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In 1835 cementum was first described. Since then, it has been an inadequately understood tissue both at the cellular and molecular level until recently. Studies over the years have confirmed that cementum is a histologically distinctive tissue. Cementoblasts are the cells that form cementum; yet, research on the proteins expressed by cementoblasts has failed to provide clear identification of proteins that are specific to cementum.10
Actually, studies on dentin and enamel revealed definite differences in both the proteins found in these tissues and the factors that control their function when they were compared to bone. However, cementum seems to possess factors in common with those related to bone and appears to be regulated by like factors developmentally.10
Some investigators proposed that factors provided by epithelial cells residing within the local environment may partly modulate the differentiation of follicle cells into cementoblasts, resulting in the deposition of cementum. Another theory suggested that epithelial root sheath cells are transformed into cementoblasts, thereby providing the appropriate matrix for the formation of cementum. Thus, the belief that follicle cells have the ability to differentiate into an osteoblast, a cementoblast, or a periodontal ligament fibroblast remains in question and is being re-examined.10
Histological evidence is mounting that the formation of cementum is integral for normal maturation of the periodontium, not only during development but also during periodontal tissue regeneration. Years of research suggest that cementum is thought to play a key role in the reparative process subsequent to periodontal disease. In spite of this, not much is known about cementoblasts, the cells that produce cementum. The majority of the current knowledge has resulted from thorough research on the histology and composition of cementum.10
Studies utilizing light and electron microscopy have contributed significantly to the classification of cementum into five different subtypes, depending on the presence or absence of cells, i.e. cellular or acellular, and the source of the collagen fibers, i.e. extrinsic or intrinsic. Unlike bone, these subtypes lack both innervation and blood vessels, and demonstrate little or no remodeling.10
In other respects cementum and bone share many similarities. First, diseases that alter the characteristics of bone frequently alter the characteristics of cementum too. Paget’s disease causes hypercementosis. Hypophosphatasia leads to an absence of cementum formation and then exfoliation of teeth. Decreased cementum is seen in hypopituitarism. Defective cementum is found in patients diagnosed with cleidocranial dysplasia.10
Second, cementum and bone share a similar composition. The composition of cementum is about 50% hydroxyapatite, 50% collagen, and some noncollagenous proteins. Proteins extracted from mature cementum stimulate cellular attachment and migration, and they also stimulate gingival fibroblasts and periodontal ligament cells to synthesize protein.10
Studies conducted on these protein extracts from mature cementum demonstrated that they contained bone sialoprotein, osteopontin, vitronectin, and fibronectin. Immunocytochemistry and in situ hybridization established that these proteins were present, and in addition identified osteocalcin, g-carboxyglutamic acid, osteonectin, proteoglycans, and several growth factors.10 In situ hybridization is a molecular biology term that describes “the use of a DNA or RNA probe to detect the complementary DNA sequence”.3 Furthermore, two extra molecules, an adhesion molecule and a growth factor, were identified that might be unique to cementum according to preliminary data.10
Cementum attachment protein may be a collagen like molecule specific to cementum. A factor originally named cementum-derived growth factor is currently thought to be an insulin-like growth factor-I–like molecule and, although unproven, may possibly possess properties similar to those of insulin like growth factor-I.10
The aforementioned factors are recognized for their potential to have a dynamic role in initiating cementum formation, while contrasting the role played by specific factors during the development of cementum compared to the repair and regeneration of cementum. Also under consideration is the obvious success of these agents in regeneration of cementum. Items for future consideration will include the significance of information derived from ongoing studies at the cellular, molecular, and tissue engineering level, up to the design of attractive therapies for regeneration of periodontal tissues.10
Events
Many of the events vital to cementum formation are well known, but the specific cells and factors necessary to the formation of this tissue during development and regeneration remain a mystery. The idyllic characteristics of mediators that could be used successfully in the regeneration of tissues are the capacity to regulate migration and attachment of appropriate cells to the healing site, followed by the stimulation of those cells for differentiation into osteoblasts, cementoblasts or periodontal ligament fibroblasts as needed.10
Of equal importance would be the ability of a mediator to stimulate mineralization via deposition of new cementum along the root surface, and to complete the formation of the periodontium by inserting periodontal ligament fibers into the cementum and opposing alveolar bone. While this simple description of events implies that “regeneration recapitulates development”, these two processes require different events and different cells in order to achieve their biological destiny.10
For example, appropriate clot formation is of paramount importance to achieve satisfactory wound healing followed by true regeneration of any tissue. Not only that, but there is a normal inflammatory response that occurs during wound healing leading to the release of various cytokines and growth factors that may not be related to the development of the tissue in question.10 However, factors thought to play a role in the regulation of tooth root cementum during development may still be valuable aids for regenerative therapy even though they may not be directly related to the regeneration of cementum.10
The definitive functions of these factors are not yet clearly understood, even though the presence of a large number of them during development and regeneration of periodontal tissues ha10s been confirmed. Additional research directed at clarifying the significance of these factors in relation to cell function will shed light on the role played by these factors in the regulation of periodontal tissues.10
Cells
Studies examining the healthy periodontium revealed that various types of mesenchymal cells are necessary to maintain a healthy periodontium. For example, the purpose of periodontal ligament fibroblasts is to provide for a functional periodontal ligament. Osteoblasts and related progenitor cells preserve the surrounding alveolar bone. Cementoblasts are cells that line the root surface; while they appear to have a restricted function in health, they may well be activated to participate in the wound healing process. Paravascular/marrow cells supply the necessary local nutrients to the area.10
In comparison, the cells responsible for the formation of periodontal tissues during development as well as during repair/regeneration are less well defined, and thus they remain the subject of concentrated study in various laboratories.10 Current evidence suggests that during development, ectomesenchymal cells, follicle cells and dental papilla cells can be stimulated to behave like cementoblasts, periodontal ligament fibroblasts, or osteoblasts.
There is also significant evidence that follicle cells and factors secreted by these cells are critical for the normal eruption of teeth, independent of root formation. Certain factors may stimulate differentiation of follicle cells and even transformation of Hertwig’s epithelial root sheath cells in order to function like cementoblasts. The exact types of cells (or cell) that have the ability to function as cementoblasts during wound healing have still not been determined.10
There is also the possibility that a little population of periodontal ligament cells found in the mature periodontium may posses the ability to differentiate in the direction of an osteoblast or cementoblast phenotype, subsequently producing bone or cementum. There is mounting evidence to suggest that periodontal ligament fibroblasts may act to inhibit mineralization.10
From this evidence we can assume that distinct cell populations may exist within the periodontal ligament region; and, that under the influence of the appropriate stimulating factors, these cells have the ability to both promote and inhibit formation of mineralized tissue. Marrow stroma and paravascular and endosteal fibroblasts are other probable sources of cementoblast or osteoblast progenitor cells. In order to design regenerative therapies that are predictable, researchers need to describe not only the cells necessary for regeneration but the trigger factors that control cell activities as well.10
Factors
The factors to be considered are those known to be related to cementum during development, maturation, and regeneration, including incompletely described molecules thought to be associated with cementum. A large number of these factors may play a part in controlling various cell activities such as:10
Adhesion and chemotactic factors
It is vital that cells posses the ability to interact with the local environment, to be attracted to the site of action, and to remain there. Cells accomplish this via receptor-ligand interactions. Researchers have identified various ligands that seem to have a significant role in attracting cells to specific sites during tissue development. One of the most extensively studied of these molecules is fibronectin. Not only does it play a role in tissue development, but it is also thought to have a significant role in the attraction and maintenance of appropriate cells at healing sites.10
Development
In situ studies have provided evidence that, during the early stages of tooth root development, cementoblasts (cells found along the root surface) express the adhesion molecules osteopontin and bone sialoprotein. However, cells that express collagen are found all through the surrounding soft connective tissue, follicle cells and periodontal ligament fibroblasts.10 In situ is a term that means “in the natural or normal place”.3
In the mature tooth, bone sialoprotein messenger RNA and protein persist in their localized position at the root surface, while the osteopontin protein is found within the periodontal ligament area. At the beginning of cementum formation, laminin has been noted on the dentin surface. It has been proposed that this protein functions to attract appropriate cementoblast-like cells to the root surface. Epithelial cells also secrete known factors and as yet unidentified novel factors that may contribute to the migration and/or adhesion of specific cells to the surface of the root.10
Enamel matrix proteins may stimulate cementoblast activity including proliferation, migration/adhesion, as well as cell differentiation, and this is the key rationale that supports the use of enamel matrix derivative. This cell differentiation would be synonymous with the recognized epithelial-mesenchymal interactions between ameloblasts-odontoblasts to secrete substances necessary for the development of both enamel and dentin.10
Various roles have been proposed for bone sialoprotein including functioning as an adhesion molecule to support applicable cells at the root surface and as an initiator of mineral formation along the surface of the root. During cementogenesis and bone formation, the temporal and spatial expression of bone sialoprotein is significantly consistent with a role for this molecule in the promotion of mineral formation.10
The phosphoglycoprotein osteopontin contains the well known adhesion domain, arginine-glycine-aspartic acid (RGD), as does bone sialoprotein. The adhesion domain RGD is directed toward specific integrin receptors in addition to other adhesion regions that act to support in vivo migration and cell adhesion.10
Bone sialoprotein is selective to mineralized tissues. In contrast, osteopontin is expressed by various cells; and it remains localized to several tissues where its function may be associated with post-translational alterations within specific tissues. In reference to bone and cementum, osteopontin is expressed during times of osteogenic and cementogenic activation in situ.10
Osteopontin has been associated with the control of ectopic crystal formation, and data advances the role for osteopontin in regulating the degree of hydroxyapatite crystal nucleation and/or growth. Studies also report the inhibition by osteopontin of apoptotic events like those associated with inflammation. This inhibitory ability may play a key role in regulating cells present at sites during the development of cementum and also during wound healing.10
Thus, while bone sialoprotein and osteopontin demonstrate a significant role in recruiting and supporting selective cells at the root surface, both may have a similarly important role in controlling mineralization along the root surface as well.10
Type I collagen is the most abundant protein found in cementum. Not only does type I collagen promote cell attachment, but it also plays a critically important role in maintaining the integrity of both soft and hard connective tissues during the processes of development and repair. Type XII collagen has a less clear role in the development and maintenance of a functional periodontal ligament.10
Researchers have established that periodontal ligament cells express type XII collagen during the later stages of root formation; however, follicle cells do not express it prior to root development. It remains unclear whether type XII collagen plays a part in the promotion of cell adhesion; but through association with type I collagen, it may promote the formation and maintenance of a functional periodontal ligament and prevent ankylosis.10
Regeneration
Recently conducted in vivo research, using rat periodontal defect models, has demonstrated that cells within the newly forming periodontal ligament express osteopontin, and cells related to the formation of mineralized tissues express both bone sialoprotein and osteopontin. Investigators have recommended studies to further evaluate the over expression or blocking expression of these molecules during wound healing in the hope that they will yield information on how these molecules function in mineralized tissues and to determine the efficacy of using these substances clinically to promote periodontal tissue regeneration.10
Maturation
In addition to the adhesion molecules under discussion above, vitronectin and cementum attachment protein are also found in mature cementum. So far the research data confirms that cementum attachment protein and type I collagen are significantly homologous.10
Determination of the definitive specificity of cementum attachment protein to cementum and whether or not it is a unique protein depend upon future research as well as the accessibility of DNA probes to identify the cells that express cementum protein for the duration of tooth root development and maturation.10
Proteoglycans are ubiquitous to all connective tissues. Proteoglycans, in addition to the classical adhesion molecules discussed here are thought to have a role in the regulation of the cell-cell and cell-matrix interactions occurring both during development and in regeneration of cementum.10
Proteoglycans are associated with various aspects of the cell including the cell matrix, cell surfaces, and cell organelles. Highly anionic molecules, proteoglycans are involved with various cell functions including hydration of tissues and storage for growth factors. They regulate growth factor activity and control the formation of collagen fibers, cell adhesion, cell growth and cell differentiation.10
Mitogens
Factors that support cell proliferation clearly provide a critical mass of cells at a specific location. This is necessary to achieve the following synthesis and secretion of molecules that create the extracellular matrix required for cell differentiation and mineralization.10
During development of the tooth root, investigators have identified mitogens including members of the transforming growth factor-beta superfamily, growth hormone, insulin-like growth factor- I, II, and parathyroid hormone–related protein.10A mitogen is a substance capable of inducing mitosis of certain eukaryotic cells.3
Studies have identified far more growth factors during odontogenesis, including fibroblast growth factor, platelet-derived growth factor, and growth hormone, but they have yet to describe their specific relationship to root formation. Even though these molecules are referred to as growth factors, their activity during tooth development does not seem to be related to proliferation.10
Certain transforming growth factor-betas and parathyroid hormone–related protein possibly provide for regulation of cell differentiation and then mineralization. In addition to these factors, investigators extracted cementum-derived growth factor from mature cementum; this factor promotes the proliferation of gingival fibroblasts and periodontal ligament cells.10
Since cementum-derived growth factor is similar to insulin-like growth factor- I, investigators have yet to determine if this molecule is unique to specific tissues. Other studies indicate that both insulin-like growth factor and growth hormone may exert independent influence on the promotion of cementogenesis, and possibly both may be able to induce expression of bone morphogenetic proteins.10
Differentiation
Investigators have increased their efforts to identify the factors that control differentiation of cells during root development into cells that can function as periodontal ligament cells, cementoblasts and/or osteoblasts, in order to provide information for the development of appropriate agents to support periodontal tissue regeneration.10
The precise mechanisms, cells or factors needed to promote formation of periodontal tissues has not yet been determined, even though the factors discussed here are likely contenders with various ones demonstrating some success clinically.10
Development
It is well known that specific molecules participate in the epithelial-mesenchymal signaling that is vital to the development of many tissues, including lung, heart, hair follicles, and the tooth crown (enamel and dentin). However, the specific mechanisms or factors utilized in the regulation of cementum formation remain elusive.10
Significant data provides evidence for the function of both bone morphogenetic proteins 2 and 4 as molecules vital to the epithelial-mesenchymal signals necessary to the formation of enamel and dentin. On the other hand, based on their expression pattern, bone morphogenetic proteins 2, 4 or 7 do not seem to provide epithelial-mesenchymal signaling molecule10s or perform any function in cememtum formation.10
However, since follicle cells surrounding first molars in mice are known to express bone morphogenetic protein-3 transcripts, it is thought that bone morphogenetic protein-3 possibly has a role in the formation of cementum.10 More recently researchers have reported the expression of bone morphogenetic proteins at later stages of root development. Particularly, only bone morphogenetic protein-3 was expressed at later stages of root development, even though bone morphogenetic proteins 2, 4 and 7 were also examined.10
In unpublished data, bone morphogenetic protein-3 was not only reported to be selective to root lining cells, but it was not expressed by cells within the periodontal ligament region. To conjecture further, bone morphogenetic protein-3 may possibly have a significant function in the differentiation of follicle cells along the cementoblast pathway.10
Several studies have utilized immunohistochemical, in situ hybridization and recombination methods to evaluate the importance of epithelial products in influencing the cementoblast phenotype. These studies have produced evidence of a role for products secreted by the epithelial root sheath in controlling the formation of cementum.10
Some of these factors have been identified and some remain elusive. Yet, it is thought that they may attract cells to the root surface, as laminin does; or they may encourage differentiation of follicle cells along the cementoblast pathway, for example, sheathlin, which is also referred to as ameloblastin and amelin.10
It is well known that alkaline phosphatase regulates the formation of mineralized tissues, as well as cementum. More recent studies including histological examination of teeth from alkaline phosphatase knock out mice provided data indicating that alkaline phosphatase may play a more vital role in regulating the formation of acellular cementum versus cellular cementum.10
Not only is this an interesting finding but it is potentially significant in the regeneration of cementum because the cementum that is formed appears to be cellular in nature. Therefore, factors that regulate the formation of cellular cementum may be dissimilar compared to those that control the formation of acellular cementum.10
Parathyroid hormone–related protein is another molecule that may be found to have a significant role in the expression of the cementoblast phenotype. Evidence is increasing to support the theory that parathyroid hormone–related protein has a role in controlling the early stages in the development and eruption of teeth.10
Rescued parathyroid hormone–related protein knockout mice exhibited characteristics of diminutive stature, cranial chondrodystrophy, and a lack of tooth eruption.10 Knockout is an “informal term for the generation of a mutant organism in which the function of a particular gene has been completely eliminated (a null allele)”.3 Although, it is still uncertain what role parathyroid hormone–related protein plays in the development of the tooth root.10
The presence of both parathyroid hormone–related protein messenger RNA and the associated parathyroid hormone/parathyroid hormone–related protein type I receptor has been documented in developing periodontal tissues. Furthermore, researchers have demonstrated that an extract of parathyroid hormone augments both tooth eruption and orthodontic tooth movement.10
Other factors including insulin-like growth factor and proteoglycans are found in developing and mature cementum. They may have more than one function, among them the supervision of mineralization and control of cell differentiation.10
In addition to these factors that we have reviewed, various studies propose that the transcription factor known as core binding transcription factor 1/osteoblast-specific transcription factor 2 is vital to the activation of osteoblastic differentiation. It is also significant that throughout tooth development in mice, mesenchymal cells (including follicle cells and cementoblasts) as well as ameloblasts all express osteoblast-specific transcription factor 2.10
Regeneration
Since some of these factors may have the potential to stimulate regeneration, various factors have been employed in models of periodontal regeneration. During wound healing, platelet-derived growth factor and platelet-derived growth factor-receptor have been localized to periodontal tissues. In this context, retaining adequate amounts of a specific factor at the local site is thought to be a limitation to current regenerative therapies. Therefore, examination of the amount of specific growth factors and their related receptors found at a specific wound healing site, such as a non-critical size defect, may assist in revealing the type and amount of essential factors necessary for stimulating regeneration at healing sites.10
Maturation
Mature cementum, similar to other mineralized tissues, is comprised of various factors, either secreted by cementoblasts or absorbed by hydroxyapatite during the process of root formation.10
Mineralization
It has been suggested that various factors play a significant role in regulating mineralization, but what remains to be confirmed is the explicit role played by individual molecules. The understanding of these molecules in the mineralization process, including cementogenesis, has been increased by studies directed at mapping the expression pattern for certain molecules developmentally, along with the use of knock-out mice.10
Development
Although it has been suggested that various factors play a role in regulating mineralization, they may also perform other functions. In regard to the concept of multifunctional roles, the projected roles for bone sialoprotein, osteopontin, and type I and type XII collagen in regulating crystal growth were addressed under the headline of adhesion molecules both during development and in periodontal tissue regeneration.10
Both osteocalcin and proteoglycans, in addition to these molecules, are thought to be significant in the regulation of mineralization in several tissues including cementum. Researchers have established that there is a selective expression of osteocalcin in root lining cells, cementoblasts; and that this profile of expression is maintained in mature tissue. In other words, periodontal ligament fibroblasts do not express osteocalcin.10
However, investigators have not yet evaluated the expression pattern for matrix “gla” protein during root development and in mature tissues. “Gla is 4-carboxyglutamic acid. A carboxylated form of glutamic acid found in certain proteins (e.g., prothrombin, factors VII, IX, and X, osteocalcin). Its synthesis is vitamin K-dependent.”3 A prominent role for osteocalcin in root development is supported by the temporal and spatial expression pattern for osteocalcin during cementogenesis and the selectivity of osteocalcin to mineralized tissues.10
Data implying that osteocalcin may have a role in controlling mineral formation comes from studies demonstrating that transgenic mice that lack the osteocalcin gene develop a phenotype characterized by enhanced mineral density.10 Transgenic is a term used in molecular biology to describe “an organism that has had genes from another organism put into its genome through recombinant DNA techniques”.3
From this we can surmise that osteocalcin may be one of the various molecules that regulate the mineral-to-ligament ratio, thus, favoring the formation of the periodontal ligament region rather than ankylosis. Formation of the periodontal ligament as opposed to ankylosis is one of the defining characteristics of true regeneration versus repair.10
Regeneration
The fact that both osteopontin and bone sialoprotein are expressed in cells and tissues related to periodontal wound healing was emphasized in the discussion of adhesion/migration factors. Osteocalcin and collagen types I, III and XII have also been linked to wound healing and/or stress to the periodontal tissues in association with orthodontic tooth movement. Proteoglycans, in addition to these molecules, probably have a vital role during wound healing in creating a balance between the formation of cementum and surrounding bone while a functional periodontal ligament is developing.10
Maturation
All of the non-collagenous molecules discussed above are contained in mature cementum. While a high percentage of type I collagen is found mature cementum, other collagens including type III and type XII collagen are found in lesser amounts. Periodontal ligament fibroblasts may produce the small amount of other collagens recognized in cementum, where formed collagen fibers are inserted into the cementum.10
Thorough comprehension of the molecular consequences secondary to the interactions between factors and their corresponding receptors is pivotal in the design of agents used to regenerate tissues. Fortunately, considerable advances have been made that enhance our understanding of the role played by regulators that control cell function in healthy and diseased states.10
Due to lack of availability of the necessary cell types, it is only recently that more studies have been carried out on cells thought to be related to root development, such as epithelial root sheath cells, follicle cells and root surface cells/cementoblasts. Conversely there is more information about cells that are related to the mature periodontium, such as osteoblasts and periodontal ligament cells.10
Emphasis of the fundamental concepts related to ligand–receptor interactions serves to stress their importance and complexities. This in turn enhances our understanding of the basic mechanisms that regulate cell activities, so necessary to the art and science of designing regenerative therapies.10
In a hypothetical model, for instance, factor A may be capable of stimulating mineralization of mature osteoblasts but not of preosteoblasts or periodontal fibroblasts. This inability to induce mineralization may be due to an absence of appropriate receptors on the preosteoblasts and periodontal fibroblasts. Hypothetically, if factor B is added, it may be able to prime these cells, enabling them to express the receptor needed to respond to factor A.10
It is with this in mind that we examine a broad overview of the current knowledge of growth factor–cell interactions, and how this may be related to the control of development and/or regeneration of periodontal tissues.10
Growth factors display many different effects on cells subject to the specific factor, the type of cell, and the stage of maturation. As an example, growth factors can stimulate DNA synthesis, regulate differentiation and modify the cytoskeleton. The local effect of growth factors is ensured by their short half-life and their relationship with extracellular matrix and growth factor–binding proteins.10
Specific cell surface receptors enable extracellular matrix molecules and growth factors to exert their effects. Binding of the cell surface receptor enables it to interact with cytoplasmic effector molecules and initiate a complex cascade of intracellular events that ultimately alter gene function.10
A prototype is a primitive form, and thus it is the first form to which subsequent individuals of the class or species conform.3 As prototypic members of a family of cell surface receptors, growth factor receptors are characterized by an extracellular binding domain, a single transmembrane portion, and a large intracellular catalytic domain.10
More specifically, the growth factor receptors that have intrinsic tyrosine kinase domains are members of the hydrophilic receptor family; they are known as receptor tyrosine kinases. Among the signaling molecules for this group of receptors are:10
The kinase domain is autophosphorylated as the ligand binds to the receptor. Dimerization of the receptor complex allows transmission of the molecular signal from the ligand to the cytoplasmic domain of the receptor. Activation of the receptor elicits intracellular cascades of protein phosphorylations that promote protein interactions. The end result of these interactions is the transmission of specific signals to the nucleus that control gene expression and ultimately alter cell function.10
The mitogen-activated protein kinase pathway serves as a good example of the complexities of these activities. Mitogen-activated protein kinase phosphorylates transcription factors resulting in the activation of specific genes.10 Mitogen-activated protein kinase is comprised of 3 isoforms: mitogen-activated protein kinase and extracellular signal–regulated kinase-1 and -2 ; it is activated by growth factors and other mitogens. Human osteoblasts, human bone marrow stromal cells, and rat and mouse osteoblastic cells all contain extracellular signal–regulated kinase-1 and -2 proteins.10
Extracellular signal–regulated kinase-2 is activated by insulin-like growth factor-I, fibroblast growth factor- 2 and platelet-derived growth factor-BB. In addition, receptor tyrosine phosphorylation stimulates interactions with various other molecules such as phospholipase-Cg, non-receptor Src family of kinases, tyrosine phosphatases and phosphoinositide-3-kinase.10
Several growth factors including members of the transforming growth factor-b super family, platelet-derived growth factor, insulin-like growth factor, epidermal growth factor, fibroblast growth factor, prostaglandin E, and parathyroid hormone/parathyroid hormone–related protein interact with periodontal fibroblasts and/or osteoblasts.10
In 1991 researchers isolated a factor from cementum that became known as cementum derived growth factor. Data from later studies revealed that cementum-derived growth factor is an insulin-like growth factor-I-like molecule. Investigators have reported that insulin-like growth factor-I enhances the effects of various growth factors and other molecules on cellular activity.10
Cementum-derived growth factor brings about a temporary increase in cytoplasmic Ca2+ concentration, promotes phosphoinositol-phosphate hydrolysis, activates the protein kinase-C cascade, and increases expression of cellular protooncogenes in gingival fibroblasts. These responses are similar to those reported with growth factors such as insulin like growth factor-I.10
Together with their complex interactions with other molecules including other growth factors and integrins, growth factors interacting with their specific receptors bring about a complex set of cell responses. Therefore, the in vitro effect of a growth factor on a specific cell can be significantly different from the in vitro effects of that factor on another cell type, or the in vivo effect on the same cell type. Growth factor regulation of cementogenesis has been examined in only a few studies, but more extensive studies have been done with animal models, versus in vitro or in situ models.10
In situ models have been used with radioautography and 14-day-old rats to evaluate the role of epidermal growth factor on differentiation of cementoblasts after injecting 125I-epidermal growth factor. The results revealed a limited effect of epidermal growth factor on cementoblast differentiation, because during differentiation of cementoblasts a very low level of epidermal growth factor–binding sites were present on the mesenchymal cells in dental follicle proper, precementoblasts and cementoblasts.10
Yet, epidermal growth factor–binding sites were noted in vivo on preosteoblasts, prechondroblasts, perifollicular cells and mature periodontal ligament fibroblasts. Therefore, the conclusion drawn from these studies is that the interactions between epidermal growth factor and epidermal growth factor-receptor are not involved directly in the regulation of cementoblast activities. Even so, they do support the theory that epidermal growth factor and related interactions with other cells during tooth/periodontal development are vital for the development of a functional periodontium.10
G-protein coupled receptors are cell surface receptors that are coupled to G-proteins (GTP-binding protein). GTP is guanosine 5'-triphosphate. It is an immediate precursor of guanine nucleotides in RNA; it is similar to ATP, and it has a crucial role in microtubule formation.3
G-proteins are “intracellular membrane-associated proteins activated by several (e.g., beta adrenergic) receptors. They serve as second messengers or transducers of the receptor-initiated response to intracellular elements such as enzymes to initiate an effect. They are also mediators of activated cell-surface receptors and their enzymes or of ion channels. They are responsible for activating a chain of events that alters the concentration of intracellular signaling molecules such as cyclic AMP and calcium. In turn, these intracellular messengers alter the behavior of other target proteins within the cell. These proteins have a high affinity for guanine nucleotides and hence are named "G" proteins”.3
G-protein-coupled receptors are linked with the capacity of growth factors to initiate the mitogen-activated protein kinase cascade and other tyrosine kinase pathways. G-proteins function as the initial effector activating substrate for multipass receptors. Adenyl cyclase, phospholipase C, phospholipase A2, phosphoinositide 3-kinase, and beta-adrenegic receptor kinase are the major downstream effector molecules controlled by G-proteins.10 “Downstream is a molecular biology term indicating portions of DNA or RNA that are more remote from the initiation sites and that will therefore be translated or transcribed later. It is a shorthand term for things that happen at a late stage in a sequence of reactions.”3
Cyclic adenosine monophosphate, diacylglycerol, inositol triphosphate and calcium are activated second messengers. They are small molecules that amplify receptor-activated signals. In addition, the mitogen-activated protein-kinase pathway is known to be activated by many growth factor–G protein interactions, such as platelet-derived growth factor and insulin-like growth factor-II.10
In the case of cementum, cementoblasts are known to express parathyroid hormone–related protein receptors. Thus, both parathyroid hormone and parathyroid hormone-related protein lead to an increased amount of cyclic adenosine monophosphate in these cells. By means of G-protein-linked receptors, parathyroid hormone/parathyroid hormone–related protein induce several parallel signaling events that include activation of adenyl cyclase (protein kinase-A pathway), phospholipase C (protein kinase-C pathway) and cytosolic free calcium transients.10
Investigators have confirmed that transforming growth factor-beta superfamily molecules create their effects by interacting with serine/threonine kinase receptors. After a group of signaling molecules known as Smads interact with and activate the receptor, they are phosphorylated selectively by bone morphogenetic proteins and form heteromeric complexes (complexes having a different chemical composition3). Smads then enter the cell nucleus and proceed to induce transcriptional activation, subsequently altering cellular behavior.10
We know that bone morphogenetic proteins perform many functions, including regulation of cell growth, differentiation and apoptosis in a variety of cell types, such as osteoblasts, neural cells, and epithelial cells. Unfortunately, the precise role that they play in the formation of cementum has not begun to be explored until now. Later, we will review their role in periodontal tissue regeneration.10
The attachment of appropriate cells on the root surface is a prerequisite for the development and regeneration of cementum. During assorted stages of root development, researchers have identified a number of adhesion molecules on the root surface, such as fibronectin, laminin, type I collagen, bone sialoprotein, cementum attachment protein, and osteopontin.10
However, they have yet to identify how important these molecules are in the control of cell adhesion and differentiation during tooth root formation. Specific growth factors are thought to promote the control of cell adhesion; and investigators have confirmed that platelet-derived growth factor- BB and insulin-like growth factor-I control the expression of integrins and proteoglycans. Numerous adhesion molecules work together with cells through specific integrins located on the cell surface. The binding of integrins with extracellular matrix stimulates signal transduction pathways and thus regulates the expression of genes.10
Focal adhesion kinases are especially fundamental to integrin signaling, and research indicates that they bind directly to integrins. Various other signaling molecules then bind to focal adhesion kinases and are phosphorylated by it, subsequently linking focal adhesion kinases to the mitogen- activated protein kinase pathway and to the 85- kDa subunit of phosphoinositide 3-kinase. It is through this series of events that the integrin signaling pathway is linked with several other pathways.10
Since only a few studies have concentrated on the role played by integrins during tooth development, the ligands and the specific integrins necessary for cell adhesion at the site of the local root surface have not yet been identified. Studies have shown that extracts of molecules from mature cementum promote adhesion of fibroblasts. These extracts also stimulate characteristic signaling events, such as activation of c-fos, focal adhesion kinases and extracellular signal–regulated kinase-2. It may be that these molecules also promote the recruiting of certain cell types to the local site during wound healing.10
If future studies can elucidate the nature of specific adhesion molecules and signaling pathways that regulate cementoblast maturation, it will further our knowledge of root development and promote the design of therapies that can activate cementoblasts.10
Preclinical and clinical advances have been accomplished toward utilizing growth factors for stimulating the regeneration of the periodontium, while emphasizing the regeneration of cementum.10
Various different agents can be categorized as regulators of periodontal tissue regeneration that stimulate formation of bone, cementum, and the periodontal ligament. They include:10
Although many published studies have evaluated the various therapies used to stimulate periodontal repair, evidence that supports these therapies are in the minority in terms of their ability to stimulate total periodontal regeneration. In order to more fully understand this process, a review of the results of preclinical and clinical studies is in order, as well as the capacity of these molecules to stimulate cementogenesis, and to govern the regeneration of bone and periodontal ligament.10
Platelet-derived growth factor alone or in combination with insulin-like growth factor-I were evaluated in some of the first in vivo research conducted on the role of growth factors in periodontal regeneration. This growth factor combination was reported to promote the formation of new bone, cementum and periodontal ligament when used to treat natural disease lesions in dogs and ligature-induced lesions in nonhuman primates.10
When platelet-derived growth factor-modulated guided tissue regeneration therapy was used in a dog model to treat class III furcation defects, new bone, cementum and periodontal ligament had begun to grow during the period of 5 and 11 weeks after the platelet-derived growth factor/guided tissue regeneration treatment. Surprisingly, when researchers applied platelet-derived growth factor directly to the tooth roots immediately after the application of citric acid to demineralize the root surface, and in the absence of a barrier membrane, extensive ankylosis resulted.10
The ankylotic union of bone to tooth, with no cementum and no periodontal ligament intervening between the bone and tooth, was present in 100% of the specimens at 5 weeks, and in 83% at 8 weeks. The investigators concluded that further research was needed, since up to this point, no other studies had reported platelet-derived growth factor-induced ankylosis.10
In 1997, the first human clinical trial to test the safety and efficacy of recombinant human platelet-derived growth factor/recombinant human insulin like growth factor-I was concluded. In this study, 38 patients diagnosed with moderate to severe periodontal disease were selected for treatment. Therapy consisted of 3 regimens with study and control groups: 10
The patients who were treated with recombinant human platelet-derived growth factor/recombinant human insulin-like growth factor-I developed 42.5% fill of the osseous defect. The control group which was treated with pooled vehicle and surgery alone developed only 18.5% fill in the osseous defect. The subjects tolerated the growth factors well and they were also demonstrated to be safe.10
Of the lesions treated, it was the furcations that responded most satisfactorily to therapy, demonstrating a nearly four-fold increase in bone volume in comparison to paired controls. The results from the human studies were highly comparable to the results from preclinical studies in nonhuman primates. Continuing studies to evaluate growth factors with various delivery systems are needed to establish the parameters for the clinical practicality of using platelet-derived growth factor/insulin-like growth factor-I to regenerate periodontal tissues in humans.10
The bone morphogenetic proteins have undergone extensive evaluation in orthopedic models for their ability to stimulate osteogenesis or new bone. Of the various bone morphogenetic proteins, the most definitively researched member of the transforming growth factor-beta superfamily for the induction of periodontal and periimplant bone regeneration is BMP 2.10
This molecule is a powerful effector in the induction of cementogenesis. Some investigators have reported that bone morphogenetic protein-2 applied via synthetic bioabsorbable polymer significantly stimulated the formation of new bone and cementum formation in periodontal tissues.10
This osteogenesis and cementogenesis were attained at 8 weeks after the application of bone morphogenetic protein-2, resulting in 95% regeneration of bone in surgically created class III furcation lesions. Unfortunately, a nearly 4-fold increase in ankylosis occurred in sites treated with bone morphogenetic protein-2 compared to vehicle treated sites. Other investigators using baboon models have reported a very strong induction of cementum and bone regeneration in class II furcation defects using a topical application of bone morphogenetic protein-4.10
In 1991, Bowers et al. conducted the first human study to use a bone morphogenetic protein to induce periodontal regeneration. They employed a single application of bone morphogenetic protein-3 (osteogenin) in combination with demineralized bone allograft in a submerged tooth model.10
Human histology revealed an increase in the bone and cementum deposited around periodontally involved submerged teeth that had been treated with bone morphogenetic protein-3. However, bone morphogenetic protein-3 did not provide significantly better deposition than the vehicle alone (bone graft group); and, in addition, pinpoint ankylosis was recorded in the submerged teeth that had been treated with bone morphogenetic protein-3 with bone grafts.10
More recently Saygin et al.10 applied bone morphogenetic protein-7/OP-1 to periodontal defects in an animal model and obtained closure of class III furcation defects. Increased induction of cellular “regenerative cementum” deposition was reported at 8 weeks after the application of bone morphogenetic protein-7/OP-1.10
However, on some root surfaces, they reported the presence of dentin resorptive pits with regenerative cementum deposition over the irregular root surface. In addition, in areas where regenerative cementum was deposited, ankylosis did not occur.10 Further examination of this phenomenon is warranted, since these findings emphasize the importance of identifying the factors that can consistently stimulate new mineralization on root surfaces.10
In 1994 Selvig et al. produced the first published report on the effect of transforming growth factor-beta in combination with insulin-like growth factor-II and basic fibroblast growth factor. Treatment consisted of the application of this growth factor combination to surgically created fenestration defects in dogs. The results failed to demonstrate any benefit from this therapy. The negative results may be due in part to the use of nanogram quantities of growth factor, which may not have been sufficient to stimulate regeneration.10
In 1998 a report was published by another group on the use of transforming growth factor-beta to achieve periodontal repair and stimulation of cementum formation in dogs. Therapy consisted of the application of transforming growth factor-beta 1 combined with polytetrafluoroethylene barriers to stimulate periodontal regeneration. No apparent benefit was derived from this combined treatment. Interestingly, they did not utilize a group treated with transforming growth factor-beta only and no barriers for comparison. The exclusion of periosteal cells by the barrier in the combination treatment may account for the poor treatment results.10
In 1999 another group reported that when bone morphogenetic protein-2 was used in combination with barrier membranes, it inhibited the induction of bone deposition around implant fixtures. Studies using orthopedic models have also yielded mixed results on the potential of transforming growth factor-beta to promote regeneration of mineralized tissues. Further research is indicated to evaluate the potential for the use of transforming growth factor-beta in reconstructive periodontal therapy.10
Enamel matrix derivative, with amelogenin being the principal protein, has been approved for human use internationally. In various preclinical and clinical settings, enamel matrix derivative has been studied to determine its potential for stimulating cementum and periodontal attachment structures.10
In 1997 investigators evaluated the capacity of enamel matrix derivative to promote periodontal wound healing in a preclinical buccal dehiscence model in monkeys. EMD treated defects were compared with carrier and ethylenediaminetetraacetic acid root conditioning treated defects for their response to therapy. Results revealed that the enamel matrix derivative treated defects demonstrated significant improvement in bone, cementum and periodontal ligament.10
Histological evaluation of enamel matrix derivative therapy in human studies has demonstrated incomplete regeneration. However, a multicenter trial consisting of 10 test centers and 107 patients demonstrated the safety of enamel matrix derivative, and a placebo-controlled study of 33 subjects with paired intrabony defects established its efficacy.10
In this placebo-controlled study, enamel matrix derivative therapy was used in conjunction with modified Widman flap surgery. They evaluated periodontal wound healing based on measurements of clinical attachment level change and subtraction radiography. Paired control defects showed no change in the radiographic bone level, while enamel matrix derivative therapy demonstrated a 66% fill in the defects at 36 months after treatment.10
The investigators concluded that the topical application of enamel matrix derivative paired with the modified Widman flap promotes periodontal regeneration that remains stable up to 36 months post therapy. They recommended expanding the studies with larger patient populations to evaluate the performance of enamel matrix derivative therapy in intrabony defects.10
The ability to direct the agents/cells to the tooth root surface is considered the rate limiting step in the re-engineering of periodontal structures including cementum. There are several factors that limit the predictable delivery of agents to the root surface and this complicates the healing process in a periodontal wound. Such factors include: 10
The various polymer delivery systems must rise to the challenge of overcoming these obstacles. So far science has succeeded in developing several biodegradable polymeric matrices used in guided tissue regeneration. However, the factors that complicate the healing in a periodontal wound impose multiple limitations that interfere with the use of these devices.10
In medicine and dentistry, materials such as copolymers of polyglycolic acid and polylactic acid have been used as vehicles to deliver bioactive molecules including growth factors, genes or cells. Research in tissue transplantation, growth factor and gene transfer has focused on the development of novel delivery systems to provide for the extended release of these agents to the target tissue (tooth root surface).10
In vitro and in vivo models have been utilized by investigators who focused their research efforts on the cellular and molecular mechanisms controlling the development and regeneration of periodontal tissues. Regenerative studies have frequently utilized animals such as rodents, canines, felines, and nonhuman primates. Investigators that concentrated on establishing the identity of the agents and mechanisms that control the development of teeth and the periodontium frequently use rodents to study molar and/or incisor development.10
In vitro models utilize cell cultures. The cells are harvested from animal tissues and cells and then manipulated by various methods to imitate the in vivo environment. For example, the cells can be grown on or within selective matrices or in a variety of co-culture models. As opposed to in vitro models, in vivo models imitate the intricacy of host-cell interactions, and they might be more accurate in reflecting human regenerative mechanisms; yet, significant limitations persist.10 Consequently both in vitro and in vivo studies are still needed because of their intrinsic and unique value. In vitro models use cell systems that supply the means needed to identify how cells respond to specific factors and the nature of the molecular factors that regulate these responses. In vivo models supply the means to provide the “proof of concept”. 10
In vivo models are limited in their capacity to design models that provide the identical type of defect found in human disease. In addition, several models use an acute defect. This defect is beneficial in establishing whether or not a factor does indeed elicit a response. However, the acute defect may not be able to imitate the response seen in chronic circumstances like those experienced by most periodontal patients.10
Furthermore, species show evidence of distinct genetic, anatomic, biochemical, immune and microbial differences. Thus, genetically engineered animals in addition to in vitro and in vivo cells are being used to circumvent these limitations. Of particular interest to us are the animal and cell models currently in use to study periodontal disease or that may be useful in future research.10
Light and electron microscopy used in some outstanding studies have yielded detailed information on cementum both during the developmental stages and during regeneration of the periodontium after disease. Enhanced knowledge regarding the factors expressed by cells related to the periodontium was also derived from immunocytochemical studies and in situ hybridization studies. Unfortunately, only indirect information was derived from these studies about the factors that are vital to the formation of periodontal tissues.10
Transgenic/knock-out animals provide another mechanism to support research efforts in identifying the role played by specific molecules in regulating tissue function. However, constraints are associated with their use because these animals do not often survive, or they may either show no phenotype or they show a complex phenotype necessitating further analysis.10
Cell cultures are a useful tool in instances where researchers can utilize advances in cell and molecular techniques to selectively manipulate specific cell types. Furthermore, scientists can isolate cells in a culture and then reintroduce them into a specific site in animals for in vivo confirmation of the cell type activity. With continued passages, cells in a culture often lose their phenotype. Thus, when working with cell cultures, it is important to be able to describe the cell phenotype at some level prior to its isolation.10
In this context, studies concentrating on identifying the factors expressed by cementoblasts and precementoblasts (follicle cells) in situ enabled cementoblasts to be isolated by researchers and cells in vitro to be analyzed to confirm that they did indeed express the same genes identified for these cells prior to their isolation.
Utilizing these tools enabled Saygin et al.10 to successfully isolate and culture murine cells from the developing root surface by means of various techniques. In their first approach, they isolated a mixed population of periodontal ligament cells and cementoblasts by means of collagenase-trypsin digestion. Although the cells did exhibit properties related to these cells in vivo, they failed to survive the continued passages.10
They then used various techniques to establish cells that could survive passage and retain their phenotype. These techniques included “immortalization of primary cell cultures using wild-type SV40, using immortalized transgenic mice, ‘immorto-mice’, and using osteocalcin promoter-driven SV40 Tag mice”. The advantage to using osteocalcin promoter-driven SV40 Tag mice is that only cells to survive in vitro are the root surface cells, cementoblasts, expressing osteocalcin; it naturally follows that periodontal ligament cells are excluded from the population.10
Researchers have also found the heterogeneous cell populations to be valuable because they mirror the local environment in vivo. If they subclone the mixed populations, they can establish both periodontal ligament cell lines and cementoblast populations. In the in vitro environment, cementoblasts express transcripts for bone sialoprotein, osteocalcin, osteopontin, alkaline phosphatase, osteoblast-specific transcription factor, parathyorid hormone/parathyroid hormone–related protein receptor 1 and type I collagen.10
Furthermore, cementoblasts respond to parathyroid hormone and vitamin D in a manner similar to that seen in osteoblasts; cementoblasts also promote both in vitro and in vivo mineral nodule formation. In addition, unpublished data reported that these cells responded to various growth factors, including platelet-derived growth factor and insulin-like growth factor. Human cementoblast in vitro models will yield more techniques to enhance our understanding of the biological behavior of periodontal tissue.10
Although various research groups have successfully isolated and characterized cultured human periodontal ligament cells, they are known to be mixed cell populations and their phenotype changes with culture. A similar method would make studies on these cells possible if they first establish the cell phenotype in vivo, and then isolate, immortalize, and subclone the human cell populations. However, science has yet to establish markers that are selective for periodontal ligament cells. Yet, immortalized periodontal ligament cell lines are obtainable, and hopefully, future studies will reveal whether these cells reflect the in vivo cell type.10
Researchers also isolated and cultured cells from a human cementoblastoma. Since a cementoblastoma is described as being poorly defined, they were not certain of the identity of the cells harvested for culture. Bone sialoprotein, cementum attachment protein, and collagen types I and V are all produced by cells cultured from a cementoblastoma. This tumor also stimulates the production of mineralized nodules in vitro.10
Cementoblasts have the potential to be an outstanding resource for human cells once their specific markers are identified. The availability of cell cultures enables scientists to concentrate on vitally important issues regarding the mechanisms and factors that control cementoblast function, including the identification of genes that are selectively expressed by cementoblasts. So it is not only the identification of the properties of cementoblasts in vitro that is important, but that these cells can be utilized for targeted gene therapy in the future.10
Defined mutations can be introduced into the mouse genome via reverse genetic techniques, including gene knockouts, and transgenesis to enhance our understanding of gene function. The usefulness of the murine models has been established by their extremely well characterized genome that enables their genes to be easily manipulated.10
The use of transgenic or knock-out animal may contribute to an increase in our understanding of the factors that regulate periodontal diseases and in particular cementogenesis. Also important in this context are animals with spontaneous mutations that may be a sign of systemic diseases or syndromes related to changes in periodontal tissues, such as Paget’s disease, hypophosphatasia, cleidocranial dysplasia, hypopituitarism, and osteopetrosis.10
When alterations in tooth structure are noted in animal models, they are most frequently referred to as a failure of tooth eruption, where the major defect is related to lack of osteoclast activity, and include op/op mice, c-fos knock-outs, src knock-outs, and osteoprotegerin ligand knock-outs.10
Replacement of the missing factors provides an important tool in the attempt to rescue and maintain animals that normally would not survive. For instance, severe variations in skeletal development and failure of the animal to survive are characteristic in parathyroid hormone/parathyroid hormone–related protein knock-out mice.10
When a transgene for chondrocytic parathyroid hormone–related protein expression was used in a rescue attempt for these animals, it failed to recover tooth eruption and root formation. Whereas, a keratin-driven transgene/ parathyroid hormone–related protein rescue attempt did succeed in correcting the defective eruption and tooth formation.10
Investigators have also concentrated on the technique of overexpression or underexpression of specific genes at various developmental stages in order to then evaluate how these manipulations affected animal function. For example, investigators have developed a transgenic mouse line to facilitate investigation of the function of amelogenin during mineralization. Researchers are hopeful that the use of this cell line in future studies may yield greater understanding of the role played by amelogenin-like molecules, including enamel matrix derivative, in regulating cells associated with the periodontium.10
Another area of intense interest is the application of molecules that are thought to induce or control periodontal regeneration. Unfortunately these molecules have short half-lives at the healing site that may diminish their effects in vivo. This prompts researchers to search for methods that increase stability in the exogenous molecules at the healing site in order to optimize wound repair.10
The capacity to transfer genetic material into specific cells provides a technique that may assist in prolonging the effect of the targeted factor. Various researchers have concentrated their efforts on establishing optimal techniques capable of delivering genes to cells for later use in the in vivo models.10
Gene transfer can be accomplished by either in vivo or ex vivo (in vitro) transfer of the appropriate transgene. In the ex vivo method of transfer, cDNA is transferred to cells in the culture; next the genetically altered cells are expanded; and finally they are dispensed to the recipient site. For instance, bone marrow cells can be harvested from an individual, transduced ex vivo, followed by reimplantation of the genetically modified cells to the target area.10
cDNA is “an acronym for complementary DNA. DNA is synthesized from a messenger RNA template; the single-stranded form is often used as a probe in physical mapping to locate the gene or can be cloned in the double stranded form. Viral reverse transcriptase can be used to synthesize DNA that is complementary to RNA (for example an isolated mRNA).3
The alternative technique is the in vivo transfer of the gene directly into the recipient tissues by the process of microseeding plasmid or viral DNA. The use of regional gene therapy to stimulate repair and formation is indeed appealing because the genes can be transported to the correct anatomic site, and selection of the appropriate vector and/or promoter will determine the duration of protein expression.10
Gene transfer technology utilizes both viral and nonviral methods. With retroviral and adeno-associated viral vectors, the transferred DNA sequences are integrated in a stable fashion into the chromosomal DNA of the target cell. Generally speaking, this method is used in the ex vivo application. Under certain circumstances, the expression of the gene is high but transient, and these instances favor in vivo transfer. The retroviral gene transfer is considered the method of choice for ex vivo applications.10
However, various characteristics of this gene transfer technique can impose limitations on its applicability, especially in the in vivo applications. Entry of the retrovirus is dependent upon the presence of the viral receptor. Also, target cell replication is required for proviral integration to take place. The production of “packaging cells” was the most significant advance in technology for viruses that are used as gene transfer vectors. The packaging cells enable high titers of replication-defective recombinant virus to be produced, free of wild-type virus (gutless viruses).10
The advantage of using adenoviruses is their ability to efficiently infect dividing and nondividing cells and to express copious quantities of gene products. Adenoviruses are transient by nature. This characteristic enables high titers of adenoviruses to be used because their effect declines gradually and persists unintegrated as extrachromosomal DNA; this is partially due to a T-cell-mediated immune response.10
The gene transfer method provides an appealing option compared to the more traditional technique in which molecules are applied topically to the complicated periodontal wound site. The concept of using a virus incorporating one or more response modifier transgenes for long-term delivery may control the cellular response in the periodontium.10
For studies of this nature, they must first establish whether putative cells harvested from the periodontium, such as periodontal ligament cells or cementoblasts, can accomplish gene transfer efficiently. While gene transfer techniques do modulate migration, proliferation, attachment, differentiation and maturation of participating cells, the mechanisms that regulate major regenerative events are still poorly understood.10
Gene therapy may be utilized to deliver the factors that regulate angiogenesis, proliferation, attachment, and differentiation of the cells into the wound site. Researchers are hopeful that gene transfer techniques will facilitate their control of periodontal regeneration and will advance their understanding of the mechanisms required for wound healing.10
This is an exciting and productive time for investigators who concentrate their efforts on refining and maximizing periodontal/implant regenerative therapies. The design of treatment modalities can now be based on sound scientific data due to the many breakthroughs contributing to our understanding of cell function regulators, in conjunction with techniques that enable scientists to engineer cells that express specific factors, and the development of more sophisticated delivery systems that control the release of cells/factors at a specific site.10
It is important to begin by investigating engineered cell types/delivery systems in both in vitro and in vivo models. This would include the evaluation of their capacity to promote cell proliferation, mineral nodule formation, specific osteoblast- and cementoblast-associated genes in vitro; as well as the in vivo formation of new bone, new root surface mineral, and a functional periodontal ligament. What has been learned from these studies has the potential to create the foundation necessary for the design of more predictable regenerative therapies than are presently available.10