoblasts, and osteoblastic cells in vitro. Researchers reported that platelet-derived growth factor augments the production of collagen type I and osteopontin in PDL cells and osteoblasts, respectively. PDL cell attachment and protein synthesis were seen to increase after the application of PDGF-BB onto demineralized dentin.15
Investigators reported the inhibition of bone nodule formation and the down-regulation of osteoblast associated genes bone sialoprotein (BSP) and osteocalcin (OCN) in cementum cell lines by platelet-derived growth factor. Moreover, they reported that the long-term delivery of platelet-derived growth factor by gene transfer resulted in the stimulation of mitogenesis and proliferation in gingival fibroblasts, PDL cells, and cementoblasts above the levels associated with the continuous application of PDGF.15
Consequently, they proposed that greater bioavailability of PDGF to various tissues might be achieved by alternative delivery methods. Thus, while platelet-derived growth factors are not involved in the differentiation of cells derived from periodontal tissues, they do enhance the migration, proliferation, and matrix synthesis of these cells.15
During gingival tissue healing, PDGF-A and B has been observed in epithelium, connective tissue, endothelium and the fibrin clot; therefore, it must have a significant role in healing of the gingival wound. Platelet-derived growth factor -a and -b receptors are not expressed in normal, unwounded tissues. After an acute tissue injury, the expression of PDGF-b receptors occurs in the cell-rich connective tissue zone of gingival wounds most significantly 3 days after injury, progressing to maximal expression at day 7. Then as the wound heals, there is a subsequent decrease in the receptor expression levels.15
Various other studies reported the same inducibility of GFs and receptor expression subsequent to cutaneous injury, with a return to normal unwounded tissues within 10 days. These studies demonstrate that tissue injury induces the production of both platelet-derived growth factor ligands and receptors.15
The insulin like growth factors (IGFS) comprise a family of growth factors consisting of insulin like growth factor-1 (IGF-I), insulin like growth factor-2 (IGF-2), and relaxin.15 Insulin like growth factors-1 and -2 are polypeptides that contain sequences very similar to insulin.3 They can stimulate the same biological responses, including mitogenesis in cell culture. There are two types of insulin like growth factor receptors on the cell surface; one closely resembles the insulin receptor which is also present.3
Various cell types produce insulin like growth factor-1, which expresses its autocrine/paracrine and endocrine functions both locally and systemically. When insulin like growth factor-1 is secreted extracellularly, it binds to insulin like growth factor binding proteins (IGFBPs). They act as buffer systems and storage pools for insulin like growth factor.15
Insulin like growth factor-1 expresses its biological activity when it binds to its receptor which is a member of tyrosine kinase family of growth factor receptors. When insulin like growth factor-1 receptor (IGF-1R) is in its active state, it forms a heterodimer comprised of 2 extracellular a and 2 transmembranous b subunits that are linked by disulfide bonds.15
When binding with the ligand at the a subunits is accomplished, the result is autophosphorylation of the intracellular tyrosine kinase domains in the b subunits. Therefore, tyrosine phosphorylation of the insulin like growth factor-1 receptor produces many docking sites for various SH-2 containing proteins. This in turn leads to signal transduction through multiple pathways.15
Insulin like growth factors demonstrate a range of biological activities, beginning with normal growth and development during early stages of embryogenesis up to the regulation of specific functions for various tissues and organs in the later developmental stages. These activities include proliferation, differentiation, transformation, and antiapoptosis.15
Insulin like growth factor-1 stimulates proliferation and differentiation of osteoblasts which subsequently increases osteogenesis. Not only does insulin like growth factor-1 increase type I collagen formation and bone matrix apposition rate, it also inhibits bone collagen degradation secondary to the blocking of collagenase activity by osteoblasts.15
Preosteoblastic and osteoblast cell lines have demonstrated upregulation of bone associated genes such as osteopontin (OPN) and bone sialoprotein (BSP). Insulin like growth factor-1 receptor stimulates cementoblasts to increase cellular proliferation and bone sialoprotein gene expression, but has no effect on osteocalcin or osteopontin gene expression.15
Insulin like growth factor-1 has also been found to stimulate periodontal ligament cell migration and proliferation, while limiting protein synthesis. Investigators could not detect the receptor for insulin like growth factor-1 in periodontal ligament or gingival connective tissue biopsies, but noted a weak expression of the receptor in regenerated tissues. However, they did detect the insulin like growth factor-1 receptor in a small subpopulation of cells from the periodontal ligament and gingiva. Therefore, in the periodontium, insulin like growth factor-1 exerts more intense effects on hard tissue cells than on soft tissue cells.15
Platelet-derived growth factor has been the subject of extensive study for its capacity to stimulate bone in the jaws, cranium, and long bone defects. Platelet derived growth factor and insulin like growth factor-1 act synergistically to stimulate osteoblast, PDL fibroblast and cementoblast DNA synthesis, and matrix production. In several periodontal disease models, platelet derived growth factor has been used concomitantly with insulin like growth factor-1 to achieve periodontal tissue regeneration.15
In naturally occurring periodontal alveolar bone defects in beagle dogs, the short-term application of platelet derived growth factor-B in combination with insulin like growth factor-1 in a gel carrier was found to promote new bone, cementum and periodontal ligament formation. When clearance studies were done on these factors, the half-life at the site of application was 3 hours for insulin like growth factor-1 and 4.2 hours for platelet derived growth factor-BB. Additionally, platelet derived growth factor and insulin like growth factor-1 were observed to promote rapid bone formation around endosseous oral implants.15
In order to evaluate the safety and efficacy of platelet derived growth factor-B combined with insulin like growth factor-1for treatment of severe periodontal bone defects in humans, investigators conducted a phase I/II human clinical trial. Investigators found no local or systemic adverse effects subsequent to the administration of these growth factors in the treatment of periodontal patients.15
Patients were treated with 150 mg/ml each of platelet derived growth factor-B and insulin like growth factor-1. At 9 months after treatment with these growth factors, significant bone fill of greater than 40% was present compared to the minimal bone fill of less than 20% following standard surgical treatment. In addition, a greater response to growth factors was noted in furcation lesions, with almost 3 mm of horizontal bone fill.15
The collective results from the animal and human studies indicate that platelet derived growth factor alone or platelet derived growth factor in combination with insulin like growth factor-1 are significant stimulants of regeneration in periodontal tissues.15
Basic fibroblast growth factor (FGF-2) is a member of the heparin-binding fibroblast growth factor family which is comprised of over 20 separate FGF proteins. Fibroblast growth factors exhibit a high binding affinity with heparin and heparan-like glycosaminoglycans (see definitions) of the extracellular matrix. In addition they share sequence similarity with about 140 amino acid residues in the core region.15
Basic fibroblast growth factor affects many cell types and tissues derived from endoderm, ectoderm and mesoderm. It is a broad spectrum and pleiotropic mitogen for growth and differentiation. Basic fibroblast growth factor promotes angiogenesis, cell proliferation, and noncollagenous protein synthesis, thereby stimulating wound healing and tissue repair.15
Mutations of fibroblast growth factor receptors are associated with bone and cartilage genetic diseases. This fact emphasizes the intense effect that basic fibroblast growth factor has on bone growth and development. Heparinases or enzymatic cleavage by proteases closely regulate the release of active fibroblast growth factors from the extracellular matrix reservoir.15
Basic fibroblast growth factor promotes human periodontal ligament and endothelial cell attachment and proliferation on dentin slices from teeth.15 Basic fibroblast growth factor is constitutively expressed, i.e. “constantly present, whether there is demand or not,”3 in human periodontal tissues. It is upregulated in early periodontal wounds for up to 7 days. Some molecules are “constitutively produced, whereas others are inducible”3.
Basic fibroblast growth factor was more intensely expressed by the periodontal ligament region than the gingival connective tissue areas. Fibroblasts and endothelial cells derived from periodontal ligament tend to highly express basic fibroblast growth factor, while the expression is less in diseased tissues.15
There is evidence from in vitro studies that basic fibroblast growth factor promotes the proliferative responses of human PDL cells, which express fibroblast growth factor receptors-1 and -2; yet it inhibits periodontal ligament cell production of alkaline phosphatase activity and mineralized nodule formation. In the in vivo situations where serum components are present, the proliferative effect of basic fibroblast growth factor-2 on PDL cells can be synergistically advanced.15
Investigators have reported that basic fibroblast growth factor demonstrates an inverse time and dose dependent effect on periodontal ligament cells by the down regulation of collagen type I expression, and the upregulation of matrix metalloproteinase-1. The high expression of basic fibroblast growth factor by periodontal ligament cells and endothelial cells supports the theory that it plays a role in PDL-mediated mitogenesis and angiogenesis during the early wound healing process.15
When other investigators evaluated the regenerative potential of basic fibroblast growth factor on surgically created alveolar defects in dogs and monkeys, they reported that FGF-2 stimulated significant PDL formation, including deposits of new cementum and formation of new bone in amounts exceeding that found in control lesions. In all of the in vivo experiments with basic fibroblast growth factor sites, no instances of epithelial downgrowth, ankylosis or root resorption were observed.15
Investigators evaluated the efficacy of basic fibroblast growth factor combined with guided tissue regeneration therapy for 90 days in critical size furcation defects in dogs. Investigators reported histological evidence of significant improvement in periodontal regeneration using a low dose (0.5 mg) basic fibroblast growth factor in conjunction with guided tissue regeneration as compared to the high dose (1 mg) basic fibroblast growth factor + guided tissue regeneration, and using only guided tissue regeneration.15
Transforming growth factor beta (TGF-b) and bone morphogenetic proteins (BMPS) are both members of the same peptide superfamily. Transforming growth factor-betas are critical to the processes associated with the regulation of somatic tissue development and renewal. The mature form of transforming growth factor beta is located at the C-terminal and contains about 130 amino acids. The active forms of the 3 isoforms of transforming growth factor beta (TGF-b1, -2 and -3) are normally composed of homodimers linked by disulfide bonds.15
Although bone cells release the active forms of transforming growth factor beta, they are stored in the extracellular matrix of bone and other tissues as latent complexes that are biologically inactive. Extracellular activation of transforming growth factor beta latent complexes serves as a highly significant mechanism in controlling the functions of TGF-b in vivo. The release of latent transforming growth factor beta from the extracellular matrix is triggered by proteolytic enzymes such as chymase, elastase, and plasmin, and this is a closely regulated process.15
Bone morphogenetic proteins (BMPS) are recognized as potent factors that induce bone growth. The first evidence of this came from a study in which investigators extraorthotopically implanted animals with demineralized bone powder and bone extracted proteins to induce bone formation. The primary biological function of bone morphogenetic proteins is development of the embryonic skeleton, and the formation of cartilage and bone (chondro-osteogenesis) in physiologic and pathologic conditions.15
Various different bone morphogenetic proteins are generated during the formation of bone and cartilage in both a spatial and temporal pattern; they regulate specific functions in this cellular process. BMP family members from this peptide superfamily perform a significant function in cell migration, proliferation, differentiation and apoptosis for a variety of cell types, including chondroblasts, osteoblasts, neural cells and epithelial cells.15
Anusaksathien et al.15 reported that there are about 20 family members obtained from bone morphogenetic protein clones. The mature form of bone morphogenetic proteins is located at the C-terminal and is comprised of approximately 130 amino acids with cysteine rich residues. Transforming growth factor beta receptors and bone morphogenetic protein receptors are transmembrane peptides containing a serine/threonine kinase domain.15
Two families of these receptors have been reported: the serine/threonine receptors type -I and -II. Transforming growth factor betas and bone morphogenetic proteins both induce a heterotetrameric receptor complex and each receptor complex is made up of two types of receptor molecules. Transforming growth factor betas and bone morphogenetic proteins first bind to the constitutively phosphorylated type II receptor. It is then that the type I receptor is recognized and recruited to the ligand/type-II receptor complex and phosphorylated by the type II receptor.15
It is through “Smads”, the downstream intracellular proteins, that the activated type I receptor initiates signal transduction. Investigators suggest that the type I receptor has an important role to play in determining the nature of the biological response to the ligand. Phosphorylated Smads form oligomeric complexes; they then translocate into the nucleus where they can regulate transcriptional responses at the genetic level.15
According to Anusaksathien et al.15 transforming growth factor beta is expressed in developing alveolar bone, periodontal ligament, and cementum during all stages of tooth development, especially in osteoblasts, PDL fibroblasts, and cementoblasts near the root apex. In rodents, the expression of transforming growth factor beta receptors type II and III was detected on the cell surface membrane and in cytoplasm in all compartments of the developing periodontium. They also reported a weak expression of transforming growth factor beta receptors I and II in fibroblasts found in normal human gingival connective and periodontal ligament tissues, but an upregulation of the receptors in regenerated tissue biopsies.15
Transforming growth factor beta has been observed to stimulate bone matrix apposition and bone cell replication in cultured fetal rat calvariae. On the other hand, while transforming growth factor beta decreases the mRNA expression of bone sialoprotein, osteocalcin, and mineralization, it increases cementoblast proliferation.15
By contrast, the ex vivo transplantation of cementoblasts treated with transforming growth factor beta-1 produced a net mineral formation. Periodontal ligament derived fibroblasts demonstrated an increase in cellular proliferation, in alkaline phosphatase, collagen and protein synthesis levels, as well as increased chemotaxis in response to transforming growth factor beta.15
The investigators evaluated the distribution of bone morphogenetic proteins -2, -3 and -7 by examining morphogenesis in the murine tooth root. They reported that the distribution of BMP-3 and 7 were localized in alveolar bone, cementum and PDL, but BMP-2 was expressed only in periodontal alveolar bone. On the basis of these results, they suggested that BMP-3 and -7 may have a function in cementogenesis and in assemblage of the periodontal ligament.15
Evaluation of the in vitro effects of BMPs on periodontal tissue cells revealed that bone morphogenetic proteins-2 and 7 did not stimulate growth change. However, by increasing the production of alkaline phosphatase, they did stimulate differentiation of human periodontal ligament fibroblasts into osteoblastic phenotypes. Bone morphogenetic proteins-2 or 3 succeeded in stimulating differentiation of the MC3T3-E1 pre-osteoblastic cell line, but failed to induce proliferation.15
However, in osteoprogenitor cells from newborn rat calvaria, bone morphogenetic protein-7 did stimulate chondroblastic and osteoblastic differentiation.15 Anusaksathien et al.15 noted that various types of recombinant bone morphogenetic proteins, including BMP-2, - 3, and -7, that were delivered by several carrier systems, have been utilized in animal models for the regeneration of periodontal tissue.
Other investigators have used either bone morphogenetic protein-2 or -7 in animal models of periodontal disease to achieve a potent induction of cementogenesis and osteogenesis. In addition, they reported evidence of bone growth induced by these bone morphogenetic proteins around endosseous dental implants and in sinus augmentation procedures. More recent studies demonstrated the potential for bone induction by these bone morphogenetic proteins in human oral reconstruction for dental implants and sinus floor elevations.15
The results from several animal and humans studies revealed a very significant impact due to bone morphogenetic proteins-2, 3, and 7 that is incorporated with resorbable carrier systems in inducing regeneration in periodontal tissues. Severe bony defects in sheep were treated with transforming growth factor beta-1 with and without guided tissue regeneration. A substantially greater amount of periodontal tissue regeneration was reported when the defects were treated with transforming growth factor beta-1 + guided tissue regeneration or transforming growth factor beta-1 alone compared to the carrier treated defects.15
There was no significant difference in cementum formation among the three groups. The results of this study on sheep revealed that transforming growth factor beta-1 did induce regenerative bone in furcation defects, but that this effect was augmented by the use of a barrier membrane.15
In a dog model of supra-alveolar critical size defects, researchers reported only a limited amount of bone and cementum regeneration. Also, there were no differences in healing response when transforming growth factor beta-1 with guided tissue regeneration, GTR plus carrier, or carrier alone were applied to the defects. According to these studies and others as well, it appears that transforming growth factor beta-1 offers only limited prospects for the regeneration of periodontal tissues.15
Enamel matrix derivative (EMD) includes proteins that belong to the amelogenin family, (the hydrophobic constituent of the enamel matrix proteins). There is evidence from early studies suggesting that enamel matrix derivative plays a role in the forming acellular cementum during tooth development; and furthermore, that it possesses the potential to stimulate the regeneration of acellular cementum in periodontal disease.15
Enamel matrix derivative is known to induce cellular proliferation, protein synthesis and mineral nodule formation in several cell types including PDL cells, osteoblasts and cementoblasts. When enamel matrix derivative was used to treat murine primary osteoblast, pre-osteoblast, and cementoblast cells, the expression of bone associated genes including collagen type I, OPN, and BSP were upregulated. However, osteocalcin (OCN) was not upregulated.15
In addition, when human pre-osteoblast (MG-63) and normal osteoblast cell lines were treated with enamel matrix derivative, researchers reported the induction of differentiation by increased alkaline phosphatase activities. Although, the in vitro formation of mineral nodules by cementoblasts was inhibited by enamel matrix derivative, mineralization was promoted when enamel matrix derivative was used to treat cementoblast implants embedded subcutaneously in scid mice.15
Compared to no serum treatment in vitro, gingival tissue and PDL derived human fibroblasts, as well as MG-63 cell lines, demonstrated enhanced cellular migration and wound closure rate in response to treatment with enamel matrix derivative. Enamel matrix derivative also induces the production of growth factors such as PDGF-AB, TGF-? and IL-6 in MG-63 and PDL cells. The results from these in vitro studies substantiate the efficacy of enamel matrix derivative in periodontal regeneration.15
Investigators evaluated the ability of enamel matrix derivative (EMD) to affect periodontal wounds in a tooth dehiscence model in nonhuman primates. There was significant enhancement of bone, cementum and periodontal ligament in defects treated with EMD, compared to treatment with a carrier and EDTA root conditioning (a chelating agent that removes magnesium and calcium3).15
In human studies on the use of EMD, human histology confirmed true regeneration in patients treated with enamel matrix derivative. Enamel matrix derivative was reported to be safe in a multicenter trial of 10 test centers with a total of 107 patients. Efficacy was demonstrated in a placebo-controlled human trial of 33 subjects with paired intrabony defects. Measurements of bone fill in defects 3 years after therapy with enamel matrix derivative confirmed the presence of significant bone fill; however, there was no change in radiographic bone level in paired control defects.15
Advanced periodontal defects in a dog model were used to test the effect of treatment with enamel matrix derivative in combination with guided tissue regeneration. After 4 months, both enamel matrix derivative alone, and EMD + guided tissue regeneration therapies resulted in similar amounts of mineralized bone and bone marrow formation with periodontal ligament fibers inserting into newly formed cellular cementum. Based on these findings, the investigators proposed that enamel matrix derivative stimulates cementogenesis during repair in periodontal wounds.15
Enamel matrix derivative was evaluated for the presence of osteoinductive, osteoconductive and/or osteopromotive properties by investigators (see definitions). Different doses of enamel matrix derivative were combined with inactive or active demineralized freeze-dried bone allograft (DFDBA) and then implanted ectopically in nude mice for a period of 8 weeks.15
Since no significant new bone formation was detected in subjects treated with varying doses of enamel matrix derivative in combination with inactive DFDBA (demineralized freeze dried bone allograft) groups, the investigators concluded that EMD is not an osteoinductive factor. Although with threshold concentrations, enamel matrix derivative is osteopromotive, partly because of its osteoconductive properties. Enhanced osteogenesis occurred only when active DFDBA was combined with high doses of EMD.15 Researchers have used biochemical extraction techniques to demonstrate the presence of growth and differentiation factors in DFDBA preparations; however, various reports indicated that some lots of commercially available DFDBA yielded unpredictable or poor bone formation.22
The collective results from various in vivo studies demonstrated that all of the growth factors mentioned above, with the exception of TGF-b1, have the capacity to induce regeneration of periodontal tissues. Bone morphogenetic proteins demonstrated the greatest potential because they are strong osteoinductive growth factors.15
In addition, compared to the regenerative potential of a single growth factor, combinations of growth factors, i.e. PDGF/IGF-I, were far more effective in stimulating regeneration. In comparison to other growth factors, high levels of FGF-2 are required for periodontal wound healing. Consequently, further studies are needed on other growth factor combinations to achieve the best regenerative outcome.15
The discipline of tissue engineering has concentrated on using biomimetic materials that simulate natural extracellular matrices to deliver cells, proteins and genes. Polymer scaffolds are made from various materials such as polylactic acid and polyglycolic acid copolymers; they have been widely used in tissue engineering. Scientists are utilizing these materials to maximize the usefulness and effectiveness of cells, proteins and genes in treating periodontal tissue defects.15
Large scale purification and production of bioactive factors together with effective methods for delivering these molecules to tissue defects are necessary for successful tissue engineering. Trials utilizing topical growth factors have revealed difficulties in maintaining therapeutic levels of proteins at the defect site, and thus failure to achieve clinically significant improvements in tissue regeneration.15
Existing protein carrier systems provide such a short duration of action of the applied growth factor that gene therapy may serve as an alternative method of targeting proteins to an oral wound. Gene therapy has been used by researchers to treat several diseases that cause significant tissue deficiencies. There are two critical steps in the approach to gene therapy: 1. Delivery of DNA to the appropriate target cells, and 2. Expression of transgene encoded proteins in a therapeutic fashion.15
A transient expression of the transgene may be optimal to restore the tissue deficiency during wound healing. Adenovirus and DNA-lipid complexes are two examples of methods of gene delivery for short-term expression of genes. Gene therapy in a chronic wound such as periodontal disease may be enhanced by an elevated and sustained production of GFs to promote tissue repair, since the regulation of wound repair occurs in a controlled fashion over a short period of time.15
For in vivo and ex vivo approaches, growth factor gene therapy for tissue engineering uses the insertion of GFs or wound-healing cytokines into transiently expressing viral vectors. Growth factor genes have been delivered by several research groups to heal skin, bone, and periodontal wounds via the use of plasmid DNA. Another method that offers promise in tissue repair is the use of biodegradable polymers to deliver PDGF DNA.15
Adenovirus has demonstrated the ability to promote high but transient transgene expression. Consequently, it has been used extensively to promote tissue repair. Also showing great promise in promoting the regeneration of soft tissue is adenoviral gene delivery of PDGF.15
Additionally, stimulation of cellular proliferation and PDGF-inducible genes can be achieved by targeting PDGF to PDL cells including cementoblasts. Impressive results have been reported by researchers who delivered BMP genes by adenovirus to orthotopic wounds by ex vivo approaches. Unique methods of transducing wounds by the in vivo microseeding technique have been developed by other researchers.15
Much work remains to be done to achieve optimal delivery of growth factors using various approaches to improve bioavailability of signaling molecules. Significant advances in materials science and gene therapy may provide the methods needed to optimize growth factor targeting to the periodontium by enhancing both the extent and duration of growth factor exposure to putative tissue repair cells. These advances portend a bright future for reconstructive periodontal therapy.15