TISSUE ENGINEERING
BIOLOGIC MODIFIERS IN PERIODONTAL REGENERATION
OVERVIEW
Periodontal diseases result in destruction of periodontal tissues, including cementum, bone, and periodontal ligament (PDL), with eventual tooth loss if left untreated. Studies targeted at understanding the disease at the cellular and Molecular level as well as clinical investigations have resulted in improved therapies for arrest of disease progression. Moreover, beyond areas of disease progression, substantial evidence exists indicating that regeneration of periodontal tissues is a viable treatment for select situation. There is a need, however, to improve the predictability of regenerative therapies. This need has led to increased efforts, among clinical and basic science researchers, to establish the specific cells, factors, delivery systems, flap design, and host responses required for enhancing the outcome of regenerative therapies.
Although significant advances have been made toward understanding the complexities involved im promoting periodontal regeneration, much remains to be elucidated including questions regarding placement of factors (e.g., within a barrier membrane? In conjuction with resorabable or nonresorbable membranes? Directly into the defect? Coating onto the root surface?). What cells should these factors be targeted to, and what activities are attractive in promotion and inhibition by these factors.
This article is limited to discussions on potential and known biologic modifiers for use in regeneration of periodontal tissues. To maintain a central theme, nonendogenous factors, such bisphosphonates and antibiotics, are excluded because these are discussed elsewhere in this issue.
For clarity, it is important first to define biologic modifiers as interpreted. Biologic modifiers are materials or proteins and factors that have the potential to alter the host tissue so as to stimulate or regulate the wound healing process. Classic examples of biologic modifiers are growth factors. These agents can act through a systematic route(e.g., hormones)or act at the local site(e.g., many polypeptide cytokines and growth factors). This article centers around biologic modifiers that may have the potential to promote regeneration of periodontal tissues(i.e., new bone, new cementum, and new connective tissue attachment) through a variety of cell-tissue interactions, including promoting (1) cell migration, (2) attachment and subsequent spreading of cells at the local site, (3) cell proliferation, (4) cell differentiation, and (5) matrix synthesis.
RATIONALE FOR USE IN DENTISTRY
The concept that biologic modifiers may serve role in promoting wound healing is not unique to dentistry. With enhancement in cellular and molecular technologies, great have been made in understanding the activities of these modifiers and also in preparing large quantities of recombinant materials.
The interdisciplinary approach to developing new agents and materials for improving tissue function has resulted in substantial progress toward restoring tissues subsequent to disease. In particular, dental procedures rank as one of the most frequent techniques used to enhance tissue deficiencies.35 Other areas that rank high are procedures to promote skin healing (e.g., burn patients and bone procedures. In the development of strategies advancing regeneration of periodontium, the periodontal field has taken advantage of approaches used for establishing directions to improve regenerative therapies for other tissues. A key factor for enhancing the predictability of regenerative therapies is an understanding of cellular and molecular events required to regenerate periodontal tissues. It is now recognized that an important link, although not exact, to understanding the requirements for regeneration of tissues is to acquire knowledge as to mechanisms involved in development of tissues. Figure I diagrams regulating events, cells, and proteins currently believed to be involved in regulating development of or regeneration of periodontal tissues. Information gained from studies targeted at understanding the mechanisms and factors controlling development of periodontal structures may p rove important for use in regeneration of such tissues, subsequent to disease. For example, data exist suggesting that dental follicle cells ( mesenchymal cells surrounding the tooth before root and PDL development) have the capacity to differentiate into osteoblasts, cementoblasts, or PDL cells, when triggered appropriately.56,57,85 Thus, it is possible that factors and proteins identified as required for development and regeneration. In fact, as discussed later, this seems to be the case for some putative biologic modifiers.
Contrasting periodontal development with periodontal regeneration, it is apparent that some common principles exist as well as some concepts that are clearly different between the two processes. Events required for regeneration of periodontal tissues are analogous with those required for normal wound healing and, for the most part, are similar to those events required for development of the periodontium. In contrast to developmental stages, however, in both wound healing and regeneration, the early events include recruitment of marrow cells and release of cell cytokines and growth factors at the healing sites. During development, it is now recognized that specific growth factors and morphogens trigger differentiation of epithelial and mesenchymal derived cells during tooth formation. The importance of these growth factors (e.g., bone morphogenetic proteins [BMPs] (see review by Thesleff and Sahlberg) for regeneration of periodontal tissues as well as the function of endogenous factors present at wound sites is currently being examined in in vivo and in vitro models. Another event considered critical for both priate cells to the site of repair or development. Once at the site, the become biologically active. That is, such cells must differentiate into osteoblasts, cementoblasts, or PDL cells must differentiate into matrix required for formation of hard and soft connective tissues. To synthesize sufficient matrix, the appropriate cells must be stimulated to proliferate at the local site. Thus, it is reasonable to imagine that many of the molecules involved in triggering dsevelopment of periodontal tissues may prove to be effective in promoting regeneration of periodontal tissues.
This article first discusses basic principles of biologic modifiers. Next, specific biologic modifiers that may have activity in regenerative therapies are presented. This is followed by a section covering results to date using biologic modifiers to regenerate periodontal tissue in vivo and in vitro models. Last, trends and emerging therapies targeted at tissue regeneration are discussed.
BASIS OF ACTION OF BIOLOGIC MODIFIERS
Mode of Action
The overall scheme of how growth factors act depends on their mode of action. To evoke a biologic effect, a growth factor must be synthesized by an originating cell, travel to its target receptor, interact with the target receptor or binding protein, and activate second messengers or terminal effectors. The mode of action is the way the biologic modifier is meant to interact with its target receptor. Hormones traditionally act in an endocrine manner whereby they are secreted by one type and travel in the bloodstream to a distant target cell to exert their actions. Examples of hormone, and luteinizing hormone. These factors have the potential for widespread effects because of their circuation in the bloodstream and availability to many different cell types and subsequently are regulated not only by their blood levels, but also by the cells that bear receptors. Local modes of action are more traditionally associated with the term growth factor and involve paracrine autocrine, juxtal line, and intracrine modes. Paracrine action involves the production of a factor by one cell, with receptors present on another cell in the local microenvironment. The biologic modifier is secreted from the first cell in a soluble manner and binds to receptors on the target cell to evoke its effects. Examples of this are the growth factors platelet-derived growth factor (PDGF) and transforming growth factor-b (TGF-b), which are produced by platelets and act on target cells such as lymphocytes and osteoblasts. Autocrine factors are those that are synthesized by one cell, secreted in a soluble form outside the cell, then bind to surface receptors on the same cell to evoke an effect. Examples of autocrine factors are TGF-a, which is produced and acts on epithelial cells, and the BMPs, which are produced and act on osteoblastic cells. Less commonly described are juxtacrine effects, which are similar to paracrine effects except that the factor produced by the cell of original cell surface bound and required cell contact by the target cell to evoke a response. An example of juxtacrine mode of action is stem cell factor. Finally, another form of autocrine action is intracrine, whereby a factor is produced by one cell and not secreted but acts intracellularly to facilitate its effects. An example of this mode of action is parathyroid harmone-related protein (PTHrP) in which a portion of the protein has been shown to translocate to the nucleus to inhibit apoptosis. Transcription factors also fit into this category.
Receptors
For a biologic modifier to exert an effect, its designated receptor must be present in sufficient quantity, orientation, and functional activity to transmit the appropriate stimuli. Growth factor receptors can be divided broadly into two categories cell surface receptors and intracellular receptors. The most common prototype growth factor receptor is the cell surface receptor, which can be further divided into three categories: (1) G-protein linked, (2) receptor tyrosine kinases, mainly bind peptide factors that are soluble in water but not easily transported across the lipophilic cell membrane. Examples of the ligands for these cell surface receptors are outlined in Figure 3. The intracellular receptors are commonly described for steroids such as vitamin D3, estrogen, and glucocorticoids. Steroid receptors have been described in both the cytoplasm and the nucleus of target cells. Additionally, intracellular receptors or binding proteins for factors that act in an intracrine manner are located within the nucleus.
Once a cell surface receptor has been bound and activated, a series of second messengers are responsible for taking the next step in evoking a biologic activity. Four main second messengers are outlined in Figure 4. Adenylyl cyclase (AC) is an enzyme activated by G-proteins in the cell membrane is response to activation of G-protein—linked receptors such as the parathyroid harmone(PTH)/PTHrP receptor. AC catalyzes the reaction of adenosine triphosphate (ATP) to cyclic adenosine monophosphate (cAMP), which activates protein kinase A to cause protein phosphorylation. G-protein-linked receptors also couple to membrane bound phospholipase C with activation of protein kinase C to evoke protein phosphorylation. The receptor tyrosine kinases and serine threonine kinases are also responsible for p hosphorylating their target proteins. Protein phosphorylation is a key component of growth factor activity and is responsible for mediating changes in cell proliferation and differentiation, which are the hallmarks of growth factor activity.
Cell Proliferation
The most fundamental proceses of tissue growth and development begin with cell proliferation. Cell growth and division is a prerequisite for regeneration and repair. In cell division, duplication of the cell occurs such that every daughter cell receives an identical copy of genetic material. Cells from different tissues grow and divide at quite different rates. For example, cells of the junctional epithelium typically have a turnover rate of 20 times days. Despite this difference, cells undergo a similar pattern of cell gele events that characterize their process of cell division . There we four main phases of the cell cycle. The two G phases represent gap phases G1 (first gap) and G2 (second gap) between the S and M phases. The cell is not actively in the cycle (i.e., it is terminally differentiate of at rest), it is considered to be in the G0 phase (exited from the cell cycle). For a ell to reenter the cell cycle from the resting G phase and hence initiate cell division, a stimulus designated as a competence factor required. Competence factors are necessary but not sufficient for the cell to enter into the cell cycle. An example of a competence factor is PDGF. After the cell has been rendered competent to undergo cell division it requires a progression factor. Progression factors are sufficient for the cells are rendered competent to progress through the cell cycle example of a progression factor is insulinlike growth factor I (IGF-I). Since a cell has progressed to the S phase, it is committed to undergo cell division, although there are growth factors that may at later stages to delay o’ block cells in the G2 phase. Progressing through the cell cycle is an obvious prerequisite for cells to multiply,.forming the basis for development and regeneration of tissues. Biologic modifiers are key regulators of this process of cell proliferation via their action at different stages of the cell cycle.
Cell Differentiation
The process of cell differentiation is also a critical component of tissue regeneration. Beyond cell proliferation, differentiation of cells into mature cells bearing the respective phenotypic and functional characteristics composing the tissue type is necessary (e.g., bone, PDL , epithelium). The process of cell differentiation has been outlined for most cell arise from a common progenitor cell, which is an undifferentiated mesenchymal cell. This cell may progress and differentiate into multiple cell types, including osteoblast, fibroblasts, adipocytes, or muscle cells with thee appropriate signals. Biologic modifiers act in this regard to stimulate or inhabit cell differentiation along the designated pathways. Certain factors may act independently at one or more stages or in concert with other biologic mediator. During development, this process of cell differentiation is exquisitely regulated; hence, the challenge with regeneration is to recreate the appropriate organization of proliferation and differentiation to result in formation of functional tissues. Table 1 lists the most common biologic modifiers, their major source(s), and primary actions.
SPECIFIC AGENTS
Platelet-Derived Growth Factor (AA, AB, BB)
PDGF, one of the first growth factors to be described, was originally isolated from platelets and found to have mitogenic activity in smooth muscle. PDGF consists of two disulfide-bonded polypeptide chains that are encoded by two different genes, PDGF-A and PDGF-B. Consequently, PDGF can exist as a heterodimer (AB) or a homodimer (AA,BB). These three subtypes can bind to the PDGF receptor that is encoded by two separate genes. Several cell types produce PDGF, including degranulation platelets, smooth muscle, fibroblasts, endothelial cells, macrophages, and keratinocytes. PDGF plays a significant role in wound healing by stimulating connective tissue growth via its mitogenic and chemotactic activities.
Insulinlike Growth Factors (I, II)
IGFs are important regulators of proliferation and differentiation in a variety of cells. IGF-I and IGF-II have 65% amino acid sequence homology and similar biologic activities; however, their synthesis is under different regulating influences. Bone cells produce and respond to IGFs, and bone is a storage house for IGFs in their inactive forms. IGFs have pleotropic effects on their target cells, including an increase in transport of glucose and amino acids into osteoblasts, an increase in breakdown. IGFs stimulate cell replication as a progression factor and are required but not limiting factors for DNA synthesis in osteoblasts. IGFs also stimulate differentiation of mesenchymal cells and enhance matrix production, including the synthesis of collagens and proteoglycans. IGFs have distrinct receptors and also have a series of binding proteins (IGFBPs), which regulate the half-lives and distribution of IGFs.
Transforming Growth Factors (a,b)
The transforming growth factors (TGFs) were first named for their ability to stimulate anchorage-independent growth of fibroblasts in monolayer. TGF-a shares structural homology with epidermal growth factor (EGF), such that they bind to the same receptor and evoke a similar biologic activity of stimulating epidermal basal cell proliferation. TGF-a shares structural homology with epidermal growth factor (EGF), such that they bind to the same receptor and evoke a similar biologic activity of stimulating epidermal basal cell proliferation. TGF-b has been the subject of wide investigation in relation to its effects on bone and other cells of mesenchymal origin. TGF-b was originally isolated as a PDGF and later was found in the largest amounts stored in bone in an inactive form. TGF-b has immunosuppressive characteristics and has also been investigated for its ability to include cartilage and new bone growth in vivo.
Fibroblast Growth Factors (Acidic, Basic)
The two major members of thee fibroblast growth factors (FGFs) are acidic FGF(aFGF or FGF-I) and basic FGF (bFGF or FGF-2). Both FGFs are heparin binding proteins and have potent mitogenic effects on cells of meodermal and neuroectodermal origin. FGFs enhance bone formation and are also angiogenic. bFGF is considered to be more potent than bFGF and may act via the stimulation o other growth factors because it has been found to stimulate TGF-b.
Bone Morphogenetic Proteins (1-15)
The BMPs have been the subject of intense investigation for more than two decades. Urist89 in 1965 reported that protein extracts from bone implanted into animals at no bone sites induced the formaton of new cartilage and bone tissue. This protein extract contained multiple factors that stimulate bone formation and was termed bone morphogenetic protein. At least 15 BMPs have been identified to date, and they are part of the TGF-b superfamily. The BMPs most widely studied include BMP-2, BMP-3a, (osteogenin), BMP-4, and BMP-7 (osteogenic protein -1 [OP-1] ).
Interleukins (1-12)
Interleukins were originally defined as factors are involved in immune cell interactions, but this definition has been extended because many of the interleukins have effects on connective tissue and other nonimmune cell types. At least 12 interleukins have been described; and the most commonly studied are interleukin-1 (IL-1), IL -2, IL -3, IL -4, and IL -6. IL -1 is produced by many cell types, including keratinocytes, macrophages, and endothelial cells. IL -1a and b bind to the same receptor and have the same biologic activities. IL -1 has also been termed endogenous pyrogen for its ability to induce fever in vivo. IL -1 induces neutrophilia, induces antiproliferative effects against certain tumor cells, and stimulates bone resorption. IL -2, also known as T-cell induces neutrophilia, induces antiproliferative effects against certain tumor cells,and stimulates bone resorption. IL -2, also known as T-cell growth factor, is produced by activated T lymphocytes and stimulates T-cell prloliferation. IL-3 is considered a colony-stimulating factor (CSF) for its ability to promote development of multipotential hematopoietic stem cells and progenitors of the granulocyte, macrophage, erythrocyte, eosinophil, megakaryocyte, mast cell, and basophil lineage. IL-4 is also known as B–cell growth, T-cell activation, and platelet production. IL-6 also stimulates bone resorption and has been implicated as a contributing factor in osteoporosis.
Colony-Stimulating Factors (G, GM, and M)
The CSFs were named for their ability to induce the development of distinct cell lineages. As described previously, IL – 3 is a CSF known as multi-CSF. IL-3 stimulates the formation of all nonlymphocyte blood cells. Granulocyte-macrophage-CSF (GM-CSF) and granulocyte-CSF (G-CSF) more specifically promote the differentiation of macrophages and granulocyes. Erythropoietin is a
cytokine produced by the kidney that stimulaes proliferation and differentiation of erythroid cells into erythrocytes. Erythropoietin was the first CSF commercially available for clinical use.
Parathyroid Hormone-Related Protein
PTHrP is a peptide growth factor with limited homology to the endocrine hormone PTH. Many cell and tissue types produce PTHrP, including keratinocytes, lactating mammary gland, and fetal parathyroid glands. PTHrP, including keratinocytes, lacting mammary gland, and fetal parathyroid glands. PTHrP has potent proliferative and differentiating characteristics and has been found to play a critical role in cartilage, mammary gland, and tooth development. PTHrP has both anabolic and catabolic effects in bone.
Epidermal Growth Factor
EGF is a keratinocyte-stimulating growth factor. Originally derived from saliva, EGF has been reported to have profound effects on tooth development. EGF is present in most biologic fluids (saliva, urine, plasma, sweat, and semen). As discussed earlier, EGF and TGF-a both bind to the same receptor and have the same biologic activity.
Adhesion Factors (Fibronectin, Osteopontin Bone Sialoprotein)
Adhesion and attachment factors are becoming increasingly important for their ability to stimulate growth and differentiation. Fibronectin is a noncollagenous glycoprotein that is a major component of serum and contains a sequence of amino acids (arginine-glycine-aspartic acid ; RGD, a domain in associated with cell adhesion. Fibronectin contains additional domains associate with cell binding. Fibronectin promotes the attachment of bone cells and likely contributes to their differentiation. Fibronectin binds to cells and to fibrin, heparin, gelatin, and collagen. Osteopontin, aslo termed Spp, BSP –1, and eta-1, is a sialoprotein component of the bone matrix. Osteopontin is thought to play a role in bone development but also in cellular transformation and metastasis. Osteopontin contains an RGD sequence and promotes the attachment of several cell types, including attachment of osteoclasts to bone surfaces. Bone sialoprotein also contains an RGD sequence and promotes cell attachment but has a more limited pattern of expression than that of osteopontin. Although the precise role for bone sialoprotein and marks a lage stage of osteoblastic differentiation and an early stage of matrix mineralization. Osteopontin, bone sialoprotein, and fibronectin are also found in odontoblasts and cementoblasts.
FUTURE PERSPECTIVES
Delivery Systems
Although the use of biologic modifiers to treat periodontal diseases has not reached the level of development necessary to ensure predictable results, knowledge of the biology involved surpassed knowledge of how to deliver these agents for optimal results. Studies focused on the biology are important; at the same time, however, studies focused on the biology are important; at the same time, however, studies to determine the mode of administration are critical and are currently under intense investigation. Items to consider regarding these materials include their biocompatibility, toxicity, ease of handling , release kinetics, and resorb ability or retrievability. Osseous grafts have been use for decades to treat periodontal defects and are a valuable source of biologic mediators. Type I collagen gels have been extensively investitgated for their space-filling properties as well as for their ability to resorb and release putative biologic mediators in wound healing situations. Collagen-based sutures and hemostatic spnges have been used extensively in medicine and dentistry. Resorbable collagen barriers have been used clinically for guided tissue regeneration procedures; however, their combination with biologic modifiers has not been explore. Another are of interest is in combination with biologic modifiers because they can be prepared reprople, a polymer of glycolic acid, is a normal product of metabolism. PGA suture material, and as implants for bone fracture fixation. The ability to impregnate these materials with biologically active factors and to control release of factors holds promise for treating periodontal defects. Polylactic acid (PLA) is more hydrophobic than PGA and is more soluble in organic solvents.copolymers of PGA and PLA have been used for many types of biomaterials, including sutures (Vicry1). Other synthetic materials are under active investigation sucha as poly (e-caprolactone), polydioxanone, and trimethylrny carbonate (for a comprehensive review see Hutmacher et al34).
Gene Therapy
The term gene therapy originally referred to the treatment of a disease by means of genetic manipulation. According to Strayer gene therapy may involve (1) supplying or increasing the expression of a mutant gene that is insufficiently expressed (e.g., to treat genetic enzymatic deficiencies); (2) blocking a gene that is detrimental (e.g., using antisense constructs to inhibit tumor proliferation); or (3) adding a foreign gene to treat a situation beyond the capability of the normal genome (e.g., introduce an enzyme into a cell or tissue that allows the tissue to become more sensitive to the effects of a pharmacologic agent). Much of the initial interest in gene thrapy centered on its potential for treating genetic diseases, such as cystic fibrosis and familial hypercholesterolemia. More recently, the potentials for gene therapy have expanded to include gene therapy for defects at local sites (e.g., bone and salivary glands).
A major consideration in evaluating the potential for gene therapy for use in periodontal regeneration is the design and construction of the targeting vectors. DNA can be transferred via (1) naked DNA, which depends on physical and chemical methods to insure uptake into cells, or (2) virus-mediated vector, which rely on viral sequences as with retroviral vectors or adenoviral vectors to infect cells with the DNA of interest. The DNA of interest is typically driven via promoter from the viral vector, for example, to result in transcription of the gene of interest. Often, the DNA is driven by a promoter that is specific to the tissue of interest to ensure expression in a designated are versus a widespread manner. For example, a keratin promoter driving a gene of interest would target expression to epithelium and not to connective tissue. Obviously, this becomes increasingly difficult when considering regeneration of the periodontium and the multiple tissue types present. It is questionable whether bone-specific promoters, such as osteocalcin and bone sialoprotein, are truly bone specific, and there are no known cementum-specific promoters to target gene expression to the cementum. Although gene therapy offers many promosing prospects for the future, developing the strategies continues to be a challenging proposition.
Cell-Based Therapy
Cell-based therapies are most commonly associated with bone marrow transplantation strategies. Bone marrow transplantation has been successfully used to treat a multitude of conditions, including genetic disorders, immune disorders, and tumors. More recently, interest has focused on marrow stromal cells as stem cells for tissues of mesenchymal origin. Hematopoietic stem cells in the bone marrow provide a continuous source of progenitors for blood cells but additionally contain cells that are stem cells for constractive tissue. Bone marrow stromal cells can differntiate in culture with osteoblasts, chondrocytes, adipocytes, or myoblasts and may also be a more natural source of biologic modifiers in the wound environment. These cells present an intriguing resource for their potential use in periodontal regeneration and are currently being explored on a basic scene level. Clinically, a significant challenge is the source of cells and the stringency of maintaining cells ex vivo before replacement in the statement site. The actual use of cell seeding in a periodontal application has been limited to a pilot report using PDL fibroblasts in beagle dogs. Althouh attractive results were presented, including complete covertase of seeded roots with cementoblasts, the study population was small, and extensive characterization would be necessary before this technique could become a clinical reality.
SUMMARY
The specific objectives of this article was to update the reader on biologic modifiers being tried or suggested for use in therapies directed at regenerating periodontal tissues. As indicated from the studies presented here , many of these biologic modifiers have significant influences on cell behaviour and show great promise for use in regenerative therapies. As discussed here, however, additional investigations are required both at the molecular level therapies. With active investigations directed toward understanding the biology of the healing site, including identifying appropriate cells to target, coupled with designing delivery systems that can control release of agents at the local site, establishing the required environment for regeneration of periodontal tissues should be feasible.
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