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While the extracellular matrix (ECM) of cementum resembles the composition of other mineralized tissues, it possesses a unique physiology and includes molecules that have not been found in other tissues. The components of cementum modulate the activities of periodontal cells, and they demonstrate selectivity for some periodontal cell types over others. Recent discoveries indicate that the ECM determines how cells respond to environmental stimuli.11
The theory follows that, in a healthy situation, the local environment of the cementum matrix probably plays a crucial role in preserving the homeostasis of cementum. Periodontal disease severely compromises the structural integrity and biochemical composition of the cementum matrix and periodontal healing generates a provisional matrix that is different from cementum.11
Grzesik et al.11 proposed that two criteria be met in order for new cementum and attachment formation to occur during periodontal regeneration: 1. the local environment must be conducive for the recruitment and function of the cells that form cementum, and 2. the wound matrix must favor repair rather than regeneration. In addition, their research shed light on the mechanism by which cementum components might regulate and participate in cementum regeneration, potential regenerative therapies based on these tenets, and cementoblast cells models.11
The term ‘periodontium’ refers to the biological structures that support the teeth, i.e. the gingiva, periodontal ligament, cementum, and alveolar bone. Many hereditary and acquired diseases affect the structure and composition of the periodontium, and among these the most significant is periodontal disease. Characteristic of periodontal disease are the destruction of soft connective tissues, loss of bone, and loss of the connective tissue attachment to cementum. If these alterations are left untreated, the result is tooth loss. The goal of periodontal therapy is the regeneration and restoration of the various disease altered components of the periodontium to their original consistency, form and function.11
By definition, regeneration of the periodontium requires the following: 11
There are new therapeutic techniques available to accomplish these objectives, namely barrier membrane use in guided tissue regeneration and the application of growth factors and enamel matrix proteins to root surfaces. However, their effectiveness, especially on the formation of attachment and new cementum, is not predictable.11
Cementum exerts a regulatory role in periodontal regeneration. The soft-tissue attachment has to be re-established in cementum; and the cementum matrix is rich in various growth factors that impact the biological activities of many cell types in the periodontium. Insufficient emphasis has been attributed to the role played by cementum in periodontal regeneration. Emerging evidence demonstrates that the local environment exerts a major regulatory role on the mechanism by which cells respond to signals and environmental cues. Grzesik et al.11 emphasized the mechanism by which the constituents found in cementum both participate in and regulate periodontal regeneration, and the potential for applying these principles to periodontal regenerative therapy.
Wound healing is comprised of three overlapping and interdependent phases. Although the general principles and events have been described by means of cutaneous wound models, they apply equally to the healing observed in surgical and periodontal disease wounds.11
When traumatic injury occurs, it damages blood vessels resulting in hemorrhage and extravasation of blood, and finally a blood clot is formed. Many polypeptide mediators are generated by the blood coagulation process and the activated complement pathway, while the blood clot acts as a provisional matrix to accommodate the migration of inflammatory cells. Bacteria, foreign particles, and dead tissue are removed from the wound by neutrophils and monocytes. Macrophages and platelets produce polypeptide mediators that orchestrate and regulate the activities of various cells that participate in the healing process.11
Within hours after the injury occurs, re-epithelialization begins as the epithelial cells closest to the wound margin migrate to the wound site. Migration and proliferation increase the amount of epithelial cells present at the wound and eventually close the break in the epithelium. At the same time angiogenesis and the synthesis of collagens and other extracellular matrix components are activated, replacing the clot with granulation tissue. Activated fibroblasts fill the wound, and some of these cells are stimulated to become myofibroblasts.11
The appearance of myofibroblasts in the wound signals the initiation of connective tissue compaction, and then the wound contracts. As contraction of the wound progresses the size of the wound shrinks, positioning the wound margins in approximation to one another. The tissue remodeling phase ensues, allowing granulation tissue to be replaced by new connective tissue. The new blood vessels created by angiogenesis are resorbed via degradation and apoptosis.11
Apoptosis is a cell biology term that means “programmed cell death as signaled by the nuclei in normally functioning human and animal cells when age or state of cell health and condition dictates. It is an active process requiring metabolic activity by the dying cell, often characterized by cleavage of the DNA into fragments that give a so called laddering pattern on gels. Cells that die by apoptosis do not usually elicit the inflammatory responses that are associated with necrosis, though the reasons are not clear”.3
When regeneration is not possible and healing occurs by repair, a fibrous scar replaces the wound. Remodeling continues during the ensuing weeks and months, and the scar tissue regains up to 70% of the original tissue strength. The migration, adhesion, proliferation and differentiation of several cell types all participate in the wound healing process. These processes are initiated by the binding of polypeptide mediators to their cell-surface receptors and of integrins to their extracellular matrix components. Within 24 hours after an injury occurs, the expression of keratinocyte growth factor is enhanced by 100-fold.11
Different growth factors are necessary for the regulation of different cell functions. A variety of growth factors and their receptors play a role in the process of re-epithelialization, such as: 11
Angiogenesis is strongly stimulated by:
Angiogenesis is inhibited by:
Platelet-derived growth factor (PDGF) is mitogenic to fibroblasts and other mesenchymal cells and stimulates cell division in these cells. As such, PDGF is a significant growth factor in wounds, and gingival epithelium provides an ample amount of this growth factor in gingival wounds. TGF-β and connective tissue growth factor (CTGF) both activate fibroblast collagen synthesis. TGF-β probably stimulates the transformation of fibroblasts into myofibroblasts. Both TGF-β and PDGF play an active role in wound contraction. 11
The active participation of integrins is required for cell migration and adhesion. The receptor that mediates the attachment of hemidesmosomes and laminin is 6β4 integrin. Dissolution of 6β4 integrin is required for the migration of epithelial cells.11
There is expression of the receptors to fibronectin and tenascin, 5β1 and vβ6 integrins, and vitronectin receptor vβ5; and relocalization of the type I collagen receptor 2β1 takes place. The rate limiting step for the formation of granulation tissue is thought to be the appearance of the integrins that bind to fibrin and fibronectin; and endothelial cells actively express the integrin 5β3 at the top of sprouting capillaries.11
Integrins are also necessary for wound contraction. Not only are alterations in integrin expression necessary, but, in order for tissue remodeling to occur, proteolysis is required to facilitate cell migration through the fibrin clot and matrix.11
During wound healing, inflammatory cells and most other cells secrete various different matrix metalloproteinases (MMPs). The fibrinolytic enzyme plasmin promotes keratinocyte migration at the leading edge in cutaneous wounds. Plasmin is activated by tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA).11
The expression of uPA, tPA, MMP-1 (interstitial collagenase), MMP-9 (gelatinase B), and MMP-10 (stromelysin-2) is stimulated or up-regulated at the wound margin. MMP-1 expression is required for keratinocyte movement to occur, and the MMP-1 enzyme also promotes cell directionality. Once re-epithelialization is complete, the MMP-1 expression is turned off.11
Additionally, the MMPs are also necessary for remodeling of the granulation tissue matrix. The substrate specificity of MMPs demonstrates extensive redundancy. However, the course of their action and the composition of matrix laid down are determined by the differences in their affinities to different substrates and their compartmentalization.11
There are several master genes that take part in the process of wound healing. These master genes code for proteins that regulate formation of the body during embryogenesis and development. These genes include homologues of TGF-β and BMPs, and transcription factors encoded by "homeobox genes". Homeobox genes are characterized by the presence of a common 61-amino-acid DNA-binding motif referred to as a "homeodomain". The proteins exert control over the expression of lineage-specific genes during normal embryonic development. In addition, they control pattern formation, cell fate, and identity.11
During wound healing and in adult eukaryotic cells, the master genes are active participants in the growth and differentiation of cells, apoptosis, and cell-cell and cell-ECM interactions. Various homeobox genes participate in angiogenesis and vascular remodeling. Regulation of master gene expression is achieved through interactions among cytokines, growth factors, ECM, and adhesion molecules. The growth factors that control their expression include TGF-β, BMPs, and FGFs. For instance, FGFs and BMPs regulate the muscle segment homeobox gene MSX and "paired" PAX.11
Alteration in the expression of these genes is seen in conjunction with metabolic and neoplastic disease. Programmed expression of master genes is probably an absolute requirement for differentiation programs related to wound healing. It is suspected that aberrations in their regulation may favor wound repair rather than wound regeneration.11
With respect to the factors that determine whether wounds heal by regeneration or repair, the degree of injury and the quantity of lost tissue that must be filled are important determinants. However, two vital factors influence the potential for regeneration v. repair: 1. Accessibility of necessary cell types, and 2. Whether or not the cues and signals needed to recruit and stimulate these cells are present. Furthermore, these two factors are not mutually exclusive. Diffusible factors such as growth factors and cytokines provide the signals; but it is the extracellular matrix that controls how the cells act in response to the signals.11
In most vertebrates, the ability to regenerate is restricted to a few tissues, for example liver and bone. In these particular tissues, regeneration may imitate the embryonic differentiation of multipotential stem cells. In contrast, mammalian tissues comprised of permanent cells such as neurons and cardiac myocytes generally heal by repair. It has been suggested that the absence of appropriate stem or progenitor cells was one of the reasons that these tissues failed to regenerate. Since a variety of adult tissues are self-renewing, it is presumed that they include a lifetime population of comparatively slow-cycling (stem) cells.11
Even non-regenerating adult tissues, including the brain, contain multipotent stem cells according to recent evidence. In non-hematopoietic tissues the stem cells are called mesenchymal stem cells or marrow stromal cells, and they can differentiate into a large number of cell types. Embryonic stem cells are thought to be nearly totipotent and malleable. Shortly after development is complete, the embryonic cells complete their diversification for the most part into the cell types that they were destined to become.11
Stem cells found in adult tissues demonstrate comparatively less malleability. Like hematopoietic and neuronal stem cells they may be pluripotent, or unipotent as in the case of epithelial stem cells that are committed to differentiate into epithelial cells. When a stem cell demonstrates a commitment to undertake differentiation, often, it proliferates rapidly at first and then differentiates, with differentiation into the final form occurring in various stages. It is presumed that these cells generate new stem cells or differentiated cells in response to specific environmental signals.11
When homeostatic conditions are normal, the most common triggers for differentiation of the stem cells are the instructive and stimulatory signals emitted by the local environment that contains the ECM (extracellular matrix) and growth factors which are sequestered in the matrix. Cross-talk with other cells of the same and different cell types is also involved in the process of differentiation and survival. An example of survival includes mesenchymal cues for epithelial differentiation and vice versa.11
Stimulus signals and directional signals for cellular migration are provided by substances found in the circulation and those in the local environment as well. These substances are comprised of growth factors, other soluble mediators, and ECM (extracellular matrix) components. They stimulate cell division and differentiation.11
So far, more than 2000 such molecules have been cloned or purified. The diversity and concentration of molecules are continuously altered during the course of wound healing. Notably, in the local microenvironment, more than one substance is present. The biological diversity of these molecules gives rise to a combined effect that may be additive, synergistic, or antagonistic.11
Extracellular matrix (ECM) is a three-dimensional structure made up of various constituents: 11
Not only does the ECM act as a substratum for cell adhesion, it also promotes the spreading of cells and organization of the cytoskeleton. Adhesion to the extracellular matrix is crucial to support transition through the cell cycle by anchorage-dependent cells. The extracellular matrix is the determining factor in the development of the three-dimensional cellular architecture, transmitting and translating the external mechanical and tensional forces in response to appropriate signals.11
Not only does the extracellular matrix function to regulate gene expression of growth factors, growth factor receptors, and other proteins, but it also determines the outcome of the cellular response to growth factors. As a primary example, cell division is suppressed and cells differentiate in the presence of extracellular matrix. Cells bind to the extracellular matrix through the influence of integrins. It is this binding that subsequently initiates a cascade of signaling reactions.11
The signaling reactions that are activated include: 11
Constituents in the extracellular matrix induce signaling pathways. These pathways then function in cooperation with substances that are activated by growth factors in modulating their biological functions. Integrin- and growth-factor-induced signals are both essential for expression of G1 cyclins and the cell cycle progression from the G0/G1 to S-phase. During the G1 cell cycle phase, integrin signals that are mediated by adhesion are required for the activation of cyclin-dependent kinase (cdk)-2and the expression of D1 and A cyclins.11
Signaling pathways that are induced by integrin- and growth-factor-receptors converge at the MAP kinases. Growth factors elicit a temporary activation of the extracellular-signal-regulated kinases 1 and 2 (ERK1/2). However, it is the extracellular matrix that provides a continuous increase in their activity; and in addition, the ECM is associated with an increase in the expression of cyclin D1. The levels of cdk inhibitors (CKI) p27kip1 (p27) and p21cip1/wafl (p21) are upregulated by the extracellular matrix, a mechanism that promotes ECM-induced cell-cycle arrest.11
The make-up of the extracellular matrix may be largely responsible for recruiting specific cell types during wound healing. For example, fibronectin and type I collagen attract most cell types by chemotaxis and also promote their cellular adhesion. However, laminin and type IV collagen are selective for specific cell types. In contrast, tenascin is anti-adhesive. Therefore, it is the composition of the extracellular matrix that controls which cells are mobilized during wound healing; and it also determines whether healing manifests in the form of repair or regeneration. Various growth factors are isolated in the extracellular matrix.11
In addition, ECM interplay can alter the binding of growth factors to their cell-surface receptors. There are multiple sites at which the extracellular matrix constituents can bind to these molecules, and the proteoglycans can interact through both the protein core as well as the glycosaminoglycan chains.11
Soluble mediators such as growth factors, cytokines, and chemokines are secreted by inflammatory cells during inflammation. As damaged tissues are biologically degraded, some mediators are also liberated and released. The molecular activity of these soluble mediators is directed towards the cells that participate in wound healing. In contrast, the activity of growth factors that are sequestered in the extracellular matrix may be restricted to the types of cells necessary to maintain normal tissue homeostasis.11
Some of the isolated extracellular matrix constituents and molecules might be tissue-specific in their distribution. The extracellular matrix components together with the available growth factors probably determine the expression of certain receptors and the induction of specific biochemical signaling events. Additionally, they will determine the specific pathway selected from among a group of several parallel pathways. They will also regulate the functional response of cells and the route of wound-healing events.11
Cell density and interaction with other cells are other factors that determine the cellular response.
There are two events that illustrate the importance of intercellular interactions: 11
The structural integrity and unique biochemical composition of the extracellular matrix are thought to be essential ingredients in the maintenance of tissue homeostasis under healthy circumstances, and also vital for regeneration after injury occurs. The local environment is important in the influence it exerts on cell behavior. This is well illustrated when fetal hematopoietic stem cells that are implanted into the adult spleen express adult progenitor phenotype cells.11
As wound healing progresses, the composition of the extracellular matrix is altered. For example, in the early stages, fibronectin, vitronectin, and types III, IV, V, and VI collagen are actively expressed during the same time that granulation tissue is actively being deposited. At the later stages in wound healing, these substances are replaced by type I collagen.11
The local environment of the healthy tissue is not imitated by the changing composition and quality of the extracellular matrix. During the course of wound healing, the type and concentration of the available polypeptide mediators change continuously. So, the destruction of the local environment and the inability to reconstitute it are thought to be important contributing factors when tissues cannot regenerate following an injury. Under these circumstances, the provisional matrix that is produced by the granulation tissue may be conducive to repair but not regeneration.11
Researchers have now confirmed that most tissues contain the cells needed for regeneration. These cells are represented by two types: the “stem cells” that are removed from terminal differentiation by several stages, and the progenitor or precursor cells in the stage immediately preceding full differentiation. Some investigators have proposed an interesting theory that the pre-existing extracellular matrix may provide signals that enable precursor cells to differentiate in healthy adult tissues, whereas the extracellular matrix that is produced by a “stem cell” and a differentiating stem cell may biologically endow the local environment to support differentiation of the next stage cell.11
After periodontal tissues are injured by infection, trauma, or other causes, the various events, growth factors, and soluble mediators discussed above also play a part in their healing process. Research has substantiated that biologically active mediators are present in the periodontium. The concentrations of these molecules differ based on their presence in the various periodontal tissues, and they appear to be present in relatively higher amounts in alveolar bone and cementum.11
These molecules are not detectable in soft tissues, especially in the gingiva. Investigators suspect that this is actually due to their low concentration rather than their nonexistence. Substances found in one periodontal structure can influence other periodontal tissues; however, in a healthy environment, these molecules are more likely to primarily affect the adjacent cells. Even under pathological conditions, these substances are likely to play a minor role, because of the destruction and biochemical changes that are present in the local environment and the relatively large amounts of serum and inflammatory-cell products.11
Anatomically cementum is a primary part of the tooth. Functionally cementum is a constituent of the periodontium, and its primary function is to provide the attachment site for the principal collagen fibers (Sharpey’s fibers) that connect the root of the tooth to the alveolar bone. For the purposes of this discussion, only those aspects of the biology of cementum that pertain to periodontal healing concern us here.11
There are three types of cementum that differ in the presence of cells and collagen fibers that have been identified in humans: 11
Acellular afibrillar cementum is comprised of mineralized matrix that lacks collagen fibrils and embedded cells, and it covers teeth both at and along the cemento-enamel junction. Its deposition is initiated at the termination of enamel maturation, continues for an indefinite period of time, and occurs in isolated patches over the enamel and dentin. Although researchers have not yet identified the cells that deposit acellular afibrillar cementum, it is thought that the matrix is laid down by connective tissue cells at the time that they physically make contact with the enamel surface.11
Acellular extrinsic fiber cementum (AEFC) provides the primary attachment function. Normally it is confined to the coronal half of the root and covers both the cervical and middle parts of the roots. AEFC is comprised of a dense fringe of collagenous fibers that insert into the dentinal matrix. It is extremely significant that they are inserted perpendicular to the root surface. Soon after the crown formation is complete, the production of acellular extrinsic fiber cementum is initiated. Its growth continues as long as the adjacent periodontal ligament is not disturbed. The cells that produce AEFC are the cementoblasts that differentiate closest to the advancing root edge.11
Cellular intrinsic fiber cementum (CIFC) is deposited on the dentin surface where there is no acellular extrinsic fiber cementum present. The formation of CIFC is initiated nearest to the advancing root edge on the forming root. Cellular intrinsic fiber cementum consists of cementocytes that are trapped in the mineralized matrix; and the collagen fibers found in CIFC are oriented parallel to the root surface. The primary function of CIFC is repair. CIFC may grow over acellular extrinsic fiber cementum and vice versa, and this development is referred to as mixed stratified cementum. This cementum is restricted to the apical portions of roots and furcations. Investigators think that its primary function is most likely the repair of previously resorbed roots.11
In rodents, cementogenesis is initiated when Hertwig’s epithelial root sheath (HERS) deposits a matrix on the dentin surface. This is followed by the disruption of Hertwig’s epithelial root sheath. Next, the ectomesenchymal cells from the dental papilla undergo migration and organization; finally, they differentiate into cementoblasts.11
The cementoblasts that produce cementum are surrounded by the HERS matrix. Differentiating progenitor cells produce the cementoblasts; and in mice the progenitor cells are believed to arise from the dental follicle. Researchers suggested that the epithelial cells of Hertwig’s root sheath probably undergo epithelial mesenchymal transformation.11
Collagens are the primary component of the organic matrix of cementum. Type I collagen is the primary type of collagen species, and it comprises 90% of all collagens. It demonstrates both structural and morphogenic roles and provides a scaffolding for mineral crystals. Type III collagen comprises about 5% of the collagen species; and its function is to coat type I collagen fibrils.11
The two major non-collagenous proteins found in cementum are bone sialoprotein (BSP) and osteopontin (OPN). These proteins remain bound to the collagen matrix, and they demonstrate cell attachment properties via the arg-gly-asp (RGD) sequence. Bone sialoprotein and osteopontin are prominently expressed in acellular extrinsic fiber cementum and acellular afibrillar cementum. Cells located along the root surface express both of these proteins during early tooth root development.11
Bone sialoprotein is expressed by root surface cells and it is also found in mature teeth. Osteopontin is found within the periodontal ligament area that is associated with mature teeth. BSP and OPN are thought to exert a major influence in the cementoblast progenitor cells differentiation into cementoblasts. Researchers believe that BSP functions to provide adhesion for the root surface cells and participates in the initiation of mineralization. BSP is chemotactic to pre-cementoblast cells and stimulates their adhesion and differentiation.11
Many cells express OPN during periods of cementogenic activity. Osteopontin controls cell migration, differentiation, and survival via interactions with vβ3 integrin. In addition, it is involved in the inflammatory process via the regulation of monocyte-macrophage activation, phagocytosis, and nitric oxide production. Osteopontin may regulate biomineralization in teeth and cementum by utilizing at least two mechanisms: controlling bone cell differentiation and controlling matrix mineralization.11
It is thought that fibronectin binds cells to the extracellular matrix. Both fibronectin and tenascin are found in the basement membrane of Hertwig’s epithelial root sheath at two different times: during odontoblast differentiation, and later at the site where the periodontal ligament attaches to the root surface. There are other matrix constituents present in cementum including osteonectin. Osteonectin is expressed by cementoblasts that produce cellular extrinsic fiber cementum and cellular intrinsic fiber cementum, osteocalcin, and laminin.11
Also sequestered in the cementum matrix are several polypeptide growth factors capable of stimulating the proliferation and differentiation of putative cementoblasts. These polypeptide growth factors include: 11
While many of these constituents are found in bone, scientists have also described molecules that are unique to cementum. One of these unique molecules is IGF-I isoform also called cementum growth factor (CGF). CGF is a 14-kDa protein which is a larger molecular size than IGF-I. The second molecule is called cementum attachment protein (CAP) which is a collagenous protein.11
CAP antibodies immunostain cementum only; and they do not immunostain other periodontal components or other tissues. Cementum attachment protein is expressed by cementoblasts in bovine tooth germs. In cementum, the CAP pattern of expression differs from that of type I collagen. Cementum attachment protein stimulates the adhesion and spreading of mesenchymal cells, however, it preferentially stimulates the adhesion of mineralized-tissue-forming cells.11
There are various diseases that affect the structure and biochemical composition of cementum. In periodontitis, the process of chronic inflammation is destructive to the gingival collagen fibers, and this destruction may extend to include the root surface. There is one significant pathological change in periodontal disease and that is the deposition of bacterial plaque substances, including their bacterial endotoxins. Attachment loss occurs in response to the destruction of connective tissue fibers.11
When the cementum surface is exposed to periodontal pockets, the damage to cementum becomes irreversible. Periodontal disease leads to changes in the biochemical composition of cementum; then the active substances are lost and the inhibitors such as endotoxins are deposited. The growth and attachment of connective tissue cells are inhibited by diseased cementum, setting the stage for the formation of an epithelial attachment. This destructive set of circumstances led to the rationale for the development of new therapeutic approaches in which diseased roots could be conditioned to stimulate connective tissue attachment.11
It is important to remember that various features of cementogenesis and cementum biology, for example, the means by which cementum attaches to dentin and the cementum apposition rate, are apparently different between species, especially between rodents and large mammals, including humans. Investigators are not certain whether these differences occur as a result of spatio-temporal characteristics such as tooth size, rate of development, etc, or if they mimic different molecular or cellular characteristics necessary to cementum formation and maintenance.11
Although much of the information is explanatory, it is especially scarce in reference to humans. It is important to solve these secrets of nature and equally important to remember that data resulting from animal models should be utilized very cautiously prior to drawing conclusions that will be applied to humans. There have been recent developments in the in vitro / in vivo animal and human models. Scientists are projecting that they will serve as outstanding systems for the clarification of interspecies differences at both cellular and molecular levels.11
The formation of new cementum and restoration of the soft-tissue attachment to cementum is a very significant objective of regenerative periodontal therapy. Cementoblasts are required for the regeneration of cementum. However, scientific knowledge is still incomplete regarding the origin of cementoblasts and the molecular factors that control their recruitment and differentiation.11
Significant clues on how cementum components control the regeneration of cementum have been derived from in vivo animal models studying cementogenesis during tooth development, the pattern of expression demonstrated by specific matrix molecules, and in vitro experiments evaluating how cementum components affect periodontal cells.11
During tooth development, dental follicle cells originating from the ectomesenchyme are thought to be the source of cementogenesis. In adult mice, cementoblasts are thought to originate as progenitor cells located in the vicinity of the periodontal ligament blood vessels or in endosteal spaces of the alveolar bone.11
Even though the formation of cementum in rodents is different from that in mammals, some researchers have proposed that the periodontal ligament may be a source of cementoblast progenitor cells in adult humans. Data from these researchers indicates that a small number of clones of cells cultured from the human periodontal ligament produce cementum-like mineralized nodules in culture and generate cementum-specific markers. Stem cells that are present in the periodontal ligament, gingiva, or alveolar bone may also be a source of cementoblasts. This may be more likely to occur when the periodontium is healing from insult or injury and the source of available progenitor cells is liable to be reduced or absent.11
However, scientists have yet to identify the molecules responsible for the recruitment of these cells and their differentiation. Scientists forecast that the mechanisms involved are probably more complicated. Various chemotactic factors, adhesion molecules, growth factors, and extracellular matrix components cooperate to achieve the recruitment, expansion, and differentiation of cementoblast progenitors. Although many of these molecules may be accessible during periodontal healing, most of them are pleiotropic (have multiple effects) and do not demonstrate evidence of cell specificity.11
Cell specificity can be accomplished in a variety of ways; for example, the ability of growth factors to target specific cell types, the uniqueness of the extracellular matrix composition, and the presence of permissive conditions for required cells and refractory conditions for all other cells. There is data indicating that constituents of cementum are capable of utilizing all of these mechanisms to control cellular activities.11
For example, cementum is known to contain molecules that more effectively stimulate chemotactic migration, adhesion, proliferation, and differentiation of certain periodontal cell types rather than others. Secondly, these substances have not been detected in other periodontal structures. Cementum also contains adhesion molecules that lead to negative selection by excluding unwanted cells. Scientists have not observed this cell specificity in other ubiquitous matrix molecules, such as fibronectin.11
Perhaps most significant is that the cementum microenvironment is endowed with all the components required for cell recruitment, proliferation, and differentiation; and molecules from the circulation are not needed. In the presence of cementum proteins alone, cells can evade cell cycle arrest and complete cell division and differentiation. Required molecules can also be found in enamel matrix extracts; however, the active constituents may or may not be the same as those found in the cementum matrix.11
Although scientists are not certain of the specific mechanism by which cementoblast progenitors are selected, it is thought to involve specific integrins and signaling events. After the progenitor cells are selected, they adhere to the root surface. Their expansion may be promoted by the presence of growth factors found in the cementum matrix. Various studies have identified growth factors in cementum including: IGF-I, FGFs, EGF, BMPs, and TGF-β.11 See Table2.
| Molecule | Biological Activity |
| Growth factors | |
| IGF-1 | proliferation, differentiation, matrix synthesis |
| FGF | proliferation, differentiation, matrix synthesis, angiogenesis |
| PDGF | proliferation, differentiation, matrix synthesis |
| TGF-β | matrix synthesis, angiogenesis, chemotaxis |
| BMPs | matrix synthesis, differentiation, bone formation |
| EGF | proliferation, differentiation |
| CGFa | proliferation, differentiationb |
| Matrix components | |
| Collagens | cell adhesion, differentiation; regulates proliferation |
| BSP | cell adhesion, differentiation, mineralization |
| OPN | cell adhesion; regulates differentiation and survival |
| Fibronectin | cell adhesion, differentiation, regulates proliferation |
| Osteonectin | regulates angiogenesis, differentiation, and proliferation |
| Cementum-attachment protein | cell adhesion, differentiationb |
Although cell selection can be accomplished at the level of adhesion, it may also occur by preferential proliferation. For example, CGF is an isoform of IGF-I. While fibroblasts respond mitogenically in a similar manner to both growth factors, osteoblasts respond better to the CGF. These findings emphasize the significance of restoring or providing the cementum microenvironment with the molecules needed to initiate and promote the formation of new cementum.11
Periodontal disease chemically alters the integrity of cementum by depositing bacterial endotoxins into it. Periodontal therapy removes the diseased cementum. Dentin, which is not covered by cementum, undergoes resorption. Treatment with root conditioning is unlikely to restore the local environment of cementum to its original composition. Rather, molecules that demonstrate poor cell specificity are exposed, especially type I collagen.11
The application of only some growth factors is probably unable to provide the complete inventory of the required molecules, as the type and concentration of growth factors change continually during the process of healing. Likewise, barrier membranes can assist necessary cells in populating a specific site, but the membranes are unlikely to create the local environment required for cellular differentiation. Supplying enamel proteins will probably stimulate early cementogenesis, but it may not be able to create an environment conducive to recruiting cementoblast progenitors in adults or promoting their differentiation.11
While extracellular matrix constituents are known to be expressed during healing of the periodontium, it is not known whether all of the molecules found in the cementum matrix are expressed as well. The temporal sequence of ECM expression is also a vital aspect in the initiation of new cementum formation. These factors may give us insight into why the currently available regenerative procedures are not able to regenerate cementum on a predictable basis.11
A succession of growth factors is accessible from both the circulatory and inflammatory cells during the process of inflammation and the wound-healing response that follow periodontal surgery. Blood clot tissue and granulation tissue form a provisional matrix that has a composition different from the cementum environment that exists under healthy conditions. Scientists report that this combination of extracellular matrix and growth factors is unlikely to be beneficial to the selection and function of cementoblast progenitors.11
If progenitor cells are unavailable and only stem cells are present, they may be able to differentiate into cementoblasts provided that events normally occurring during the early stages of cementogenesis are repeated and providing that the requisite signaling molecules are unlikely to be present in the wound-healing environment. Therefore, it would seem that the precise combination of extracellular matrix constituents and growth factors are essential for the induction of new cementum formation.11
One important fact deserving of emphasis is that, in early and moderate periodontitis, the acellular cementum found on the coronal half of the root is adversely affected; later the damage progresses to involve the cellular cementum in the most advanced and furcal lesions. During successful regeneration these surfaces are almost always covered by cellular cementum, but the question remains as to whether this is adequate.11
The cells of the gingiva, periodontal ligament, and alveolar bone are also actively influenced by growth factors and adhesion molecules present in cementum. As such, the constituents of cementum may have the potential to play a role in the regulation of homeostasis and regeneration of these tissues.11
Yet, since the growth factors found in cementum remain bound to the cementum matrix, this may not be significant under conditions of health. Their relative concentrations will probably be less than those available from the blood and inflammatory cells even if they are released by the inflammatory process. Thus, whatever contributions the cementum molecules make to the regeneration of other periodontal tissues will probably be marginal or insignificant.11
New approaches have emerged for the development of treatment modalities designed to restore attachment that possibly utilize the application of cementoblastic cells. Viable cementoblasts and/or periodontal ligament cells located near the cementum are thought have a vital role in regenerating the periodontal attachment tissues.11
Studies have confirmed that when an avulsed tooth is replanted into the tooth socket very soon after it was avulsed, or if the tooth is placed in an environment that promotes cell survival until replantation, the cementum-generated attachment is re-established. However, if viable cells are not present when the avulsed tooth is replanted, the healing process is often impaired and uncompromising complications such as ankylosis and root resorption have a greater potential to develop.11
This suggests that, in addition to the cementum matrix, the viable cementoblasts and/or intact molecules associated with them are probably involved actively in recruiting the cells that differentiate into cementoblasts and then form new cementum. Most significantly, this new cementum is vital for the regeneration of structurally and functionally sound attachment.11
Well accepted studies support this approach to the investigation of cementoblastic cells as a likely supply of factors that are distinctively involved in the healing/regeneration of cementum.
Recently, various research laboratories reported that they have successfully isolated and propagated both animal and human cells that exhibited an apparent cementoblastic phenotype in vitro. After being transplanted into immunodeficient mice in vivo, these cells have demonstrated the capacity to form histologically evident cementum-like tissue in reproducible studies.11
One of the most significant benefits to be derived from establishing these combined in vitro / in vivo models is the increased understanding that they will provide about the relationship between osteoblasts and cementoblasts. Compared to the base of knowledge on bone, very little is known on the cellular and molecular level about the specific mechanisms that participate in the maintenance of cementum structure and function in humans.11
Much of the available information on cementum is an amplification of that pertaining to bone, the general assumption being that the physiology of cementoblasts closely approximates that of osteoblasts. While this may be true in a wider sense, there are distinct differences between the structure and function of these two tissues. It is possible that the specific mechanisms of differentiation of osteoblasts and cementoblasts respectively are being directed by different factor(s). This model is especially important in developing methodologies that will regenerate cementum utilizing cell-based therapies.11
In contrast to cementum, bone is constantly remodeled by osteoclasts and supplemented by bone marrow. Bone is deposited onto the root surface by osteoblasts or osteoblastic precursors that are either delivered or targeted to the tooth root surface. This is thought to lead to osteoclast recruitment and subsequent bone marrow development. Finally, a significant alteration of the local environment in the periodontium would result and could lead to root resorption, ankylosis, and subsequent loss of attachment.11
Therefore, the in vitro/in vivo models may support significant advances in the development of relatively straightforward and reproducible methods of recruiting cells capable of cementoblastic potential and differentiation, while excluding undesirable osteoblastic precursors. In addition, utilization of these systems provides for direct in vivo experimentation with human cells. The fact that cementum is thought to manifest major differences between species in various aspects of its physiology may render this especially significant.11
Cells with a cementoblastic phenotype can be cultured, thus offering the potential to develop more far reaching treatment. This more radical therapy could potentially be utilized in cases where nearly all of the cementum in situ and the adjacent soft tissues have been destroyed secondary to pathology. In this set of circumstances, re-establishment of the correct microenvironment alone will not be adequate to allow regeneration because the number of accessible cementoblast precursors/progenitors is either too low or totally absent in the diseased or injured area.11
In principle, it is possible to isolate cells that make cementum from a relatively small specimen, expand these cementogenic cells in culture, and eventually re-transplant them back into the same patient from whom they were originally harvested. When dissected fragments of healthy tooth root cementum are treated with collagenase, primary human cementoblasts can be grown from them proficiently.11
Grzesik et al.11 recommended using standard culture medium supplemented with 10% fetal bovine serum to maintain these cells. This allows them to acquire significant numbers (in the range of 108-109) of committed cementogenic cells from a single tooth. The drawback of this method is the need for the extraction of healthy teeth for the future cultures. It remains unclear whether or not it is possible to reproducibly obtain cementogenic cells from the teeth of aged and/or diseased patients using this method. Doubts arising over this issue somewhat impede the progress of this cell-transplantation approach.11
If cells with cementogenic potential or cells capable of being induced toward cementoblastic differentiation could be harvested from sources other than cementum, i.e. periodontal soft tissues such as periodontal ligament and gingiva, or even more distant sites such as bone marrow and fat, this scenario would be much more feasible. Utilizing sources other than cementum to isolate cells may necessitate the imposition of two strict conditions: excluding the unwanted cell lineages and inducing differentiation of the cementoblastic lineage.11
The exclusion of unwanted cell lineages can be achieved at three different time frames: by selecting the specific cells before culturing begins, by selecting the cells during the culture expansion, and finally by selecting the cells before placing them into the defect. While molecules such as CAP have demonstrated promise, osteogenic precursors are currently the only defined molecules known to discriminate between the cementogenic and non-cementogenic.11
The induction of differentiation along the cementoblastic lineage could potentially be achieved by pre-treating the cells in vitro or in vivo or both with a specific inducing factor, i.e., by applying an inducing stimulus simultaneously with delivery of the cells to the patient. However, currently there is no known defined molecular factor that is capable of specifically inducing the cementoblastic phenotype.11
Currently, the use of cells harvested from soft tissues for cell therapy to treat cementum loss remains a highly hypothetical option, as the methodology to meet the criteria of these two conditions is not available. Initially, scientists would have to solve the two foremost problems associated with the selection of cells that have an adequate differentiation potential. They would also have to test the general feasibility of this complicated methodology.11
Undoubtedly, the newly developed in vitro/in vivo combined models of cementogenesis will greatly facilitate these goals. Investigators can utilize both the in vitro and in vivo conditions to significantly evaluate the effect of any factor and its relationship to the cementoblastic phenotype, at least in principle. The fact that some well known molecules, including pharmacological agents, are able to induce cementoblastic differentiation cannot be excluded. For example, it has been demonstrated that cyclosporin A, a known immunosuppressant, is capable of inducing the development of cementum in gingival rat tissues.11
Likewise, the in vitro/in vivo system may be useful in evaluating the mechanisms of action of some developed and currently tested compounds such as Emdogain that apparently promote the growth of cementum. Scientists must first identify the critical molecules for cementum regeneration, and then develop a comparatively simple delivery system if these molecules are to be used for periodontal regenerative therapy. In the in vivo settings, bioactive molecules must be present in a specific location and must remain bio-available and bioactive for extended periods of time. These biological requirements pose one of the primary difficulties when applying bioactive molecules in the in vivo settings.11
The anatomy of cementum is unique, i.e., “cementum is a mineralized ‘interface’ tissue connecting mineralized dentin and non-mineralized fibrous periodontal ligament”.11 In the case of cementum regeneration, its anatomy suggests that to be successful, a delivery system must reconcile these two very different environments. The deposition of regenerated cementum must be restricted to the tooth root surface only, without jeopardizing the integrity and structure of the periodontal ligament. This in itself may pose an additional complication. Targeting and binding bioactive molecules to the mineralized surface of the tooth root would be one possible method.11
Some of the growth/differentiation factors, such as BMPs, demonstrate an affinity to the mineral found in bones and teeth; but there are also many factors that do not manifest this preference. The moderate and unpredictable therapeutic effects of many of those substances tested so far can be accounted for by the lack of efficient and stable binding of biologically active molecules to mineralized tooth root surfaces.11
In the past 10 years, investigators have discovered several proteins that bind mineral with high affinity. They have confirmed a link between this activity and the acidic motifs (poly-aspartate and poly-glutamate “stretches”) present in these molecules. It may be possible to achieve cementoblastic differentiation via fusion proteins consisting of a bioactive part(s) (corresponding to factor[s] promoting cementoblastic differentiation, such as the CAP. Efficient targeting to the tooth root surface could be achieved via the mineral-binding domain derived from a different protein.11
Integrin-matrix interactions are fundamentally important to almost all of the facets of cellular metabolism, and even more so for the healing, regeneration, and maintenance of the structure and proper functioning of tissues and organs. Also of importance is the significant and clear positive correlation between integrin ligation and the survival of cells. The receptor-binding domains have been mapped for most of the integrin ligands. The synthetic peptides that correspond to these sequences are biologically active.11
Investigators have confirmed that cementoblastic cells express several integrins as well as their natural ligands (e.g., collagens, BSP, OPN, CAP), suggesting that these molecules may be significant in the regulation of cementogenesis. In addition, this suggests that targeting integrin receptors may facilitate the stimulation of cementum regeneration.11
For example, Grzesik et al.11 recently confirmed that synthetic peptides that contain the integrin-binding RGD sequence and the polyglutamate domain obtained from BSP can be bound easily to synthetic hydroxyapatite/tricalcium phosphate ceramic. When they utilized ceramics such as these as a carrier to transplant cementogenic cells into immunodeficient mice, Grzesik et al.11 reported in unpublished data that cementum formed more easily and copiously than when they used unaltered ceramics.
Their results suggest that integrins may be directly implicated in certain facets of cementum formation, and, as the numbers of active adhesive integrin ligands are increased on mineralized surfaces the formation of cementum by committed cementogenic cells is enhanced.11 Grzesik et al.11 are conducting research to evaluate whether this method can be used to direct bioactive peptides toward naturally occurring mineral matrices of bone and dentin in an experimental setting.
In effect, the science of periodontal tissue regeneration that utilizes cell-and matrix-based therapies is still in its infancy. Nevertheless, more recent developments in basic science illustrate that these methodologies are highly feasible and worth further exploration.11