Biology of periodontal regeneration

Introduction:

The ultimate goal of periodontal treatment is regeneration. Regeneration can be defined as the reproduction or reformation of organs or tissue that have been lost or injured as a result of a wound or infection. Periodontal regeneration results in functionally aligned periodontal ligament fibers between newly formed bone and the root surface. Regenerative periodontal procedure involves the creation of new alveolar bone, cementum, and periodontal ligament.

The regeneration of periodontal tissues requires a conducive biological environment which induces the differentiation of undifferentiated cells to make required structures. Various molecules such as growth factors and cytokines play a vital role in regeneration. A thorough knowledge of these molecules as well as their functions is required to understand the biological mechanisms involved in regeneration.

The events that take place during wound healing play an important role in regeneration. Type of injury and events that take place during healing determine whether repair or regeneration will take place. Let us first try to understand the healing process before we discuss regeneration at molecular level.

General overview of wound healing:

Wound healing has three principal phases: inflammatory, proliferative, and remodelling. The phases of wound healing are overlapping, but are described in a linear fashion for the purpose of clarity. Healing is also described as healing by primary intention or secondary intention. In healing by primary intention, the wound edges are apposed and there is minimal tissue loss. Healing by secondary intention is characterized by edges that are separated with a more extensive tissue loss. Healing by primary intention results in a small scar, whereas healing by secondary intention is followed by a large scar.

Inflammatory phase:

The inflammatory phase is characterized by hemostasis and inflammation. It begins at the time of injury and lasts for 24 to 48 hours. The healing process cannot proceed until hemostasis is accomplished. Collagen exposed during wound formation activates the clotting cascade (both the intrinsic and extrinsic pathways)(read more in “The clotting mechanism and bleeding disorders”). The end product of the hemostatic process is clot formation which is primarily composed of fibrin mesh and aggregated platelets along with embedded blood cells. Fibrin mesh present in the clot acts as wound matrix onto which fibroblasts and other cells migrate as the healing process proceeds. Platelets form the initial thrombus release growth factors that induce the chemotaxis and proliferation of neutrophils and macrophages, which co-operate to remove necrotic tissue, debris, and bacteria from the wound. Macrophages and neutrophils release gelatinases and stromelysins (MMP’s) that aid the diffusion of inflammatory cells into the wound during the inflammatory phase. Elastase and gelatinase, released by neutrophils, also remove debris from the ECM. Vascular endothelial cells release MMPs that aid the migration of angiogenic cytokines (VEGF, TGF-α, TGF-β, TNF-α, aFGF and bFGF) into the wound 1.

The degranulation products of platelets play very important role during initial inflammatory process. Alpha granules of platelets contain a variety of immunomodulatory and proteinaceous factors that are involved in both the early and late phases of healing. These include factors like albumin, fibrinogen, fibronectin , IgG, and coagulation factors V and VIII, as well as platelet-derived growth factor (PDGF), transforming growth factor α and β (TGF-α and TGF-β), fibroblast growth factor-2 (FGF-2), and platelet-derived epidermal growth factors (EGFs), and endothelial cell growth factor.

Monocytes in the nearby area are attracted to the area and transform into macrophages, usually around 48 to 96 hours after injury. Macrophages then become the prominent cell during this time period and release various growth factors and cytokines that change the relatively acellular wound into a highly cellular environment. Macrophages secrete important cytokines like IL-4, IL-5, IL-8, IL-12, IL-15 which play an important role in activation of  T-cell and B-cell mediated immune response.

Regulatory mechanisms ensure that growth factors and cytokines are present within the wound for sustained periods. For instance, lymphocytes are activated by macrophages to produce cytokines such as interferon-γ (INF-γ) that act back on macrophages and monocytes (in a paracrine manner) to release other cytokines such as tumour necrosis factor-α (TNF-α) and interleukin-1 (IL-1).

Wound healing & factors involved in wound healing

Wound healing

Proliferative phase:

During this phase epithelialization, angiogenesis, granulation tissue formation, and collagen deposition take place. Fibroblasts proliferate to become the dominant cell of the proliferative phase. They migrate into the wound site from the surrounding tissue, become activated, and begin synthesizing collage. They proliferate in response to growth factors and cytokines that are released from macrophages, platelets or mesenchymal cells, or those that have been stored in the fibrin clot. In addition to chemotactically drawing fibroblasts into the wound, PDGF, FGF and EGF induce fibroblast activation and proliferation. Starting at day 3 or 4 collagen is deposited, net collagen deposition is positive until day 21. After this day, collagen deposition is in balance with collagen resorption and no further net collagen deposition occurs. Platelet-derived growth factor (PDGF) and epidermal growth factor (EGF), derived from platelets and macrophage are the main signals for fibroblast proliferation and maturation. PDGF expression by fibroblasts is amplified by autocrine and paracrine signaling. TGF-β, INF-γ, IL-l and TNF-α either stimulate or reduce fibroblast proliferation, depending on their concentrations in the wound.

Remodelling phase:

It begins at about 2 to 3 weeks and can last up to 2 years. Re-organization and remodelling of collagen already deposited takes place during this period. Matrix metalloproteinases (MMPs) are intimately involved with the breakdown of collagen molecules. MMPs are zinc dependent endopeptidases that cleave most macromolecules within the ECM. There are nine members of the MMP family, four of which are involved in wound repair 3. These are interstitial collagenases, stromelysins, gelatinases and membrane type metalloproteinases. Osteoblasts assisted bone formation and re-modelling take place during this period.

Healing in the Periodontium:

Healing in periodontium is in many ways similar to healing in other parts of body but at the same time it has some fundamental differences. Dentogingival junction is a unique structure associated with teeth. It is considered as the battle field of bacterial and host immune response. So, during inflammation of periodontium its continuity is first to be lost. Maintenance of its integrity is essential in order to preserve the periodontal ligament and underlying bone.

Instrumentation done during surgical or non-surgical therapy, leads to injury in the already inflamed dentogingival tissues. The healing following these therapeutic measures is mainly dependent upon the cellular and molecular processes. The cellular events that take place during healing are similar to that explained in previous section with the exception that there is a mineralized tissue located at the junction of epithelium and connective tissue in periodontal wounds.

Research done on periodontal wound healing has provided us information regarding tissue response and how it can be modulated to achieve regeneration of lost periodontal structures. Guided tissue regeneration is an example of modulation of tissue response during healing.

With this basic information regarding wound healing process, lets now discuss the cellular and molecular basis of periodontal regeneration.

Cellular and molecular basis of periodontal regeneration:

To regenerate periodontal tissues a combination of cellular and molecular activities are required. Cells such as epithelial cells, osteoblasts and fibroblasts play an important role in post-operative healing. As described in the previous section the activated inflammatory cells produce various mediators which play a specific role during healing process. Molecules such as growth factors, adhesion molecules and structural proteins play an equally important role. The signalling molecules that have been most intensively investigated in periodontal regeneration include bone morphogenetic proteins (BMPs, e.g. BMP-2, BMP-7, & BMP-12) 3-12, transforming growth factor (TGF)-β1 13, 14, platelet-derived growth factors (PDGFs, such as PDGF-BB) 15-18, insulin-like growth factor (IGF)-1 16, 19-20, and basic fibroblast growth factor (b-FGF) 21-24. Following is the list of cellular and molecular components of periodontal regeneration.

Essential components of periodontal regeneration

Cells Fibroblasts Responsible for formation of collagen fibers of gingival and periodontal ligament
Osteoblasts Osteoblasts responsible for alveolar bone formation
Cementoblasts Responsible for new cementum formation on root surface
Molecules Cytokines     IL-1α
IL-1β
TNF-α
IL-4
IL-6
IL-8
IL-10
Growth factors    Fibroblast growth factor-1 and -2
Insulin-like growth factor-I and II
Bone morphogenetic proteins
Epidermal growth factor
Platelet-derived growth factor
Adhesion molecules  Fibronectin
Laminin
Osteopontin
Bone sialoprotein
Collagens
Cementum attachment protein
Structural proteins Types I, III, V, XII and XIV collagens
Proteoglycans,
Hyaluronan
Osteocalcin
Non-collagenous proteins
Tenascin
Osteonectin
Dentin/enamel matrix proteins
Note: The above list comprises of well studied components of periodontal regeneration. There are many others, on which research is going on or still required.

Let us discuss these components in detail,

Cellular components:

Fibroblasts:

Fibroblasts synthesize the extracellular matrix and collagen, the structural framework (stroma) for tissues, and play a critical role in wound healing. The main function of fibroblasts is to maintain the structural integrity of connective tissues by continuously secreting precursors of the extracellular matrix. Fibroblasts are derived from primitive mesenchyme, like all connective tissue cells. They play a vital role in periodontal regeneration as these are responsible for periodontal ligament formation.

Fibroblast activation:

Normal fibroblasts are embedded within the fibrillar extracellular matrix (ECM) of the connective tissue and constitutively express vimentin and fibroblast-specific protein 1 (FSP1). The activated fibroblasts typically contain a large oval euchromatic nucleus with one or two nucleoli, rough endoplasmatic reticulum, and a prominent Golgi apparatus. Activation of fibroblasts takes place when tissue injury occurs. The injured epithelial cells and infiltrating mononuclear cells such as monocytes and macrophages release various factors like transforming growth factor-β (TGF-β), epidermal growth factor (EGF), platelet-derived growth factor (PDGF) and fibroblast growth factor 2 (FGF2), which also activate fibroblasts. In addition, fibroblasts are activated by direct cell–cell communication and contacts with leukocytes through adhesion molecules such as intercellular-adhesion molecule 1 (ICAM1) or vascular-cell adhesion molecule 1 (VCAM1).

The activated fibroblasts secrete various mediators which play a key role in connective tissue remodelling. These include proteases such as matrix metalloproteinase-2 (MMP2), MMP-3 and MMP-9. In addition to these, activated fibroblasts secrete various growth factors such as transforming growth factor-β (TGF-β), insulin-like growth factor (IGF), nerve growth factor (NGF) and keratinocyte growth factor (KGF) which can induce proliferative signals within adjacent epithelial cells.

Osteoblasts:

Osteoblasts are specialized stromal cells that are exclusively responsible for the formation, deposition and mineralization of bone tissue. Bone formation is characterized by a sequence of events starting with the commitment of osteoprogenitor cells and their differentiation into pre-osteoblasts and then into mature osteoblasts whose function is to synthesize the bone matrix that becomes progressively mineralized. It is a strictly regulated process.

Regulation of osteoblast activity:

Various factors, cytokines and molecules have been demonstrated to facilitate the proliferation and differentiation of osteoprogenitors to pre-osteoblastic and mature osteoblastic cells. Bone Morphogenic Protein (BMP) is a family of proteins that can act on early osteoprogenitors to instigate their differentiation to pre-osteoblastic cells 25. The most important regulatory system in bone formation and resorption is RANK (receptor activator of nuclear factor κB)/ RANKL (RANK ligand)/OPG (osteoprotegerin) system. Detailed description of this regulatory system has been given in “Osteoimmunology of periodontal diseases”.

Cementoblasts:

Cementoblasts play a central role in periodontal development and regeneration, and in particular the formation of root cementum. The mineralized tissue-forming progenitor cells are believed to give rise to the cementoblastic and osteoblastic lineages, but the true origin of cementoblasts is still controversial. Cementoblasts resemble bone-forming osteoblasts but differ functionally and histologically. Mature cementoblasts produce matrix proteins common to osteoblasts, suggesting that cementoblasts are uniquely positioned osteoblasts. There are many proteins present in the matrix produced by cementoblasts including osteocalcin, osteopontin, bone sialoprotein, E-cadherin and ameloblastin etc. More information on cementoblasts is available in “cementum in health and disease”.

Cytokines:

IL-1 :

IL-1 is a representative proinflammatory cytokine that regulates many aspects of the immune and inflammatory responses. There are two IL-1 ligands with agonist activity, IL-1α and IL-1β, which are produced by various kinds of cells such as neutrophils, monocytes/macrophages and fibroblasts. Both IL-1α and IL-1β bind to the same receptor and have similar, if not identical, biological properties. IL-1α and IL-1β expression was strongly enhanced during wound healing 26. IL-1α plays important role during wound healing. It is known to be constitutively produced by epidermal keratinocytes under normal conditions, and injection of this cytokine enhances wound reepithelialisation 27. IL-1 receptor knocked out mice exhibited significantly delayed oral healing 28.

IL-1β and TNF-α are the key mediators of the inflammatory process, contributing to the reparative phase either directly by influencing endothelial and fibroblast functions or indirectly, by inducing additional cytokines and growth factors.

TNF-α:

Tumor necrosis factor α (TNF-α) is a pleiotropic cytokine produced by a variety of cell types including macrophages, T cells, mast cells, and keratinocytes. In various concentrations it has inflammatory, cytolytic, mitogenic, antitumor, and possibly angiogenic or antiangiogenic effects; therefore it is likely to affect wound healing. There are two types of receptors for TNF-α encoded by distinct genes: TNF receptor with a molecular mass of 55 kDa (TNF-Rp55) and one with a molecular mass of 75 kDa (TNF-Rp75) 29. TNF-Rp55 mediates various activities of TNF-α, including cytotoxicity, fibroblast proliferation, and induction of superoxide dismutase whereas TNF-Rp75 mediates thymocyte and cytotoxic T cell proliferation 30.

Interleukin 4 (IL-4):

IL-4 is secreted by Th2 lymphocytes, mast cells and macrophages, which stimulates a number of fibroblast activities such as migration, proliferation, and synthesis of the extracellular matrix via this secretion 31. A late increase of IL-4 is found at the wound site, correlating with the down-regulate expression of several other cytokines 32, 33.

Interleukin 6 (IL-6):

It has mitogenic effects on epithelial cells and has chemoattractive effect on neutrophils 34. IL-6 is also known as a potent stimulator of fibroblast proliferation 35 and it is important in inhibiting extracellular matrix breakdown during proliferation 36. It has been reported that a complete lack of IL-6 caused impaired wound healing 34. IL-6 performs an important function in acquired immunity by promoting specific differentiation of T and B cells. As for T cells, IL-6 together with transforming growth factor (TGF)-β has been shown to be essential for Th17 differentiation from naïve CD4-positive T cells, whereas IL-6 inhibits TGF-β-induced regulatory T cell (Treg) differentiation.

Interleukin 8 (IL-8):

IL-8 is secreted by epithelial cells and macrophages. The expression of IL-8 is triggered by IL-1 and TNF-α 37 and correlates strongly with neutrophil infiltration in the wound site 38. IL-8 is a major chemoattractant for polymorphonuclear leukocytes. There are many receptors on the surface membrane capable of binding IL-8; the most frequently studied types are the G protein-coupled serpentine receptors CXCR1 and CXCR2. It is important to note that IL-8 can be secreted by any cells with toll-like receptors that are involved in the innate immune response.

Interleukin 10 (IL-10)

It is an anti-inflammatory cytokine involved in the limitation and termination of the inflammatory process. As a factor produced by T helper 2 (Th2) cells, IL-10 inhibits the production of cytokines by Th1 cells 39. IL-10 regulates growth and/or differentiation of immune cells, epithelial cells, and endothelial cells and inhibits infiltration of neutrophils as well as the expression of several cytokines 40. An increased expression of IL-10 was shown to correlate with impaired wound healing 41. IL-10 can also contribute to the maintenance of bone mass through inhibition of osteoclastic bone resorption and regulation of osteoblastic bone formation.

IL-10 can downregulate the synthesis of proinflammatory cytokines and chemokines, such as IL-1, IL-6, and TNF- α 42, 43. Therefore, it can also be regarded as an important regulator of bone homeostasis, in homeostatic and inflammatory conditions

Role of growth factors in periodontal regeneration:

Over the last 20 years, there has been an immense increase in our knowledge about growth factors, cell adhesion molecules and cytokines, with a significant improvement in the understanding of the cellular and molecular biology of periodontal regeneration. Growth factors, cell adhesion molecules and cytokines play a vital role in periodontal regeneration.

So, what are growth factors? Growth factors are naturally occurring substance capable of stimulating cellular growth, proliferation and cellular differentiation 44.  Growth factors act as signaling molecules between cells. Following is a brief description of growth factors which have been our focus of research,

Platelet-derived growth factor:

Platelet-derived growth factor (PDGF) was purified from platelet extracts and is characterized as a mitogen for fibroblasts 45. It was originally purified from human platelets, but recently has been found to be produced by various other cells, for example, monocytes, megakaryocytes, vascular endothelium, smooth muscle cells, and transformed cells 46, 47. In humans, several dimeric isoforms are produced from four different genes, namely PDGF-A, -B and, more recently, -C and -D. It has important roles during wound healing. Until now, five dimeric compositions have been identified: PDGF-AA, -BB, -AB, CC, and –DD 48. As PDGF is a dimeric ligand, it forms a complex with two PDGF receptor molecules during intracellular signalling.

Platelet-derived growth factor (PDGF) in vitro stimulates DNA synthesis and chemotaxis of fibroblasts and smooth muscle cells and stimulates collagen, glycosaminoglycan, and collagenase production by fibroblasts. PDGF has been shown to potentiate regeneration of periodontium in naturally occurring periodontitis in dogs 49 as well as experimental periodontitis in monkeys 50.

Fibroblast growth factor-1 and -2:

Fibroblast growth factors (FGFs) play an important role not only during normal development but also during wound healing. To date, twenty three distinct FGFs have been discovered, numbered consecutively from 1 to 23. Out of these FGF-1 (Acidic FGF/ aFGF) and FGF-2 (Basic FGF/ bFGF) have been extensively studied for periodontal regeneration. Both FGF-1 and FGF-2 were initially isolated from bovine pituitary extracts based on their stimulation of [3H] thymidine incorporation in 3T3 fibroblasts 51, 52.

FGF-1 (Acidic FGF/ aFGF):

The human FGF-1 is a 155 amino acid protein. It is a well known fibroblast activator, which acts through four specific cell surface receptors, among which, fibroblast growth factor receptor 4 (FGFR4) is highly specific. It is also known as heparin-binding growth factor (HBGF-1).  aFGF is considered to function in several important physiological and pathological processes, such as embryonic development, morphogenesis, angiogenesis and wound healing.

FGF-2 (Basic FGF/ bFGF):

Human FGF2 occurs in low molecular weight (LMW) and high molecular weight (HMW) isoforms. LMW FGF2 is primarily cytoplasmic and functions in an autocrine manner, whereas HMW FGF2s are nuclear and exert activities through an intracrine mechanism 53. FGF-2 contains four cysteine residues at amino acids 26, 70, 88 and 93. While the cysteines at 26 and 93 are conserved, those at 70 and 88 are absent or located elsewhere in other FGFs. It was initially identified as a mitogen with prominent angiogenic properties, but now it is well recognized as a multifunctional growth factor. It produces its biological effects in target cells by signaling through cell-surface FGF receptors.

Functions:

Major functions of fibroblast growth factors (FGFs) include cell proliferation, cell migration, cell differentiation and angiogenesis. It is one of the main growth factors playing vital role during wound healing.

Insulin like growth factor:

The insulin like growth factor family has three peptide hormones or growth factors: insulin, IGF-I, and IGF-II having approximately 50 percent of their amino acids in common. Insulin is synthesized in the beta cells of the pancreas as proinsulin, which is cleaved to form insulin and C peptide.  The IGFs, which are synthesized primarily by the liver, retain the C peptide and have an extended carboxy terminus 54.

Insulin, IGF-I, and IGF-II bind specifically to two high-affinity membrane-associated receptors that are tyrosine kinases. These growth factors stimulate DNA synthesis and regulate development and differentiation in a large variety of cell types, thus playing a vital role during wound healing. IGF-I is found in substantial levels in platelets and is released during clotting along with the other growth factors present in platelets. It is a potent chemotactic agent for vascular endothelial cells. IGF-I released from platelets or produced by fibroblasts may promote migration of vascular endothelial cells into the wound area resulting in increased neovascularization. IGF-II has similar effects to IGF-I but many studies suggest that IGF-II is not as potent as IGF-I.

Bone morphogenetic proteins:

Bone morphogenetic proteins (BMPs) represent a unique set of differentiation factors that induce new bone formation. According to Bowers and Reddi 55,  BMPs are proteins found in high amounts  in bone tissues and are considered as responsible for inductive  and regenerative abilities of demineralized bone grafts used in  periodontal therapy. BMP’s have a great potential in periodontal regeneration. A lot of research has already been done on their role during wound healing. A detailed description of BMP’s is available in “Bone Morphogenetic Proteins”.

Epidermal growth factor:

Epidermal growth factor is a small protein with only 53 amino acids. Cells that respond to EGF have receptors on the cell membrane that recognize the factor. The binding of the growth factor to the receptor initiates a cascade of molecular events involving the MAPK/ERK pathway that will eventually lead, among other effects, to cell division. The cell membrane receptor for epidermal growth factor (EGF) belong to the family of growth factor receptors which possess intrinsic protein tyrosine kinase activity. The binding of EGF to the extracellular ligand-binding domain of the receptor activates its cytoplasmic tyrosine kinase, resulting in rapid autophosphorylation as  well as the phosphorylation of various cellular substrates 56-58. This factor has been shown to activate cell division thereby promoting cellular proliferation.

Application of growth factors in periodontal regeneration has been our focus in recent past. A detailed discussion is required to thoroughly understand the structure and functions of these molecules because in future we are going to see a lot of research in this field. A detailed description of growth factors has been given in “Growth factors in periodontal regeneration”.

Various growth factors, their sources and their effects

Growth Factor

Source

Effect

Fibroblast growth factors 1, 2, 4 Macrophages, endothelial cells Fibroblast  proliferation and angiogenesis
Transforming growth factor- α Macrophages, keratinocytes Re-epithelialization
Transforming growth factors β1, 2 Platelets, macrophages Fibroblast and  macrophage chemotaxisExtracellular matrix synthesisSecretion of protease inhibitors
Insulin-like growth factor Plasma, platelets Endothelial and fibroblast proliferation
Platelet-derived growth factor (isoforms AA, AB and BB) Platelets, macrophages, keratinocytes Fibroblast and macrophage  chemotaxisFibroblast proliferationMatrix synthesis
Epidermal growth factor Platelets Re-epithelialization
Keratinocyte growth factor Dermal fibroblasts Keratinocyte  proliferation
Interleukin 1-α and 1β Neutrophils Activate growth factor expression in macrophages, keratinocytes and fibroblasts
Tumor necrosis factor-α Neutrophils Activate growth factor expression in macrophages, keratinocytes and fibroblasts

 

Adhesion molecules:

Fibronectin :

Fibronectin (FN), a ubiquitous and abundant extracellular matrix (ECM) protein, is secreted by cells as a soluble dimer and is subsequently assembled into insoluble multimeric fibrils at the cell surface. It is a glycoprotein found at a size of 220-250 kD subunits lined by two disulfide bonds. This molecule is involved in adhesion and attachment of variety of cells. In addition to its cell attachment functions, Fibronectin may involve interactions with collagen, heparin and other cell surface glycosaminoglycans. It is a major component of extracellular matrix and is an important component of basement membrane. Besides acting as a barrier to macromolecules and cells, the basement membrane also provides a substratum for some cell types, supporting spreading, differentiation, and migration 59.  

Fibronectin plays an important part during wound healing. It promotes the spreading of platelets at the site of injury, the adhesion and migration of neutrophils, monocytes, fibroblasts, and endothelial cells into the wound region, and the migration of epithelial cells through the granulation tissue. At the level of matrix synthesis, fibronectin appears to be involved both in the organization of the granulation tissue and basement membrane. During tissue remodelling, it acts as a nonimmune opsonin for phagocytosis of debris by fibroblasts, keratinocytes, and under some circumstances, macrophages. It also enhances the phagocytosis of immune-opsonized particles by monocytes 60.

Laminin:

Laminin is the most abundant glycoprotein in the basement membrane. It is both structurally and biologically active component of basement membrane. Laminins are large (400-900 kDa) heterotrimeric molecules composed of one α, one β and one γ subunit in a cruciform or T-shaped appearance. To date, five α, three β and three γ chains have been characterized. They represent the products of distinct genes that evolved by duplication and recombination of ancestral α, β and γ genes, hence they share sequence similarity. Currently, the trimers are named according to the composition of the α, β and γ chains and more than 15 different laminin isoforms, with various arrangements of laminin subunits, have been identified 61-63. Laminin-111 is one of the best characterized laminins and is composed of the α1, β1, and γ1 chains 61Laminin 5 is known to induce the adhesion, spreading and migration of keratinocytes. The laminin-332/integrin α6β4 interaction is believed to support directed migration of keratinocytes 64. The exact role of the laminins during wound healing is not clear. Several studies have demonstrated that keratinocyte migration is necessary during human wound re-epithelialisation 65, 66, and laminins play important role in this process.

Know more………..

Role of integrin receptors:

Integrins are a family of cell adhesion molecules (CAM’s) that mediate the communication between the intracellular and the extracellular compartments. These receptors are formed by a non-covalently associated glycoprotein complex of two distinct polypeptide chains, called, α and β. In human cells there are at least three major subfamilies of integrin receptors, which are defined by their component β chains.The first subfamily comprises at least six related complexes, each consisting of a β1 chain with a distinct companion α chain. Members of the β1 subfamily include receptors for fibronectin (α3β1, and α5β1), laminin (α1β1, α3β1 and α6β1) and collagen types I and IV (α1β1, α2β1 and α3β1). The β2 subfamily is involved in leukocyte-leukocyte and leukocyte-endothelial cell communications. The β 3 integrin subfamily mediates the adhesion of platelets with fibrinogen and other ligands.In inflammation, wound healing and thrombosis, integrins and other CAM’s mediate the interactions among the injured tissue and circulating cells.

 

Osteopontin:

 Osteopontin is a secreted phosphoprotein containing the arginine-glycine-aspartate (RGD) tripeptide integrin binding motif. It is also known as bone sialoprotein I (BSP-1 or BNSP). It is a highly negatively charged, extracellular matrix protein that lacks an extensive secondary structure. It is composed of about 300 amino acids and is expressed as a 33-kDa nascent protein; there are also functionally important cleavage sites. Osteopontin is biosynthesized by a variety of tissue types including fibroblast,  preosteoblasts, osteoblasts, osteocytes, odontoblasts and some bone marrow cells etc. Osteopontin has been implicated as an important factor in bone remodeling. It also binds to several integrin receptors including α4β1, α9β1, and α9β4 expressed by leukocytes. It has chemotactic properties, which promote cell recruitment to inflammatory sites.

Bone sialoprotein:

Bone sialoprotein is a highly glycosylated and sulphated phosphoprotein that is found almost exclusively in mineralized connective tissues. The polypeptide chain of BSP has a molecular weight of 35 kDa. BSP belongs to the SIBLING (Small Integrin-binding LIgand N-linked Glycoprotein) gene family 67  and displays several characteristics typical of these proteins. BSP is expressed by several cell types associated with mineralized tissues but is expressed in abundance by osteoblasts. It binds to several distinct ECM constituents with diverse biological roles, including collagen 68, 69, factor H 70, matrix metalloproteinases 71, hydroxyapatite 72 as well as integrins present on numerous cell types 73-75

Collagens:

Collagens are a group of closely related proteins that comprise the most abundant proteins found in mammals representing 25–35% of the total body protein 76. To date, at least 28 different types of collagen encoded by 45 genes have been identified 77, 78

Collagen is composed of three chains, wound together in a tight triple helix. A repeated sequence of three amino acids (glycine, proline and lysine) forms this sturdy structure. The prerequisite for the assembly into a triple helix is a glycine residue, the smallest amino acid, in every third position of the polypeptide chains resulting in a (Gly-X-Y)n repeat structure. The α-chains assemble around a central axis in a way that all glycine residues are positioned in the center of the triple helix, while the more bulky side chains of the other amino acids occupy the outer positions. Three α chins may be identical (homotrimers) as in collagens II, III, VII, VIII, X, and others or by two or more different chains (heterotrimers) as in collagen types I, IV, V, VI, IX, and XI.

Collagen has many properties which make it a suitable component of connective tissue. For example, it has RGD (Arg-Gly-Asp) and non-RGD domains which bind cell surface-associated integrins 79, 80, which facilitates cell migration 81 , attachment 82-85, and differentiation 86.

Cementum attachment protein:

Cementum attachment protein is a collagenous protein that is expressed in the matrix of tooth cementum. This protein binds with high affinity to non-demineralized root surfaces, hydroxyapatite and fibronectin. It promotes attachment of mesenchymal cells and may function in cementogenesis. This protein is capable of recruiting cementoblasts on the root surface during periodontal wound healing thereby initiating new cementum formation. Integrin α5β1 is responsible for mediating the attachment to cementum attachment protein of the periodontal-derived cells, human gingival fibroblasts and human periodontal ligament fibroblasts.

Structural proteins:

Structural proteins are components of basic framework of the connective tissue. Three major components of ground substance are,

  • Proteoglycans (glycosaminoglycans with proteins): chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate, heparan sulfate, heparin.
  • Glycosaminoglycans (non-sulfated and sulfated): hyaluronic acid.    
  • Glycoproteins: fibronectin, laminin, etc.

Proteoglycans are responsible for the highly viscous character of the ground substance. Proteoglycans consist of proteins (~5%) and polysaccharide chains (~95%), which are covalently linked to each other. The polysaccharide chains belong to one of the five types of glycosaminoglycans, which form the bulk of the polysaccharides in the ground substance. Hyaluronan (or hyaluronic acid) is the dominant glycosaminoglycan in connective tissues which serves as a “backbone” for the assembly of other glycosaminoglycans in connective tissue. The remaining four major glycosaminoglycans are chondroitin sulfate, dermatan sulfate, keratan sulfate and heparan sulfate.

The collagen and non-collagenous proteins in the basic framework of the ground substance are formed during healing process. A continuous remodelling of the tissue takes place during healing period resulting in an organised structure.

Intracellular signalling mechanism:

All the growth factors, cytokines and other chemical mediators, act via a process known as receptor-mediated signal transduction. This process is activated by the binding of ligands such as growth factors, and cytokines to specific receptors. Before we discuss these intracellular signalling cascades, we should know about three general modes of signaling, named autocrine, paracrine and endocrine signalling.

Autocrine signaling:

In this signalling, cells respond to the signaling molecules that they themselves secrete. In this manner, an autocrine loop is established. Autocrine growth regulation can be seen in proliferation of antigen-stimulated lymphocytes.

Paracrine signaling:

In this case one cell type produces the ligand, which then acts on adjacent target cells that express the appropriate receptor. The responding cells are in close proximity to the ligand-producing cell and are generally of a different type. Paracrine signaling is commonly seen in connective tissue repair during wound healing, in which a factor produced by one cell type (e.g., a macrophage) has a growth effect on adjacent cells (e.g., a fibroblast).

Endocrine signaling:

In this type of signaling, one cell type produces the ligand, which then acts on distant target cells that express the appropriate receptor. Hormones synthesized by cells of endocrine organs act on target cells distant from their site of synthesis, being usually carried by the blood. Growth factors and cytokines also systemic effects.

Receptors and Signal Transduction Pathways:

The binding of a ligand to its receptor triggers a series of events by which extracellular signals are transduced into the cell resulting in changes in gene expression. Following signal transduction pathways have been described in case of growth factors and cytokines intracellular signaling,

Receptors with intrinsic tyrosine kinase activity:

This mechanism of activation is found in most growth factors such as EGF, TGF-α, HGF, PDGF, VEGF, FGF, c-KIT ligand, and insulin. Here, the receptor for the ligand has an extracellular ligand-binding domain, a transmembrane region, and a cytoplasmic tail that has intrinsic tyrosine kinase activity. When the ligand binds to the receptor, dimerization of the receptor, tyrosine phosphorylation, and activation of the receptor tyrosine kinase result. The active kinase then phosphorylates, and thereby activates, many downstream effector molecules. PI3 kinase pathway, MAP-kinase pathway and IP3 pathway are involved in downstream signaling and transcription factor activation in the nucleus.

Receptors without intrinsic tyrosine kinase activity that recruit kinases:

This signaling mechanism is found in many cytokines, such as IL-2, IL-3, and other interleukins; interferons α, β, and γ; erythropoietin; granulocyte colony-stimulating factor; growth hormone; and prolactin. These receptors transmit extracellular signals to the nucleus by activating members of the JAK (Janus kinase) family of proteins. After activation, JAK’s attach to and activate cytoplasmic transcription factors called STATs (signal transducers and activation of transcription), which directly shuttle into the nucleus and activate gene transcription.

Intracellular signalling cascade

Intracellular signalling cascade

G protein–coupled receptors:

Various ligands including chemokines, vasopressin, serotonin, histamine, epinephrine and norepinephrine, calcitonin, glucagon, parathyroid hormone, corticotropin, and rhodopsin signal through this mechanism. These receptors transmit signals into the cell through trimeric GTP-binding proteins (G proteins). They contain seven transmembrane a-helices. Binding of the ligand induces changes in the conformation of the receptors, causing their activation and allowing their interaction with many different G proteins. Activation of G proteins occurs by the exchange of GDP, present in the inactive protein, with GTP, which activates the protein. Precipitation of 3′,5′-cyclic adenosine monophosphate (cAMP) has multiple effects intracellularly ultimately causing activation of transcription factor in the nucleus.

Conclusion:

The above description regarding the biology of periodontal regeneration is just an overview of the events that take place during periodontal wound healing and all the components that are required during periodontal regeneration. Presently, a lot of research work is going on at molecular level to find out efficient and cost effective methods to deliver biologically active molecules like growth factors at the site of healing to achieve regeneration of the lost tissue. With the background present discussion we can now go ahead with discussion on our present status of molecular research in periodontal regeneration. As already stated, growth factors play a very important role in periodontal regeneration. A detailed description of growth factors application and its limitations is available in “Growth factors in periodontal regeneration”.  

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