Cells of the Periodontium1

A very brief overview of the gross anatomy, the histological features and their functions must be understood before the molecular processes that determine these features are discussed. A brief description of the pathological changes affecting the structures will follow, after which the molecular process that mediate these changes will be discussed.

Functionally, the marginal gingiva forms the soft tissue wall of the V-shaped gingival sulcus that is formed by the tooth on one side and the gingiva on the other. The gingival sulcus can be deflected away from tooth by a blunt instrument such as the periodontal probe. In health, the gingival sulcus has a probing depth of about 2–3 mm. The sulcus is filled with the inflammatory exudate called the gingival crevicular or sulcular fluid (GCF). The gingival crevicular fluid has in addition to other constituents, factors important for the defense mechanism of gingiva. The need for defense arises from the fact that the sulcus is the primary plaque forming domain and is the portal of entry for periodontopathogenic bacteria. The gingival fibers help brace the marginal gingiva and prevent it from getting overly displaced from the tooth. Marginal gingiva is thus equipped with cellular and extracellular matrix elements that grant both esthetic and functional characteristics.

The dimensions of the attached gingiva vary from the anterior to the posterior teeth. The width of the attached gingiva refers to its apico-coronal dimension and varies from 3.5–4.5 mm in the anterior teeth to 1 mm in the premolar region. The width is often least in the premolar regions due to the presence of accessory frenae in those areas. In the palatal surface, the attached gingiva blends almost imperceptibly to the palatal mucosa. Lingually, the attached gingiva gives way to the lingual mucosa and is dimensionally much lesser than on the buccal surface.

The width of attached gingiva increases with age and in supraerupted teeth. This increase in dimension occurs as a result of an increase in the height of the alveolar process which in turn is the result of passive eruption. The width of the attached gingiva is reduced either due to gingival recession or periodontal pocket formation.

The mucogingival line remains stationary throughout life as it is a genetically determined landmark that separates not only the attached gingiva from the alveolar mucosa, but also the alveolar process from the basal bone. Any change in dimension is thus, a direct result of changes occurring in the gingiva.

The other dimension that may play a significant role in the maintenance of the periodontal health is the thickness of the gingiva. Gingival phenotype or biotype has been classified by Eger & Muller into thick and thin or Class I, IIA and IIB. Thick gingival phenotype seems to be more conducive to periodontal health. A thin phenotype predisposes to gingival recession and increased tendency to gingival inflammation.

The functional relevance of attached gingiva relates to its firm attachment to the underlying bone. It prevents deflection of marginal gingiva due to the effects of lip musculature and frena. By preventing this deflection, it also helps avoid food entrapment 3between tooth and gingival sulcus, thus aids plaque maintenance. The dense collagenous structure of the attached gingiva also aids in slowing down the inflammatory spread from the marginal gingiva.

The clinical features that have been described above are all a result of molecular processes that occur within the cell. These processes and the changes they undergo during pathology are described in subsequent chapters.

Sulcular and junctional epithelium are both non-keratinized. Sulcular epithelium lines the gingival sulcus. Junctional epithelium has special features to allow for adhesion to tooth. The junctional epithelium along with the gingival fibers forms the dentogingival unit which provides attachment to the tooth. In addition, junctional epithelium is also expected to exhibit defense capability against the invading microorg-anisms. The non-keratinized epithelial cells which are not in close contact with each other allow diffusion of the sulcular fluid from the connective tissue to fill the sulcus. The crevicular fluid with its flushing effect and anti bacterial properties helps keep the pathogenic processes in check.

Plaque related gingival inflammation can be broadly of two types-edematous or fibrotic. Edematous inflammation results in bluish red color, soft consistency and a smooth surface that bleeds on probing. Fibrotic inflammation causes gingiva to remain predominantly pink in color with firm consistency, retaining its stippling and perhaps some change in contour due to enlargement.

Gingivitis is a reversible condition and the pathology may be self-limiting as long as only the gingiva is involved. The factors that trigger the change from gingivitis to periodontitis have not yet been fully elucidated but are related to the host mediated response. Periodontitis is today, known to be a result of an exaggerated response to specific bacteria present in biofilm such as Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tanerella forsythus, etc. The distinctive changes in the periodontium occur as a result of host mediated damage. The end result of these changes is apical migration of the junctional epithelium leading to pocket formation. The molecular basis behind the process of apical migration is described in later chapters.

Cementum has been classified into acellular and cellular cementum. Acellular cementum is present in the coronal 2/3rd of the root and is characterized by the lack of cells. Cellular cementum is present in the apical 1/3rd of the root surface and is characterized by the presence of the cementum forming cells called cementoblasts.

Cementum is also classified based on the fibers present in its matrix into intrinsic and extrinsic cementum. Extrinsic cementum consists of fibers that arise from the periodontal ligament and are inserted perpendicularly to the root surface. These fibers are mineralized and are called Sharpey's fibers. Sharpey's fibers are arranged perpendicular to the root surface and in regular bundles corresponding to the principal fibers of the periodontal ligament. Intrinsic fibers are secreted by the 5cementoblasts and are present within the matrix. These fibers are randomly arranged and are not present in bundles like the extrinsic fibers.

Cementum was for long considered to be an inert mineralized tissue due of its avascular nature and lack of turn over. It is now recognized that, cementum is associated with several biological proteins that are involved in the attachment as well as homeostatic mechanisms.

The biological characteristics of the acellular and cellular cementum are yet to be clearly delineated and offers considerable scope for further research. Similarly the origin and factors that affect the differentiation of 6cementoblasts are yet to be fully elucidated. Currently available information about these factors has been described in later chapters.

The principal fibers are made of type I collagen. In addition to type I, the periodontal fibers have other collagens like type III, XVIII etc. In addition to collagen, periodontal ligament also has oxytalan fibers which belong to the elastin network (elastin, elaunin and oxytalan). Periodontal ligament also has a characteristic distribution of glycoproteins and proteoglycans present in its extracellular matrix.

Cellular elements present in the periodontal ligament are fibroblasts, osteoblasts, undifferentiated mesenchymal cells and the ECRM. The role of each of these cells, their unique features and the molecular process each of them undergo are described later in this chapter.

The alveolar process is in most parts dictated by teeth. The shape of the root, alignment of teeth and their position in the arch determine the shape and size of alveolar bone. The bone cells are osteoblasts, osteoclasts and osteocytes, and together they regulate bone formation. These cells and their regulating functions are described later in this chapter. Bone loss is perhaps one of the most definitive features of periodontitis.

The characteristic features of each of these tissues have been described briefly. The intracellular process that goes towards formation and maintenance of these tissues in health, the changes they undergo during pathology and their potential use as markers of periodontal disease forms the rest of the book.

All cells are enclosed by a plasma membrane within which is the cytoplasm or the cytosol of the cell. Each cell has several structures called organelles that are required for synthesis, transport of substances (esp. proteins) and to perform important functions that are required for basic cell survival. The important organelles of the cell are-cytosol, nucleus, Golgi complex, endoplasmic reticulum, ribosomes and lysosomes. These organelles have been briefly discussed so as to offer an understanding of the common processes that are involved in production of structural molecules that are relevant to the periodontium.

The ability of cell to expand and adapt to their external stimuli is dependent on transfer of ions and solutes in and out of the cell. A rigid impermeable barrier would not allow exchange of material between the cell and the external environment. For this purpose, the plasma membrane needs to be semipermeable that would retain essential intracellular substances at the same time allow transfer of solutes that are required to maintain homeostasis.

Unlike other membranes within the cell, plasma membrane needs to maintain its rigidity to prevent any distortion due to external pressure. This rigidity is conferred by the cytoskeleton.

All biological membranes that surround the cell and other intracellular organelles have a common general structure – an outer membrane and an inner membrane (Fig. 1.2). The lipid bilayer confers impermeability to the plasma membrane. This lipid bilayer has a hydrophilic end that is exposed to the external environment and a hydrophobic end that faces the interior of the cell. Apart from the lipids, the chief constituents of the plasma membrane are proteins.

The presence of the glyco lipids on the apical surface of the epithelial cells grants these cells their polarity. Cell polarity is important for the motility of the keratinocytes as we shall see in following chapters.

The lipid bilayer is not similar to each other and there is considerable asymmetry in their protein binding capability. This asymmetry has biological effects that are functionally relevant to the cell. There are specific proteins that bind to phospholipids that are asymmetrically distributed on each of the lipid layer, e.g., protein kinase C (PKC) binds to the hydrophobic tail of the lipid monolayer, specifically to phosphotidyl serine. The negative charge produced by the phosphotidyl serine is required for this binding to take place. On the other hand, phosphotidyl inositol is not present in the hydrophobic portion of the lipid bilayer. Following signal activation, this molecule transforms its binding to the hydrophilic portion of the membrane.

Transmembrane proteins are usually present in two forms-the α helix and the β sheet. The α helix may be a single pass structure, i.e. they may cross the plasma membrane just once, e.g. receptor tyrosine kinases like the epithelial growth factor receptor, fibroblast growth factor receptor etc. They may otherwise be a multiple pass transmembrane structure like the G protein coupled receptors that run back and forth across the membrane seven times.

The β sheet proteins form transport channels that are mostly involved in transport of ions and other molecules across the cell. Transport proteins are classified into two main categories-

The most important source of ATP is the dietary carbohydrates. These carbohydrates, are broken down to smaller subunits before they can be utilized by the cell through various metabolic processes.

Carbohydrate metabolism differs according to the presence and absence of oxygen. In the absence of oxygen, they undergo a pathway called the anaerobic glycolysis, whereby glucose is broken down into acids such as lactic acid. This process is generally not a rich a source of ATP. Oxidative phosphorylation, on the other hand, results in metabolism of glucose with formation of pyruvate as an end product of this metabolism. Several ATP molecules are produced during these reactions and these can be utilized for energy dependent processes within the cell. Even this process is however, not entirely energy efficient, as the pyruvate that is formed is not normally utilized for other purposes. Utilization of pyruvate and production of further molecules is possible only through the presence of specialized structures in the cell called the mitochondria.

Ultrastructurally, the mitochondria have two membranes called the inner and outer mitochondrial membrane with an intervening space labelled as the membrane space. The inner mitochondrial membrane encloses the mitochondrial matrix. The outer membrane is, by and large, a smooth structure but it is intersperced by large aqueous channels at 12several points. These large channels that are formed are called porins (Fig. 1.3). Porins are freely permeable to transport of most proteins that are of a relatively low molecular weight, i.e. lesser than 5000 Da. These proteins freely cross the outer membrane but are trapped within the membrane space.

The inner membrane is not a smooth structure like that of the outer membrane. They are thrown into several infoldings called the cristae. The inner membrane and the mitochondrial matrix are the structurally important regions of the mitochondria. The structural relevance of the matrix results from the fact that it is the site of the ‘citric acid cycle’ or ‘Kreb cycle’. Kreb cycle is, perhaps, the most important carbohydrate utilization process of the cell resulting in the formation of the carbon dioxide and water. During this process, several molecules of ATP are generated and this is then utilized for the energy requirement of the cell.

The other important feature of mitochondria is the fact that it is an important source of extra nuclear genetic material. While the overwhelming majority of the genetic material is stored in the nucleus, eukaryotic cells can exhibit DNA in other intracellular organelles. Among these organelles, mitochondria store a large amount of DNA, RNA polymerases and other nuclear proteins. The reason for such a large presence of genetic material in the mitochondria is currently unknown. However, it has been well characterized that inheritance of mitochondrial DNA does not follow mendelian laws like the nuclear DNA. Because of its nature of inheritance, the mitochondrial DNA lead to progeny with same haploid characteristics as that of the parent. This feature has been used to identify early genetic damage that occurs as a result of exogenous damage through radiation or chemicals. Oxidative damage, that is cellular damage that results from excessive production of reactive oxygen species, can be first recognized from mitochondrial DNA. Mitochondria are thus, not only the energy power house of the cell, but also an important source of extra nuclear genetic material.

The functions of the nucleus and the cytosol are, interdependent on each other. Optimal function of one organelle is dependent on proteins/macromolecules present in the other. In order to maintain homeostasis, there is a need for constant communication between the two 13compartments. Therefore, the nucleus and the cytosol are always organized in a sort of two way traffic system. The importance of this two-way communication can be illustrated from the following examples. Important proteins like histones and RNA polymerases that are required for nucleic acid regulation within the nucleus are normally present in the cytosol. Similarly, protein synthesis begins in the nucleus and is completed in the cytoplasm. This bidirectional trafficking is made possible through a network of open channels. Transport, both in and out of the nucleus occurs through an active transport process.

The nucleus is covered by the nuclear envelop that separates the nucleus and cytosolic components into two. The nuclear envelope has an outer nuclear membrane and an inner nuclear membrane which are structurally dissimilar to each other (Fig. 1.4). The outer nuclear membrane is continuous with the membrane of the endoplasmic reticulum at various points. The outer nuclear membrane has numerous ribosomes embedded on its surface. Inner nuclear membrane, on the other hand, has specific proteins that can bind to genetic material and transport them to the cytosol. The space between the inner and outer nuclear membrane, called the perinuclear space is continuous with the lumen of the endoplasmic reticulum.

The nuclear membrane is lined by large open structures called the ‘nuclear pore complexes’. These nuclear pore complexes are built by proteins called nucleoporins that are arranged in a symmetrical octagonal manner. Usually, the greater the transcriptional activity of the cell, the greater the number of pores in the nuclear envelop. Each nuclear pore is composed of one or more aqueous channels. These channels allow smaller molecules (less than 5000 Da) to pass through freely. This passive diffusion allows molecules such as Histones to pass into the nucleus. Histones, as we shall see in detail later, are required to pack DNA to form chromatin. This passive diffusion allows a large number of histones to be transported into the nucleus in a short period of time. In a cell that is undergoing active synthesis, about 100 histones must be transported into the nucleus every minute. The larger molecules, however, cannot use this transport mechanism as the small pores do not allow them to pass freely from one side to the other, e.g. the RNA polymerases that have to enter the nucleus and the large ribosome bound proteins are far too large to passively diffuse through these pores. As these macromolecules are important for the normal functioning of the genetic transfer and protein synthesis, this requires an alternate transport mechanism. This is achieved through an active transport process.

Similarly transport of proteins outside the nucleus occurs though nuclear export proteins. These proteins are encoded by a set of genes that exhibit structural homology to that of the import proteins. Functionally, too, they are very similar to import proteins and the only difference they exhibit is in the direction of movement, i.e., they move molecules from inside to outside the nucleus. This bidirectional active transport is an energy dependent process that requires activation of GTP/GDP. This energy is produced by a molecule called Ras. Ras is a monomeric GTPase, i.e., it can split GTP to form GDP and Pi and in the process releases energy. Ras is present in both cytosol and nucleus and is essential for active transport across the nucleus. Ras is present as bound to GTP or GDP and like other signal molecules, alternates between conversion from an active to inactive state. This conversion is brought about by GAPs that convert Ras-GDP to Ras-GTP. GAP's are present in significantly higher concentration in the cytosol. In contrast, the GEF's convert the GTP-Ras to GDP-Ras and is present more in the nucleus. They promote the import and export in the appropriate direction.

For practical purposes, the Golgi complex may be divided into two cis face and a trans face both of which are divided into two distinct compartments.15

Protein synthesis begins in the nucleus and they are then secreted into the cytosol through the nuclear pores. The endoplasmic reticulum selectively captures these secreted proteins from the cytosol and internalizes them for further processing. These proteins are of two types, the transmembrane proteins and the water soluble proteins. The transmembrane proteins remain attached to the membrane of the endoplasmic reticulum, whereas the water soluble proteins cross the membrane barrier and enter the lumen. Functionally, these proteins differ in that the transmembrane proteins reside either in the endoplasmic reticulum itself or become an integral part of the membrane of other organelles, while the water soluble proteins remain in the lumen of other organelles or are secreted outside the cell.

Normally most proteins are transported into organelles after the process of translation is completed. Otherwise, the enzymes of the cytosol are capable of proteolysis of the polypeptide chains. In order to avoid this, 18proteins are folded and processed by molecules called chaperons that preserve the integrity of the proteins. Transport of proteins into most organelles is then a post-translational process. In contrast proteins are transported into ribosomes as a part of the translational process.

Ribosomes are classified into membrane-bound and free, both of which are structurally and functionally similar. Assembly of proteins, however, occurs in the ribosomes that are attached to the endoplasmic reticulum. Transport of proteins into the endoplasmic reticulum occurs when they are still in the stage of polypeptide chains and is thus a co-translational process. A single mRNA chain is bound by several ribosomes and the structure thus formed is called polyribosome. Polyribosomes are attached to the endoplasmic reticulum at its cytosolic face and they eventually form the protein chain. Polypeptide chain requires specific signal sequences that have to be recognized by the endoplasmic reticulum before they can be internalized. The polypeptide sequence is recognised by the presence of signal recognition peptide (SRP). This SRP is recognized by the SRP receptor that is present in the membrane of the endoplasmic reticulum. The SRP binds to the endoplasmic reticulum signal sequence as soon as the peptide has emerged from the ribosome. This binding creates a halt in the protein synthesis and during this pause the ribosome binds to the endoplasmic reticulum. As a result, the polypeptide is not released into the cytosol, thereby preventing proteolytic destruction. The processing of protein is different between transmembrane proteins depending on whether they are single pass or multiple transmembrane proteins. On entering the endoplasmic reticulum lumen the proteins undergo a process of glycosylation to prevent their degradation.

The smooth portions of the endoplasmic reticulum are also involved in protein synthesis, but in a different manner. The smooth areas are those regions in which vesicles are formed that help transport the proteins synthesised in the endoplasmic reticulum to the Golgi complex. In addition to this, the smooth endoplasmic reticulum is important in cells that are actively involved in synthesis of steroids. The smooth region seems to be necessary for incorporation of various enzymes that are required to process cholesterol to form steroids.

In summary, the process of protein synthesis begins in the nucleus, with the conversion of DNA to RNA (transcription). Thereafter, mRNA is converted to the polypeptide chain that is represented by the DNA (translation). The polypeptide chain is transported out through the nuclear membrane pores. It is then processed in the ribosome/ endoplasmic reticulum complex. After this step, it is stored and transported via the Golgi complex into various areas in the cell.

After this brief description of the important organelles of the cell, the rest of the chapter will focus on various cell types of the periodontium.

Structurally, keratinocytes are like any other cell in that they have organelles like nucleus, cytosol, ribosomes, Golgi complex etc. The plasma membrane of the keratinocytes is thrown into infoldings on their apical surface. These cytoplasmic extensions aid in the locomotion exhibited by the cells. In gingiva, this movement is essential to replenish the constant shedding of cells that occur in the coronal areas of the gingiva that is exposed to masticatory stress. Intracellular organelles that are present in keratinocytes but not in the other cells of the periodontium, include melanosomes, which are the pigment bearing granules in the keratinocytes. The formation of these granules and their transport into the coronal areas are described in subsequent chapters.

Keratinocytes originate from the ectodermal germ layer. Their differentiation occurs from basal to coronal layers from the epidermal proliferating unit (EPU) present in the basal layers. Keratinization process requires presence of sulfhydryl groups, calcium influx into the cell. Cytokeratins are the characteristic feature of all epithelial cells. The basal cytokeratins are K5 & 14, the simple epithelial keratins are K6, 16, etc. The keratinocytes that are differentiating terminally to produce keratinized epithelium shows K1 & 10. Cytokeratins are also described in detail in later chapters. Other proteins present are the filaggrin, loricin, both of which aid in the process of keratinization.

Gingival keratinocytes, unlike their dermal counterparts, e.g. are also involved in defence mechanisms of gingiva. For defence purposes, keratinocytes of the gingival epithelium secrete antimicrobial peptides. The important among them are defensins. The defensins are classified into α and β defensins. For similar reasons, epithelial cells are associated with inflammatory cells (neutrophils, dendritic cells) and their mediators (IL-8, ICAMs) that modulate immunological reactions that may be required for homeostasis of the gingiva.

The difference between the two fibroblast populations is related to the functions they are expected to perform. PLF's are primarily responsible for anchoring teeth to bone, absorb mechanical forces and maintain tissue homeostasis during tooth movement. Gingival fibroblasts on the other hand, contribute to immune regulation in addition to production and maintenance of the ECM of gingiva.

PLF's originate from stem cells that are present in the paravascular spaces. From these spaces, cells proceed in their differentiation pathway until they become fully functional fibroblasts by moving towards the extracellular spaces. The differentiating cells move in a direction from the central towards the peripheral areas of the PDL space. Similarly, these cells exhibit directional motility in an apicocoronal direction. Gingival fibroblasts, are derived from the undifferen-tiated progenitor mesenchymal cells that are present in the follicle and more importantly from the ridge mucosa.

The fibroblasts are, however a heterogeneous lot with different sub-populations of cells present in both the gingiva and periodontal ligament. Some of these heterogeneous populations present differences in their morphometry. The PLFs have been shown to exhibit almost 4 to5 different (morphological) shapes ranging from round to stellate shaped. These differences are dictated largely by their differences in functionality and stage of differentiation.

The greater expression of the contractile elements in the PLF's membrane grants these fibroblasts a greater ability to contract collagen. This greater expression of contractile proteins would have functional relevance to the PDL as they are better equipped to absorb mechanical stress without being displaced from the socket. The stretching and elongation of the collagen fibers can be thus offset by the contraction of the PLF's.

Similarly, the cytoskeleton related proteins such as Transglein and Desmoplakin are expressed to a greater extent in the PLFs. Transglein is a surface protein that is thought to be associated with and upregulated in cells undergoing shape transformation. They are found usually associated with the actin filaments. Along with the contractile elements, these proteins help the PLFs change shape at will. This is an important functional adaptation that enables PLFs to accommodate the stretching and compression of the collagen bundles along which these cells are lined.

A greater expression of the cell cycle associated proteins such as Cyclins observed in the GFs are thought to aid in this increased proliferation. This greater proliferation has an important clinical relevance in that these cells are able to provide enhanced wound fill. Greater proliferation is linked to several genetic and environmental factors as distinct subsets within the gingiva exhibit greater capacity to proliferate.

On the other hand, apoptosis occurs at a much lower rate in the PLF's when compared to the GFs. The presence of the SFRP (Secreated Frizzled Related Protein) that activates the WNT pathway may act along the GBP(i) especially to retard apoptosis. The longer cell cycle of the PLFs means that these cells have the ability for greater protein synthesis and thus matrix turnover:

Cultured gingival and periodontal fibroblasts differ in their protein synthesis. Periodontal fibroblasts are thought to exhibit greater quantities of protein related to extracellular matrix, especially collagen I, sulphated glycosaminoglycans, fibronectin, vitronectin and laminin when compared to the gingival fibroblasts. Gingival fibroblasts are capable of greater expression of angiogenic factors Ang-1, VEGF, when compared to periodontal fibroblasts. Periodontal fibroblasts show greater expression of the negative regulator of angiogenesis namely the pigmented epithelial derived growth factor (PEDF). Among the various non-collagenous proteins secreted by the fibroblast, gingival 22fibroblasts show the greater expression of the elastin protein, secreting elastin, elaunim and oxytalan. The periodontal fibroblasts are capable of secreting only the oxytalan fibers.

Both fibroblasts are involved in catabolism of connective tissue elements such as collagen. Fibroblasts are involved in phagocytosis of collagen by two different mechanisms.

The periodontal fibroblasts are also capable of exhibiting greater alkaline phosphatase (ALP) activity than the gingival fibroblasts. Alkaline phosphatase is an essential requisite for formation of mineralized tissue as it is necessary for conversion of organic phosphates to inorganic phosphates prior to formation of hydroxyapatite

Periodontal ligament fibroblasts can also transform to bone forming cells when they are exposed to constant mechanical stress. The periodontal ligament fibroblasts exhibit special features so as to facilitate their osteoblastic behavior. The periodontal ligament fibroblasts, exhibit a baseline expression of Cbfa1, even under unstimulated conditions. Cbfa1 is a master switch that regulates osteoblastic differentiation. The baseline Cbfa1 expression of the periodontal fibroblasts is definitive indication of the ability of these cells to undergo osteoblast differentiation. In addition, these cells can also exhibit a higher level of bone morphogenic proteins (BMPs)-2 and 4, both of which are required for bone formation, than the gingival fibroblasts. These cells are also capable of higher expression of bone related proteins such as osteopontin (OPN) and osteonectin (ON). However, Bonesialoprotein (BSP) the initial nucleator of hydroxyapatite crystals is minimally expressed by the periodontal ligament fibroblast. Osteocalcin (OCN), the regulator of crystal formation is however highly expressed in periodontal ligament.

The epithelial mesenchymal inter-relationship that goes towards formation of enamel/dentine with the inductive role played by odontoblasts in formation of enamel, is clearly understood. However, it was realized in recent years that epithelium may play a role in formation of cementoblasts as well. The cells of the HERS secrete proteins that have been collectively termed as the enamel matrix proteins, the principal proteins amongst which are amelogenin, sheathlin and tubulin. These enamel proteins play an inductive role in differentiation of developing undifferentiated mesenchymal cells to form cementoblasts. These enamel derivatives form the matrix and secrete inductive factors that cause undifferentiated mesenchymal cells to form cementoblasts.

In recent years, another hypothesis regarding the origin of cementoblasts has been proposed. Cell culture studies have revealed that there is a propensity for some cell types to undergo transformation into a cell of a different germ line, e.g. Epithelial cells cultured in collagen gels have been shown to transform to cells that resemble myofibroblasts. Other cells have also shown a similar propensity and this process is thought to be important in the wound healing process. This phenomenon has been termed 24epithelial-mesenchymal transformation or trans-differentiation.

Bosshardt and Nanci have proposed that the cells of Hertwigs epithelial root sheath may show a similar effect and transform to form cementoblasts. Earlier investigators reported that these cells have shown secretory vesicles on their surface that face the forming cementum that were thought to secrete the amelogenin related proteins. These authors suggest that these cells show a change in morphology as cementum formation proceeds. This has led them to propose that it is actually the HERS cells that trans-differentiate to form cementoblasts. Further strength to this theory has come from studies that have reported that REE (the resting cells of the HERS) seems to be involved in cementum repair. These cells have also shown evidence of presence of bone related proteins such as osteopontin, bonesialoprotein and the related BMPs such as BMP-2.

The morphology of the cementoblasts that lay down AEFC is similar to the PLFs, so much so that they are virtually indistin-guishable from each other. As suggested earlier, heterogenecity of the PLFs means that there are several cell types that are structurally distinct from each other. Cementoblasts resemble only those PLFs that line the cementum surface and seem to be regulating cementogenesis.

Under the SEM, the cementoblasts has been shown to have cytoplasmic extensions that have been differentiated into wing like processes and finger like processes. The wing like processes encircle the extrinsic fibers while the finger like processes line the cementum surface and are involved in intrinsic fiber production. The intrinsic fibers are parallel to the root surface and are thought to form a link between the extrinsic fibers and the root surface.

Amelogenins are one of the principal local regulators of AEFC.

Amelogenin gene consists of 7 exons and introns in between this and an active ‘amino’ terminal end that is designated as the TRAP (tartarate rich amelogenin protein). TRAP has the ability to upregulate pathways that are involved in Cementoblast differentiation, for eg., Cbfa1, BSP, etc. Another member of this family LRAP is also known to upregulate the Cementoblast differentiation process.

Other splice variants of the Amelogenin gene designated A+4 and A-4 are known to differentially regulate the mineralization process. A-4 is specifically thought to upregulate Cbfa1 expression, therefore helping the mineralization process while A+4 causes collagen II expression regulating osteoblast differentiation.

Cementoblasts, like the osteoblasts, show surface integrins that are required for attachment to collagen. Mineralization is a process that occurs after cells are adherent to collagen I. The surface integrins are α₂β₁ and those that are attaching to the bone associated proteins. Numerous β₃ integrins are present on the surface of cementoblasts. The α_vβ₃ integrins help them attach to osteopontin, bonesialoprotein and osteonectin.

There are several proteins in the matrix that are unique to cementum. The keratan sulphate rich lumican is present in greater concentration in cementum than in bone. This proteoglycan is secreted by the cementoblasts. However, the process involved in the regulation of their secretion is yet to be fully understood. Additionally, cementoblasts secrete and respond to a unique protein called the cementum attachment protein (CAP). This CAP is thought to play a role in recruitment of cementoblasts to the developing cementum. Further, CAP is important for insertion and retention of periodontal ligament fibers. CAP is secreted by the cementoblasts and again the genes regulating this process and the growth factors responding to their activation is not yet fully understood.

In any case, there is still some work to be done before the complex mechanisms that underlie the origin of cementoblasts, the protein they secrete and their ultimate 26organization into the mineralized tissue can be fully unravelled. It is therefore, no surprise that of all the tissues of periodontium, cementum continues to be the least regenerated.

Bone is a mineralized tissue that is in a constant state of flux. Since it is bone that is responsible for all the mechanical forces applied, it is important that it is a dynamic structure that is capable of constant change. The alveolar bone is always exposed to masticatory forces, necessitating constant formation and resorption. This is made possible only through the presence of osteoblast/osteoclast coupling reaction. In other words, these two cells are dependant on each other for their existence and for efficient functioning. Alveolar bone, in addition, also has to contend with a constant inflammatory reaction that originates from gingiva. The inflammatory reaction of the alveolar bone has to be tightly regulated to prevent excessive loss of bone. Both osteoblasts and osteoclasts have to act in tandem to form and maintain homeostasis of bone.

The definitive transformation of BMSC's into future osteoblasts occurs when they are in the presence of Runx2/Cbfa1. The runt homology regions have the requisite genetic material that can result in the activation of its own gene. On activation of the Cbfa1 gene, other genes that are associated with bone formation can further be activated. Important among these are the ColI gene, the ALP gene and genes for specific bone related proteins such as OPN, BSP and importantly OCN. There are other genes that are required for osteoblast activation in their early stage. Important ones are the TAZ gene; Dlx homeobox genes that help transform the early uncommitted progenitor cells to become committed osteoblast progenitors. Once the cells become committed osteoblasts, other factors such as the Cbfa1 and osterix are responsible for the differentiation of these cells to form the fully developed osteoblast.

Lining cells are osteoblasts that have a flat shape and limited intercellular organelles within it.27

Once these signals are in place, fusion of multiple cells must occur for the formation of the multinucleated osteoclasts. Fusion consists of two processes:

RANK is activated through its ligand RANKL. RANKL is produced from the osteoblasts/stromal cells. Upon RANK/RANKL binding, activation of osteoclasts occurs and subsequently leads to bone resorption. On the other hand, osteoprotegrin (OPG) is also produced by various mesenchymal cells and acts as a soluble decoy receptor for RANKL. OPG binds to RANKL but prevents downstream activation. As down stream signaling does not occur, there is no activation of transcription factors and therefore no osteoclastogenesis. Apart from the osteoblasts, other source of RANKL is the activated T cell. The link between the immunological reactions and osteoclasto-genesis has been identified in recent years. This understanding led to the development of a new branch “Osteoimmunology”. This finds relevance in several diseases such as rheumatoid arthritis, where the activation of T cells in synovial spaces is thought to lead to excessive RANKL production and osteoclast activation, leading to bone loss observed in this disease.

The RANK/RANKL/OPG axis has special significance in the periodontium. The periodontal ligament fibroblasts have been demonstrated to produce high levels of OPG 28in health. This expression of OPG prevents excessive osteoclastogenesis and thereby prevents root resorption. In periodontal disease, it has been observed that T cells present in the gingival tissues can produce RANKL. It is thus thought possible that osteoclastogenesis observed in periodontal disease is a direct result of RANKL production by the T cell. Either way, it is clear that osteoclast development and activation is atleast in part dependant on signals produced by the osteoblasts. Conversely, the osteoblast activation is also dependant on osteoclasts. The fully developed osteoclast has abundant lytic enzymes like the TRAP. This acid phosphatase results in demineralization of the mineralized tissue. This results not only in bone loss, but also release of inductive factors present in the matrix including the BMPs. BMPs are important for osteoblast development and are entrapped in the bone matrix. Liberation of BMPs from the mineralized matrix, results in further osteoblast differentiation and this activates Cbfa1 and other genetic factors responsible for osteoblast development and maturation.

Bone, in addition to providing mechanical rigidity to enable locomotion, is also an important store house of calcium. Calcium signaling is important as a second messenger for a number of cellular reactions that are important to sustain life. When serum calcium levels undergo any change, the calcium required for replenishing the reduced calcium is obtained from bone. On the contrary, when excess calcium is present, it is excreted by the kidney with sufficient reservoirs stored in bone.

Parathormone is the major hormone that regulates serum calcium levels. The receptor for parathormone is present on osteoblasts and not the osteoclasts. In response to the parathormone stimulus, osteoblasts release greater quantities of RANKL resulting in osteoclastogenesis. The mandible may also serve as a reservoir even though it may not be the primary calcium storing organ like the long bones. Thus, in conditions like hyperparathyroidism, the mandible may also lose mineralized tissue, resulting in the condition called Paget's disease of the bone (osteitis fibrosa cystica).

Locally generated inflammatory mediators such as IL-6 do not affect the osteoclasts directly. The IL-6 receptor is present on the osteoblasts, which on activation results in increased production of RANKL which subsequently results in osteoclastogenesis. In summary, both systemic and locally generated signals that result in formation and destruction of bone do so through stimulation of the osteoblast/osteoclast coupling behavior.

Osteocytes present in the mineralized matrix embedded in ‘cases’ that are called lacuna. These cells have long cytoplasmic processes called canaliculi through which they communicate with each other and the other bone cells. Through these communications, they are in a position to direct osteoblast/osteocyte activity on 29demand so as to regulate mineralization. Osteocytes are generally identified by the presence of MEPE, Sclerostin/E11/qp28 all of which are specific to these cells.

These communications are thought to allow the osteocytes to transmit signals they receive from the external environment to the other cells of bone, thereby making them respond in order to maintain homeostasis of the bone.

For a long time, it was thought that osteocytes are quiescent cells that do not play any specific role within bone. It is now realized that osteocytes have three important functions:

Osteocytes have surface receptors such as Integrins (α_vβ₃); gap junction proteins connexins and canaliculi that respond to fluid movement that corresponds to mechanical stress.

On loading, both anabolic and catabolic responses occur.

The two primary genes that respond to mechanical loading are

The Wnt catenin pathway that increases mineralization.and

LRP 5 which is an important positive regulator of mechanically stimulated gene activity. Sclerostin, on the other hand is the most important negative regulator that responds to membrane loading.

Osteocyte is hence, far from a passive cell, it plays an important role in mechano-sensation and regulation of mineralization.

In addition to these important cells that go to form gingiva, periodontal ligament, cementum and alveolar bone, there are a number of other cells that play a role in maintaining the homeostasis of the periodontium. Important among these cells are the inflammatory cells, both of innate and adaptive immune systems. Inflammatory cells and their actions are beyond the scope of this book. Other cells, unique to the periodontium are the epithelial cell rests of malassez, melanocytes and the undifferentiated mesenchymal cells.

Several theories have been forwarded regarding their function or the lack of it. It has been proposed that epithelial cell rests of malassez play an important role in the maintenance of the periodontal ligament space. In physiological conditions, it is therefore thought that these cells that are devoid of any mineralization propensity may contribute to maintaining a non-mineralized area. Some authors have shown that these cells may lead to ankylosis of teeth in isolated areas.

Structurally, epithelial cell rests of malassez are similar to epithelial cells anywhere else. The cytokeratin profile of these cells is similar to that of junctional epithelium, with a high expression of K5, K6, K7, K8, K16 and K19, all of which are characteristic of simple and differentiated epithelium. However, these cells are of ectomesenchymal origin and hence retain some characteristics of odontogenecity. Therefore, it has been reported that these cells show expression of BMP2, 4 and also some bone related proteins such as osteopontin and bonesialoprotein. It has consequently been argued that these cells may be involved in regeneration of periodontium, especially cementum. These cells are involved in pathogenesis of several disorders including pocket formation. The presence of c-met receptors in the epithelial cell rests of malassez suggest that these cells can respond to the inflammatory cytokine, Hepatocyte growth factor. This scatter factor is thought to be capable of aiding migration of these cells and hence aid in the pathogenesis of pocket. Also, these cells have been reported to be involved in production of neuropeptides such as substance P, VIP and CDGRP. These neuropeptides are involved in vasodilation and other proinflammatory responses.

These cells are also thought to be responsible for cementum repair as they are found in areas of resorption when mechanical injury was created in experimental animals. In this experimental report, these cells were found to exhibit the presence of BMP2 – an osteoblast inducing factor. It has been suggested that cells retain the capacity to differentiate into cementoblast and lay down matrix.

In summary, the epithelial cell rests of malassez are thought to play a role in physiological maintenance of periodontal ligament space; may play a role in pathogenesis of pocket formation and probably may mediate process involved in regeneration.

Understanding the structure, biology and functions of each of these cell types is an essential step to our understanding of the physiology of the periodontal tissue. The role each cell plays in maintaining the harmony of periodontal tissue needs to be clearly delineated. It is only then that complete regeneration, that remain an elusive goal till now, may become a reality.

Stem cells are characterised by their ability to divide continuously, in theory, an unlimited number of times. In practice, stem cells do have a finite number of divisions. They are also characterised by a slow turnover rate. The ability to divide continuously when compared to adult stem cells is conferred by the higher expression of teleromase enzyme. The best characterized stem cells are the bone marrow stem cells (BMSC) and the MSCs. BMSC's are identified by the presence of STRO-1 and CD172. BMSC's differentiate into adipocytes, monocytes, osteoblasts or blood cells based on the availability of the transcription factors.

The presence of stem cells in periodontal ligament has been under investigation for a very long time. Progenitor cells in the paravascular spaces of the periodontal ligament have demonstrated the capacity to differentiate into osteoblasts and periodontal ligament fibroblasts. Stem cells have been recently identified in the periodontal ligament with the marker STRO-1. These cells have shown to differentiate into adipocytes, fibroblasts and osteoblast and form mineralized nodules. It is this ability of the stem cells to differentiate into adult cell types that make these cells an attractive option for use in tissue engineering techniques and regenerative procedures.

The lack of appropriate delivery systems, i.e., vehicles through which stem cells can be implanted to the desired site limit their clinical use to date. The development of better biological systems that provide the right matrix/ growth factors / adhesion molecules that can deliver these stem cells into vertical defects can result in optimal regeneration. Although much work remains to be done, it is clear that identification of stem cells have paved the way for development of newer techniques in periodontal regeneration.

A thorough understanding of these cellular changes would also help evolve strategies that could revert these pathological changes. Knowledge of cellular behavior is also important to devise newer treatment strategies that involve tissue engineering techniques. Stem cell research is currently thought to be the most promising method for regenerating lost tissue parts.