Regeneration of periodontal tissues: cementogenesis revisited

Transcription

1 Periodontology 2000, Vol. 41, 2006, Printed in Singapore. All rights reserved Copyright Ó Blackwell Munksgaard 2006 PERIODONTOLOGY 2000 Regeneration of periodontal tissues: cementogenesis revisited MARGARITA ZEICHNER-DAVID Virtually all types of periodontal disease are caused by periodontal pocket infections, although several other factors, including trauma, aging, systemic diseases, genetics, etc., can contribute to the destruction of the periodontium (1, 18, 31, 52, 60, 107, 128, 127, 194). Repair of the periodontium and the regeneration of periodontal tissues remains a major goal in the treatment of periodontal disease and is an area still in need of major research attention, as recently stated by the American Academy of Periodontology (260). In general, to achieve complete tissue regeneration and repair, it is necessary to recapitulate the process of embryogenesis and morphogenesis involved in the original formation of the tissue. In the case of the periodontium, complete periodontal repair entails de novo cementogenesis, osteogenesis and the formation of periodontal ligament fibers. Current strategies for periodontal repair are based on anti-infectious measures such as scaling and root planing, guided tissue regeneration (with or without bone grafts) or the use of growth factors, none of which fully restore the architecture of the original periodontium. Several different approaches involving tissue engineering are currently being explored to achieve complete, reliable and reproducible regeneration of the periodontium. As tissue engineering is defined as the science that develops techniques (based on principles of cell and developmental biology) for fabricating new tissues to replace or regenerate lost tissues (205), it is important to understand the formation of specific tissues, the physico-chemical characteristics of the tissues and the molecular events leading to the normal function of the tissues. Development of the periodontium The periodontium can be defined as Ôan intricate mosaic of cells and proteins that is primarily responsible for the attachment of teeth in the oral cavityõ (144). Several excellent reviews have been published describing the embryonic lineage of the principal periodontal tissues (cementum, periodontal ligament, gingiva and alveolar bone), as well as the cells and extracellular matrix components of the periodontium (10, 13, 14, 21, 19, 46, 45, 51, 71, 80, 82, 144, 158, 185, 186, 193, 212, 214, 243, 244, 245). Formation of the periodontium is initiated with the process of root formation where, following crown formation, the apical mesenchyme continues to proliferate to form the developing periodontium, while the inner and outer enamel epithelia fuse below the level of the cervical enamel to produce a bilayered epithelial sheath, termed the Hertwig s epithelial root sheath. As these cells divide, there is an apical migration of the Hertwig s epithelial root sheath cells through the underlying dental ectomesenchymal tissues, dividing them into the dental papilla and the dental follicle (Fig. 1). As the root develops, the first radicular mantle dentin is formed and the epithelial sheath is fenestrated. It is believed that cells of the Hertwig s epithelial root sheath migrate away from the root into the region of the future periodontal ligament where they re-associate to form the Epithelial Rest of Malassez. However, not all Hertwig s epithelial root sheath cells migrate into the periodontal ligament site; a few undergo apoptosis and some remain in the root surface (108). Although it is accepted that the Hertwig s epithelial root sheath plays an important role in root development, the precise nature of its role remains controversial. In 1940, Schour & Massler suggested that the major function of the Hertwig s epithelial root sheath was to induce and regulate root formation, including the size, shape and number of roots (244). Other investigators suggested that the role of the Hertwig s epithelial root sheath was to induce the differentiation 196

2 Regeneration of periodontal tissues: cementogenesis revisited Fig. 1. Root development and periodontium formation. Histological sections of 7-day postnatal mouse mandibular molars showing the initial development of the root by formation of the Hertwig s epithelial root sheath. At the 14-day postnatal time-point, apical migration of the roots continues, and there is formation of the periodontium with cementum, periodontal ligament and bone. Am, ameloblasts; C, cementum; D, dentin; Ds, dental sac; HERS, Hertwig s epithelial root sheath; Od, odontoblasts; PDL, periodontal ligament. of odontoblasts to form the root dentin (183, 182, 222, 243, 251), or to differentiate dental sac cells into cementoblasts (181). The current notion states that Hertwig s epithelial root sheath cells produce the basement membrane containing chemotactic proteins, which serve to direct the migration of precementoblast cells (140, 141, 182, 235, 251) and to induce cementoblast differentiation (191, 232, 234). Amongst the basement membrane molecules are several extracellular matrix proteins, growth factors, enamel proteins and adhesion molecules, such as a collagenous-like protein, known as cementum attachment protein (CAP), which has chemotactic potential capable of recruiting putative cementoblast precursors (11, 149, 156, 196, 275). In the second stage of cementogenesis (when the tooth reaches occlusion and cellular cementum is formed), the proliferation of cells of the Hertwig s epithelial root sheath is considerably reduced, and some cells are entrapped in the newly formed mineral where they may influence phenotypic changes in the dental sac cells (252). It is also suggested that Hertwig s epithelial root sheath cells undergo epithelial mesenchymal transformation to become functional cementoblasts in charge of producing the acellular cementum (251, 275). The gingival tissues appear to be derived from both the oral mucosa and the developing tooth germ (135). It has been suggested that the dental follicle (connective tissue surrounding the developing teeth) gives rise to the fibroblasts forming the periodontal ligament as well as to the alveolar bone and cementoblasts (45, 136, 186, 243), all of which have a common neural crest origin (34). Therefore, it is postulated that there are different types of cementoblasts: those originating from the Hertwig s epithelial root sheath via epithelial mesenchymal transformation and which form the acellular cementum; and those derived from the dental follicle, which form the cellular cementum (9, 19, 105, 251, 275). It is also believed that progenitors for periodontal ligament, osteoblast and cementoblast cells adopt a paravascular location in the periodontal ligament, and these cells, which exhibit some features of stem cells, can regenerate functional tissues when the need arises ( , 195). Periodontal 197

3 Zeichner-David ligament stem cells have recently been isolated from the human periodontium (162, 224, 225). The Epithelial Rest of Malassez cells remain in the periodontal ligament throughout life, suggesting that they have important, although yet unknown, functions, rather than just being leftover structures. Roles attributed to the Epithelial Rest of Malassez cells range from bad to good. The Epithelial Rest of Malassez cells are held responsible for the formation of periodontal cysts and tumors as a result of periapical inflammation associated with pulpal necrosis (26, 57, 77, 176, 226, 242). It has also been suggested that Epithelial Rest of Malassez cells contribute to the formation of the periodontal pocket because of their continuum with the junctional epithelium (176, 238). Some studies report the ability of Epithelial Rest of Malassez cells to resorb bone and extracellular matrix, and thus implicate the cells in root resorption (15, 75, 122). On the other hand, it has also been suggested that the cells of the Epithelial Rest of Malassez may protect the root from resorption (259). The finding of Epithelial Rest of Malassez cells being closely associated with neural endings suggests that they have a role in the development of periodontal ligament innervation (126). Studies performed with 1-hydroxyethylidene-1,1-bisphosphonate, a drug that interferes with homeostasis in the periodontal ligament, showed a severe reduction in the width of the periodontal ligament with the development of ankylosis, which was repaired after discontinuing the administration of 1-hydroxyethylidene-1,1-bisphosphonate (261). As the study did not detect a change in the number of Epithelial Rest of Malassez cells posttreatment, it was suggested that cells of the Epithelial Rest of Malassez are unlikely to play an important part in the homeostasis of, and may not be a prerequisite for, the repair and maintenance of the periodontal ligament. On the other hand, the Epithelial Rest of Malassez cells secrete hyaluronic acid, which contributes to the formation of the loose connective tissue characteristics of the periodontal ligament (155). Cells of the Epithelial Rest of Malassez react to mechanical stress, like that associated with orthodontic tooth movement, by increasing their proliferation rate and cell size (27), and thereby help to maintain the space between the periodontal bone and cementum to avoid ankylosis (134). The increased activity of the Epithelial Rest of Malassez cells is consistent with their putative role on collagen turnover in the periodontal ligament, which is accelerated during tooth movement (241), and during cementum repair in areas of root resorption (24). It is suggested that the Epithelial Rest of Malassez cells may negatively regulate root resorption and induce acellular cementum formation (56). In addition, cells of the Epithelial Rest of Malassez may help in cementum repair because of their ability to activate matrix proteins, such as amelogenin, which are also expressed during tooth development (76, 81). In summary, based on the information presented, it appears that the developed or ÔadultÕ periodontium retains its potential for repair/regeneration in the form of cells of the Epithelial Rest of Malassez, progenitor cells and stem cells, which can be induced to differentiate into cementoblast, osteoblast or periodontal ligament cells to regenerate periodontal tissues. Molecular factors involved in periodontal development It is well known that tooth development is regulated by temporal- and spatial-restricted reciprocal epithelial mesenchymal interactions. A number of genes that play a crucial role in tooth development have been identified and include growth factors and their receptors, such as transforming growth factor b-1 and )2, bone morphogenetic protein-2 and )4 (BMP-2, )4), activins, fibroblast growth factor-4, )8 and )9 (FGF-4, )8, )9), hepatocyte growth factor, and midkine and transcription factors, such as the homeobox genes (Msx1, Msx2, Dlx1, Dlx2, Dlx3, Otlx2, Barx1), Pax genes (Pax9 and Pax6), and Lef1, Gli2/Gli3 and Shh (40, 100, 192, 249, 274). It has been documented that growth factors are involved in establishing the presence, number, site, size or shape of teeth. The availability of knockout mice has provided critical information on some growth factors that are determinants of early tooth development. However, little information is currently available on the growth and transcription factors involved in the later stages of tooth development, such as root development. Although one can assume that the same epithelial mesenchymal interactions will take place between the Hertwig s epithelial root sheath and the underlying ÔrootÕ mesenchyme, and all or some of the same growth factors will be involved in root formation, these issues have been only minimally addressed. Transforming growth factor b-1 and its receptors (58, 59), and BMP-2, )3 and )7 (249), have been identified in cementoblasts, periodontal ligament and alveolar bone, and BMP-2, )4 and MSX-2 have been reported in the Hertwig s epithelial root sheath (266). Fibroblast growth factor-2 (143), receptors for epidermal growth factor (42) and growth hormone (270) have been detected in periodontal tissues. However, the published studies are all descriptive and do not provide 198

4 Regeneration of periodontal tissues: cementogenesis revisited information as to the function of these growth factors in periodontium development. Furthermore, the transforming growth factor-b1-knockout mouse displays no apparent defects in tooth and root development (39), thus excluding a role for this factor in these processes. On the other hand, by using transgenic mice that express the BMP inhibitor, noggin, driven by the keratin 14 promoter (K14-noggin), we recently demonstrated that BMPs are important for proper root morphogenesis. When the function of BMPs is repressed, the transgenic mice demonstrate a delay in tooth development, lack of enamel formation and abnormally shaped roots (198). Insulin-like growth factor-i receptor has been demonstrated in the Hertwig s epithelial root sheath, and in vitro experiments suggest that insulin-like growth factor-i receptor plays a role in the proliferation and elongation of the Hertwig s epithelial root sheath, which is critical for root development (55). Transcription factors associated with root development include two members of the homeobox family of transcription factors: Dlx2 and Dlx3. The expression of Dlx2 by the Hertwig s epithelial root sheath during root development was demonstrated using Dlx2/LacZ transgenic mice (132). Although these studies are only suggestive of a role of Dlx2 in root development, it was of interest that the Dlx2 knockout mice showed normal teeth, while the Dlx1/Dlx2 knockout mice lacked maxillary molars (253). The involvement of Dlx3 in root development comes from the phenotype expressed by patients affected with the genetic disease, trichodento-osseous syndrome, which presents root defects as well as defects in hair, bone and enamel. A deletion of 4 bp in the Dlx3 gene, which causes a frameshift mutation and premature codon termination, resulting in an altered protein, were identified in a family with tricho-dento-osseous syndrome (199). We recently reported the importance of the Nfi-c transcription factor in root development. Nfi-c knockout mice appear normal, except that they exfoliate their teeth shortly after eruption. These mice show a lack of roots of both mandibular and maxillary teeth, and therefore their teeth have no bone attachment. Histological analysis indicated a normal crown, enamel and dentin formation, and although there is initial formation of the Hertwig s epithelial root sheath and a budding root, no further development occurs of the roots, cementum and periodontal attachment apparatus (239). Cementum composition In order to understand the process of cementogenesis, it is important to determine the composition of cementum. As in bone and dentin, the major component of cementum is collagen (16). The expression of noncollagenous proteins that stimulate cell migration, attachment, proliferation, protein synthesis and mineralization during root formation has been reported by several investigators (38, 142, 147). In the early stages of root development, immunohistochemical techniques have shown the expression of multifunctional proteins, such as laminin and fibronectin (140). These proteins, as well as other proteins extracted from cementum (173), are initially believed to function as chemo-attractants. Laminin and fibronectin can also function as adhesion proteins, together with tenascin (137), bone sialoprotein (38, 142), osteopontin (25), and a 55-kDa cementumattachment protein (196, 263). The presence of other bioactive proteins, such as enamel-like proteins (235, 234), osteonectin/sparc (201), and mitogenic factors (157, 269), have also been reported in the cementum. In addition to these proteins, cementoblasts synthesize and secrete several glycosaminoglycans (such as chondroitin-4-sulfate, chondroitin-6-sulfate and dermatan sulfate, and collagen fibrils), which are present in the cemento dentinal junction (88, 264, 265). It has been suggested that cementoblasts exhibit an osteoblast-like, rather than an odontoblastlike, phenotype (25). Odontoblast, osteoblast and cementoblast cells express several matrix proteins, such as osteopontin, bone sialoprotein (BSP), osteonectin, osteocalcin, matrix Gla protein (208) and dentin-matrix-protein 1 (DMP-1) (106). The presence of osteocalcin in cementum is more controversial. Bronckers et al. (25), using immunohistochemistry, reported the presence of osteocalcin on the cellular intrinsic fiber cementum (CIFC) and associated cementoblasts (mature), but not in the acellular cementum and its associated cementoblasts. Tenorio et al. (246) reported the presence of osteocalcin in acellular extrinsic fiber cementum (AEFC) but not in the associated cementoblasts, while CIFC and associated cementoblasts stained weakly. Bosshardt & Nanci (20) used two different antibodies (OC1 and OC2), which gave different results: OC1 showed reactivity with acellular cementum, while OC2 was negative. Similarly, the presence of DMP-1 has been associated with acellular cementum (275) and cementocytes, but not with cementoblasts (255). It has been suggested that acellular cementum is a unique tissue, while cellular cementum and bone share some similarities, although there are still morphological, functional and biochemical differences between the two tissues (19). The presence of cementum-specific proteins remains questionable, although some putative 199

5 Zeichner-David cementum-specific proteins have been invoked: a 55-kDa CAP (263); a mitogenic factor (167); and a 72-kDa protein, CEM-1 (235). However, as the characterization and the sole expression by cementoblasts of these proteins have not been determined, the possible existence of cementum-specific proteins remains unknown. It has been reported that cementoblasts and cementocytes produce high levels of the GLUT-1 monosaccharide transporter, while osteoblasts or osteocytes do not express this protein. These data suggest that GLUT-1 may play a role in cementogenesis and could serve as a biomarker to differentiate between cells of cementoblastic and osteoblastic lineage (124). However, the observed differences in GLUT-1 are quantitative, and GLUT-1 is present in many different cell types. Recently, we reported the isolation of a cementoblastoma-derived protein, CP-23, that is expressed by cementoblasts and some precursor cells present in the periodontal ligament, but not by osteoblasts. The function of the CP-23 protein is currently unknown; however, given its nuclear location, it may be required for cementoblast differentiation and may be used as a marker for cementoblast cells (3). The CP-23 protein is also expressed by Hertwig s epithelial root sheath cells (275). Based on our current knowledge of the development of periodontal tissues, several strategies exist for targeting regenerative therapy, ranging from inducing their own ÔregenerativeÕ mechanisms using molecular approaches, or utilizing cells to repopulate and recapitulate the developmental process. Strategies for periodontal regeneration/repair The process of periodontal tissue regeneration starts at the moment of tissue damage by means of growth factors and cytokines released by the damaged connective tissue and inflammatory cells. It is well accepted that in order to improve periodontal healing, root planing or root conditioning is a necessary antecedent to mesenchymal cell migration and attachment onto the exposed root surface. Acid treatment, in particular with citric acid, has been found to widen the orifices of dentinal tubules, thereby accelerating cementogenesis and increasing cementum apposition and connective tissue attachment. However, a systematic review performed by Mariotti (145) suggested that the use of citric acid, tetracycline or EDTA to modify the root surface provides no clinical significant benefit for regeneration in patients with chronic periodontitis. Conversely, when periodontal ligament cells are removed from the cementum or are unable to regenerate, bone tissue invades the periodontal ligament space and establishes a direct connection between the tooth and the wall of the alveolar socket, resulting in ankylosis. The ankylotic, nonflexible type of tooth support can lead to loss of function and resorption of the tooth root (13). Can guided tissue regeneration and bone grafting regenerate cementum? Nyman et al. (174), using MilliporeÒ membranes, introduced the concept of a membrane barrier, which excludes the apical migration of gingival epithelial cells and provides an isolated space for the inwards migration of periodontal ligament cells, osteoblasts and cementoblasts. Guided tissue regeneration was successfully used to aid in the regeneration of lost periodontal tissues caused by periodontitis (67). The first guided tissue regeneration membranes were nonabsorbable and made of polytetrafluoroethylene, such as Gore-TexÒ. Studies on experimentally induced periodontal defects in monkeys suggested that guided tissue regeneration was capable of inducing the formation of new bone and cementum (4). The second generation of guided tissue regeneration used absorbable membranes made of collagen or polylactic and citric acid (28, 159), which eliminated the need for surgical membrane retrieval (66). Recent systematic reviews indicate that, in the treatment of intrabony and furcation defects, guided tissue regeneration is more effective than open flap debridement. Various barrier types yielded no systematic difference in clinical outcome, but barrier types could explain some heterogeneity in the results. Overall, guided tissue regeneration is consistently more effective than open flap debridement in the gain of clinical attachment and reduction of probing depth in the treatment of intrabony and furcation defects (99, 163). The use of grafting material in combination with guided tissue regeneration seems to improve clinical outcomes for furcation, but not for intrabony defects, when compared with the use of barrier membranes alone. It has also been questioned whether guided tissue regeneration produces true cementum regeneration or only cemental repair. The newly formed cementum has been characterized as a cellular cementum that is usually poorly attached to the dentin surface (125). It is suggested that periodontal healing with guided tissue regeneration therapy occurs in two stages. The first stage comprises an initial healing phase with the formation of a blood clot, transient root resorption/demineralization, 200

6 Regeneration of periodontal tissues: cementogenesis revisited deposition of acellular cementum on the root surface and formation of connective tissue. The second phase comprises a remodeling process, which will result in a regenerated cementum similar to pristine cementum as maturation proceeds over time (69). In conclusion, several clinical studies have demonstrated that guided tissue regeneration is a successful treatment modality for periodontal reconstructive surgery and it has become an accepted procedure in most periodontal practices, either by itself or in combination with other treatment modalities. Autologous bone grafts to repair periodontal osseous defects have been used for many years and different approaches have been the subject of several reviews (165, 209). Bone repair can also be achieved using ceramic materials such as Bioglass, which is a bone-bonding bioactive material that has been widely used for bone healing (110). Studies in monkeys suggested that PerioGlasÒ (synthetic bone particulate) can achieve superior bone repair and cementum regeneration and retard epithelial down-growth compared with other, similar materials (50, 109). Additionally, these materials can be used as scaffolds or to deliver other bioactive molecules to enhance their function. The use of bone grafts, powders or ceramics is quite prevalent in many dental practices. A recent systematic review on the efficacy of bone replacement grafts compared with other interventions in the treatment of periodontal osseous defects was performed by Reynolds et al. (202). Meta-analysis indicated that for the treatment of intrabony defects, bone grafts are effective in reducing crestal bone loss, increasing bone level, increasing clinical attachment level, and reducing probing depth compared with open flap debridement procedures. Histological studies showed that demineralized freeze-dried bone allografts support the formation of a new attachment apparatus in intrabony defects; however, the available data indicate that alloplastic grafts support periodontal repair rather than regeneration, and that the best treatment is a combination of bone grafts with barrier membranes. Nevertheless, these strategies are directed mainly to enhance alveolar bone and periodontal ligament repair and have the problems that they do not address cementogenesis and therefore do not completely regenerate the architecture of the original periodontium. Molecular approaches for cementum regeneration Advances in our knowledge of developmental biology, and of the growth factors that initiate and regulate tooth development and tissue repair, suggests the use of some of these factors for periodontium regeneration (37, 61, 68, 71, 118, 116, 117, 128, 170). Some attachment proteins, such as fibronectin (29, 206, 262) or CAP (156, 196), are able to enhance fibroblast migration, attachment and orientation of the connective tissue to the root surface. New strategies, utilizing growth factors to induce cell migration, proliferation and differentiation, were developed to repopulate the damaged periodontal tissues with periodontal ligament cells (32, 247). It is believed that growth factors play important roles in modulating the proliferation and/or migration and/ or differentiation of structural cells in the periodontium (58, 86, 97, 197, 230). It is suggested that growth factor molecules are produced during cementum formation and then stored in the mature cementum matrix with the potential to induce periodontal repair or regeneration when needed (236). Large-scale production of recombinant growth factors has facilitated in vitro and in vivo studies to determine the efficacy of growth factors in periodontal tissue regeneration. Amongst the growth factors currently being used are platelet-derived growth factor, insulin-like growth factor (36, 63, 92, 138, 188, 210), transforming growth factor-b1 (146), basic fibroblast growth factor (213), dexamethasone (211) and BMPs (121, 205, 211). However, problems in applying these growth factors for periodontal repair include the nonspecific activity of some factors on different cell lineages in time and space, and the rapid loss of growth factors applied topically (13, 138). It has been shown that both platelet-derived growth factor and insulin-like growth factor-1 can stimulate the proliferation and chemotaxis of periodontal ligament cells, and that the combination of platelet-derived growth factor and insulin-like growth factor-1 can further increase the mitogenic effect (23, 44, 175). In addition to the mitogenic activity, platelet-derived growth factor also appears to stimulate collagen synthesis in periodontal ligament cells (146). Furthermore, dexamethasone has been shown to exert the same effect as insulin-like growth factor-1 on periodontal ligament fibroblasts, gingival fibroblasts and pulp fibroblasts, and may substitute for insulin-like growth factor-1 in the platelet-derived growth factor stimulation of cell proliferation (210). In addition to the previously described effects, platelet-derived growth factor has the capacity to significantly negate and reverse the inhibitory effects of lipopolysaccharide on the proliferation of human gingival fibroblasts. Lipopolysaccharide from a variety of gram-negative bacteria is known to inhibit 201

7 Zeichner-David gingival fibroblast proliferation and synthesizing activity, has been implicated in periodontal inflammation and may also be responsible for delayed wound healing following periodontal therapy (12). In vivo studies using the beagle dog (natural periodontal disease) and the nonhuman primate (ligatureinduced attachment loss) models showed that the application of platelet-derived growth factor/insulinlike growth factor-1 resulted in significant amounts of new bone and cementum formation (138, 210). Treatment with insulin-like growth factor-1 alone did not significantly alter healing compared with controls, while treatment with platelet-derived growth factor alone showed significant regeneration of attachment. Although there are differences in the response to platelet-derived growth factor/insulin-like growth factor-1, depending on which animal model is used (the osseous response in dogs appears to be greater than that of the nonhuman primate, while new attachment formation appears to be greater in the nonhuman primate than in the dog), there is consistency in promoting periodontal regeneration (63, 64). Rutherford et al. (211) showed that platelet-derived growth factor and dexamethasone, combined with a collagen carrier matrix, induced regeneration of the periodontium in monkeys. It has also been shown that the combination of platelet-derived growth factor and guided tissue regeneration work better than either of the two modalities alone (36, 188). Clinical trials in humans using platelet-derived growth factor/insulin-like growth factor to treat periodontal osseous defects showed that only high doses of these factors gave rise to a statistically significant increase in alveolar bone formation (92). When platelet-derived growth factor was used in combination with bone allografts to treat Class II furcations and interproximal intrabony defects, histological evaluation showed regeneration of new alveolar bone, cementum, and periodontal ligament (30, 171). Platelet-rich plasma is a fraction of plasma that contains platelet-derived growth factor and transforming growth factor-b (180). An alternative to the use of recombinant growth factors is the use of a platelet gel in combination with demineralized freeze-dried bone allografts (5, 43). The limitations of topical protein delivery to periodontal osseous defects include transient biological activity and bioavailability of platelet-derived growth factor at the wound site. To overcome these limitations, studies have used genetic engineering to transduce cells derived from the periodontium, using adenovirus carrying the platelet-derived growth factor gene to promote sustained release and ensure biological activity (7, 6, 65). The potential use of gene therapy in vivo to stimulate periodontal tissue regeneration has been studied in large tooth-associated alveolar bony defects in rats. The results showed that the direct gene transfer of platelet-derived growth factor-b stimulates the regeneration of alveolar bone and cementum (104). As stated above, some members of the BMPs are normally expressed during the development of the periodontium, such as BMP-3 and BMP-7/OP-1, which have been localized immunologically in alveolar bone, cementum, and periodontal ligament, whereas BMP-2 was only localized in the alveolar bone (249, 266). Although the exact role of BMPs in the development of the periodontium has not yet been determined, these proteins are good candidates for stimulating periodontal regeneration because of their ability to promote not only osteogenesis but also cementogenesis. The expected role of BMPs in stimulating intramembranous bone formation without an endochondral intermediate may provide greater osteogenic potential than autogenous bone or other bone substitutes (121, 118, 119, 170, 205, 240). Studies indicate that recombinant BMP-2 exerts no effect on the growth and differentiation of human periodontal ligament cells in vitro; however, BMP-2 stimulates alkaline phosphatase activity and parathyroid hormone-dependent 3,5 -cyclic adenosine monophosphate (camp) accumulation, which are early markers of osteoblast differentiation. Nevertheless, BMP-2 produced no mature osteoblasts, as measured by expression of osteocalcin, and also inhibited 1,25(OH)2D3-induced osteocalcin synthesis in these cells (123). In vitro studies using mouse-derived dental follicle and periodontal ligament cells suggest that BMP-2 induced dental follicle cells to differentiate towards a cementoblast/osteoblast phenotype but had no effect on periodontal ligament cells (278). Paradoxally, BMP-2 was found to inhibit cementoblast cell mineralization in vitro by decreasing the expression of BSP and collagen type 1 (279). In studies of BMP-2 on early wound healing in a rat model of periodontal regeneration, the connective tissue attachment was found to be similar in animals receiving BMP-2 and in controls. However, BMP-2 induced bone formation at some distance from the defect, which indicates the need for a suitable delivery system to maintain the BMP-2 at the site of implantation (120). Other studies suggest that the effects of BMPs may be influenced by certain factors, such as root surface conditioning, delivery systems, masticatory forces, etc., and that BMP-2 stimulates the proliferation and migration of cells from the adjacent 202

8 Regeneration of periodontal tissues: cementogenesis revisited periodontal ligament into the wounded area, promoting new cementum formation (119). The expression of both BMP-2 and BMP-7 during periodontal tissue morphogenesis suggests that optimal therapeutic regeneration may require the combined use of the two BMPs. BMP-7-treated molar furcation defects in baboons resulted in substantial cementogenesis, while BMP-2 showed limited cementum formation but greater amounts of mineralized bone and osteoid; however, the combined application did not enhance alveolar bone regeneration or new attachment formation over and above that obtained by separate applications of the two BMPs (207). Recently, it was shown that the application of a synthetic BMP-6 polypeptide to a periodontal fenestration defect in rats resulted in increased formation of new bone and cementum (93). Perhaps the use of other members of the BMP family, such as growth and differentiation factor-5, )6, and )7, might provide better and more complete regenerative outcomes. These factors have been detected during the process of periodontal development at the surfaces of alveolar bone, cementum and periodontal ligament fiber bundles (223). Limitations for the regular use of BMPs are the need for high doses, non-specific activity on different cell lineages in time and space, and the rapid loss of topically applied growth factors (13, 138). Some of these problems can be overcome by the use of gene transfer technology. Jin et al. (103) used adenoviruses containing BMP-7 to transduce dermal fibroblasts that were then used to treat mandibular alveolar bone defects in a rat wound repair model. Their results showed chrondrogenesis, with subsequent osteogenesis, cementogenesis and bridging of the periodontal bone defects, suggesting that this genetic engineering approach may be useful in alveolar bone regeneration. A recent literature review (62) concluded that although promising, there were insufficient data at the present time to conduct a meta-analysis on the effect of growth factors for periodontal repair, and pointed to the need for more clinical trials. Do enamel-associated proteins regenerate cementum? Based on the presence of enamel proteins in acellular cementum (133, 235, 182, 233), it was thought that these proteins may play a role in the repair/regeneration of periodontal tissues destroyed by periodontal disease (78). This idea was tested by adding enamel proteins or purified enamel matrix derivative to surgically produced periodontal defects in monkeys, followed by histological analysis that showed almost complete regeneration of acellular cementum, firmly attached to the dentin and with collagenous fibers extending towards newly formed alveolar bone (79). These studies resulted in a new therapeutic preparation to treat periodontal disease, consisting of hydrophobic enamel matrix proteins extracted from porcine developing enamel, which has been marketed by Biora, Inc., under the name of EmdogainÒ. In the past 8 years, the use of enamel proteins for inducing the formation of cementum, bone and dentin has generated numerous in vivo and in vitro studies, as well as clinical trials, resulting in almost 300 publications. In vitro studies, animal studies and clinical trials are all being conducted simultaneously (60, 70, 83, 154). In vitro studies, using periodontal-associated cells such as periodontal ligament fibroblasts, osteoblasts, cementoblasts, gingival fibroblasts, gingival epithelial cells, etc., have been conducted in an attempt to understand the molecular and cellular mechanisms involved in the process of enamel matrix derivativeinduced tissue regeneration. In order for enamel matrix derivative to regenerate periodontal tissues, it will need to exert an effect on proliferation, migration, attachment and/or differentiation of the surrounding periodontal cells, and most studies have measured these parameters, as shown in Table 1. Few studies have tested the effect of enamel matrix derivative on cell migration, but available data suggest an increased migration of periodontal ligament cells, osteoblasts, gingival fibroblasts and dermal fibroblasts in response to enamel matrix derivative, with the exception of one study that found no effect on periodontal ligament cells (184). Most studies on the effect of enamel matrix derivative on cell attachment, which generally included periodontal ligament cells, found an increase in cell attachment (184). However, one study found the enamel matrix derivative to have no effect on cell attachment of gingival fibroblasts (256). A number of studies, which measured the effect of enamel matrix derivative on cell proliferation, have found an increase in cell proliferation in the presence of enamel matrix derivative. However, the proliferative effect was not found in two studies using periodontal ligament cells (41, 256), in two studies using osteoblast cell lines (215, 268) and in one study using gingival fibroblasts (256). Several studies found an inhibition of cell proliferation when epithelial cells were used (112, 139, 273). These data may explain the clinical observation that application of enamel matrix derivative suppresses the down-growth of junctional 203

9 Zeichner-David Table 1. In vitro studies on the effect of enamel protein derivative on cells Cells Species Migration Attachment Proliferation Differentiation Mineralization Reference Periodontal ligament cells Human ND ND ND AP increase osteoblast Yes (166) Human No effect No effect Yes increase No (Type I col) ND (184) Rat (primary) ND ND Yes decrease No Col, AP (95) Human (primary) ND ND Yes increase Yes less AP cementoblast ND (33) Human (primary) Yes ND Yes increase ND ND (203) Human (primary) ND Yes No difference ND ND (41) Human (primary) ND ND Yes increase Yes, increase IGF-1 and TGF-b1. No effect on bone phenotype ND (178) Hu (primary) ND ND ND Increase matrix (versican, biglycan, decorin, hyaluronan ND (73) Hu (primary) ND Yes No effect Increase AP and TGF-b1 ND (256) Hu (primary) Yes ND Yes increase ND ND (89) Hu (primary) ND Yes Yes increase Increase camp, TGF-b1, IL-6, PDGF-AB ND (139) P (primary) ND Yes Yes increase Increase OPN ND (204) Mo (cell line)** ND Yes Yes increase Inhibits Col I, de novo expression BSP and OCN, increase BMP2 ND (273) Mo (cell line) ND Yes Yes increase Inhibits Col I, de novo OCN and BMP3 ND (273) Osteoblasts Hu (ROS17/2.8) ND ND ND BSP increase ND (227) Hu (primary) ND ND Yes increase More FGF2 and COX2; less AP and MMP1 ND (161) Mo (ST2) ND ND No effect Yes AP ND (268) Mo (KUSA/A1) ND ND Yes increase Yes AP, Col, OPN, TGF-b1, OCN and MMPS Yes- more (268) Mo (primary) ND ND Yes increase ND ND (101) Mo (primary) ND ND ND Increase Col, IL-6 and PGHS-2; no effect on OCN and IGF-1 ND (102) Mo (MC3T3-E1) ND ND Yes increase Increase OPN and less OCN (254) Hu (2T9 pre-osteoblasts) ND ND Yes increase No effect ND (215) Hu (MG63 osteoblast like) ND ND Yes decrease Yes, increase AP, OCN, TGB1 ND (215) 204

10 Regeneration of periodontal tissues: cementogenesis revisited Table 1. Continued Cells Species Migration Attachment Proliferation Differentiation Mineralization Reference Hu (primary) ND ND Yes increase Yes, increase AP, OCN, TGB1 ND (215) Hu (MG63) Yes ND Yes increase ND ND (89) P (primary) ND Yes Yes increase Increase OPN ND (204) Hu (Ros17/28)* ND ND ND BSP increase ND (228) Gingival fibroblast cells Rat ND ND Yes double Faster osteogenic Yes more (115) Hu (primary) Yes ND Yes increase ND ND (203) Hu (primary) ND ND ND Increase matrix (versican, biglycan, decorin, hyaluronan ND (73) Hu (primary) ND No effect No effect Increase AP and TGF-b1 ND (256) Hu (primary) Yes ND Yes increase ND ND (89) Rat ND ND Yes increase More ECM No (115) Rat ND ND No difference ND ND (72) P (primary) ND Yes Yes increase Increase OPN ND (204) Dental follicle Mo (SV40) ND ND Yes increase More OPN, Less OCN Inhibits (74) Cementoblasts Mo (SV40) ND ND Yes increase Decrease Ocn Inhibits (254) Mo (OCCM-30)* ND ND ND Decrease BSP Inhibits (258) Mo (OCCM-30)ND à ND No effect Decrease OCN, increase OPN and OPG Inhibits (17) Fibroblasts Mo (L929) ND ND No difference ND ND (72) Rabbit Yes vascular endothelium. Growth factors (160) Human (primary) Yes ND Yes increase ND ND (203) Mesenchymal stem cells Hu (C2C12) ND ND ND Yes increase AP. Osteoblast phenotype ND (177) Epithelial cells Hu (HELA) ND ND Inhibited Increase camp and PDGF-AB ND (113) Hu (SCC25) ND ND Inhibited Increase p21waf1/cip1; decrease CK-18 ND (113, 112, 114) ERM P (primary) ND Yes Yes increase Increase OPN ND (204) Endothelial cells Hu (HUVEC) Yes increase ND No effect ND ND (271) AP, alkaline phosphatase; BMP, bone matrix protein; BSP, bone sialoprotein; Col, collagen; Hu, human; IGF-1, insulin-like growth factor; IL-6, interleukin-6; MMPS, matrix metalloproteinases; Mo, mouse; ND, not determined; OCN, osteocalcin; OPN, osteopontin; OPG, osteoprotegerin; P, pig; PDGF-AB, platelet derived growth factor AB; PGHS-2, prostaglandin G/H synthase 2; TGF-b1, transforming growth factor b-1. *Mouse recombinant amelogenin. Mouse recombinant ameloblastin. à Mouse leucine rich amelogenin peptide (LRAP). 205

11 Zeichner-David epithelium onto dental root surfaces, a process that frequently interferes with the formation of new connective tissue attachments (79, 78). The majority of available in vitro studies have analyzed the effect of enamel matrix derivative on gene expression and differentiation, and most of these studies found either an increased or a decreased expression of certain transcription and growth factors, extracellular matrix proteins or mineralization-associated proteins in the cells tested. Where mineralization was measured, it was found that enamel matrix derivative induced mineralization of periodontal ligament cells (166), increased mineralization of osteoblasts (268) and gingival fibroblasts (115), decreased mineralization of cementoblast cells (254) and inhibited the mineralization of dental follicle cells (74). Differences in results amongst studies can be explained by differences in sources and concentrations of enamel matrix derivative and in the cell preparations used. Most studies employed primary cell cultures derived from different patients, which probably contained mixed populations of a variety of cells present in the periodontium. Nevertheless, taken together, these studies suggest that enamel matrix derivative can act as a multipurpose growth factor capable of stimulating the proliferation of mesenchymal cells while inhibiting the cell division of epithelial cells, and can stimulate attachment and phenotypical changes in some cells, while inhibiting matrix production in others. Given the widespread use of EmdogainÒ, and the fact that it is made from an extract of enamel proteins, it is important to identify the actual protein responsible for its function. Studies by Maycock et al. (148) found that, in addition to amelogenin, EmdogainÒ contains metalloproteases and serine proteases. Studies by Kawase et al. (114) demonstrated that porcine enamel matrix derivative contains transforming growth factor-b1 (or a transforming growth factor-b-like substance), and that the action of enamel matrix derivative is mediated by the smad- 2 signaling pathway. In addition, a neutralizing anti-transforming growth factor-b immunoglobulin blocked the action of enamel matrix derivative on epithelial cells, although it failed to block completely enamel matrix derivative-induced fibroblastic proliferation, suggesting the presence of more than one growth factor. Iwata et al. (98) isolated the inductive activity of enamel matrix derivative by using chromatography and characterized it as being BMP-2 and BMP-4 using specific antibodies. Furthermore, in the presence of noggin (an inhibitor of BMPs), enamel matrix derivative lost its inductive activity, indicating that BMPs are the molecules responsible for enamel matrix derivative activity. Although these studies suggest that the action of EmdogainÒ is a result of the presence of contaminating growth factors, other studies have shown that pure recombinant enamel proteins indeed have activity as inducers. The results obtained in our laboratory indicate that mouse recombinant amelogenin can increase attachment and proliferation of mouse periodontal ligament cells in vitro (272, 273). Furthermore, a post-translational modified recombinant ameloblastin, another enamelassociated protein, had an effect similar to that of amelogenin on periodontal ligament cells. Both recombinant amelogenin and ameloblastin can change the phenotype expressed by periodontal ligament cells by inhibiting the expression of collagen type I and inducing de novo expression of osteocalcin. Amelogenin also induced the expression of bone sialoprotein and BMP-2, while ameloblastin induced the de novo expression of BMP-3 (273). These results indicate that both enamel-associated proteins have a modulatory effect on the expression of BMPs, suggesting that perhaps these proteins exert their signaling effect by means of BMPs. Recombinant mouse amelogenin improved osteoblast adhesion (90), and increased the expression of bone sialoprotein and decreased the formation of mineralized nodules in cementoblasts (258). A leucine-rich amelogenin peptide, which exhibited no effect on cell proliferation, down-regulated osteocalcin and up-regulated osteopontin in a dose- and timedependent manner, and inhibited the capacity to form mineral nodules (17). Taken together, these reports point towards a growth factor activity for enamel proteins that may be of importance in periodontal tissue regeneration. Several clinical trials have shown an increase in periodontal attachment and bone formation in individuals treated with EmdogainÒ (54, 85, 87, 154, 179, 200, 217, 216, 218, 219, 277). However, in many of these studies, the results were no better than those obtained with other previously established treatments, such as guided tissue regeneration, which yields better outcomes in the management of deep intrabony periodontal defects (84, 187, 218, 221, 231). Histological studies revealed that treatment with EmdogainÒ is unpredictable, resulting in the formation of cellular cementum rather than acellular cementum, and this cementum was only partially attached to the root surface, similar to the cementum formed with the use of guided tissue regeneration. Furthermore, more bone regeneration occurred by using a guided tissue regeneration procedure than 206

12 Regeneration of periodontal tissues: cementogenesis revisited EmdogainÒ (216, 219, 218). Other studies showed no evidence of improvement in radiographic bone level, and surgical re-entry found new tissue with a rubbery consistency and that was not mineralized (189, 190). Experiments in rats, using a wounded rat periodontium model followed by immunohistochemical analysis, showed that EmdogainÒ does not affect the expression of differentiation markers or bone matrix protein synthesis in the repopulation response of wounded rat molar periodontium (35). Systematic studies, using literature reviews and meta-analysis, suggest that treatment with enamel matrix derivative results in significant variations in clinical outcomes (107). Although EmdogainÒ is able to significantly improve probing attachment levels and pocket depth reduction, some studies found no evidence of clinically important differences between guided tissue regeneration and EmdogainÒ (47, 62) and reported that guided tissue regeneration is more predictable for cementum and bone regeneration (257). Although animal histological studies with surgically created defects suggest that enamel matrix derivative induces the formation of acellular cementum and promotes attachment of the supporting periodontal tissues, human histological studies have questioned both the consistency of the histological outcomes and the ability of enamel matrix derivative to predictably stimulate the formation of acellular cementum (107). It appears that following treatment with enamel matrix derivative, a bone-like tissue resembling cellular intrinsic fibrous cementum is formed (22). Despite the mixed results obtained from both in vitro and in vivo studies, new applications of EmdogainÒ are continuously being reported. Some studies suggest that it has the ability to induce the formation of reparative dentin in pulpotomized teeth (94, 96, 168, 169). It is being used to coat titanium implants with mixed results; one study suggests that there is enhanced formation of trabecular bone (229) while the other found no effect (53). It has also been suggested that enamel matrix derivative can combat bacteria in postsurgical periodontal wounds, which otherwise could hamper wound healing and reduce the outcome of regenerative procedures (8, 172, 220, 237). More recently, an acceleration of skin wound PLF PL-7 DPM Control HERS-CM Control HERS-CM Control HERS-CM 14 days 21 days 28 days 35 days Fig. 2. Effect of Hertwig s epithelial root sheath-conditioned media (HERS-CM) on periodontium-associated cell mineralization. HERS-CM was prepared by growing the cells in Dulbecco s modified Eagle s minimal essential medium (DMEM) supplemented with 10% fetal calf serum (FCS) and 100 U/ml of penicillin/streptomycin. Cells were incubated at 39.5 C in a humidified atmosphere of 95% air and 5% CO 2 for 7 days, after which the media were collected, the protein concentration determined and then lyophilized. Periodontal ligament fibroblasts (PLF), cementoblasts (PL-7) and dental papillae mesenchyme fibroblasts (DPM) were prepared from Immortomouse (275). Cells were grown in differentiation conditions (DMEM supplemented with 10% FCS, 100 U/ml of penicillin/streptomycin, 50 mg/ml of ascorbic acid and 2 mm sodium b-glycerophosphate), with or without (controls) 100 lg of HERS-CM proteins. At different time-points of culture, cells were fixed with 70% methanol and 30% acetic acid and stained with Von Kossa to determine mineralization. 207

13 Zeichner-David healing in the presence of enamel matrix derivative was reported (160). Cellular tissue engineering for cementum regeneration It has long been recognized that a recolonization of periodontal ligament cells onto the root surface is necessary for periodontal ligament regeneration (129, 174). One therapeutic approach proposed the removal of autologous cells from the patient s periodontal ligament, culture of the cells in vitro, to place them back onto the exposed root coated with chemoattractant factors, and then to cover the area with an artificial basement membrane (247). A pilot study was carried out with four patients, using hydroxyapatite as a vehicle for cell delivery. After 6 months, the treated patients exhibited greater pocket reduction and clinical attachment gain, and less gingival recession, than control patients; however, both groups showed good fill of the osseous defects studied (48, 49, 91). Lekic et al. (130) tracked the fate and differentiation of rat periodontal cells and bone marrow cells transplanted into periodontal wounds in rats using cells constitutively expressing b-galactosidase as a marker. Labeled cells were localized in the periodontal ligament and regenerating alveolar bone and it was suggested that, following a cyclical process of growth and development, both cell types were able to differentiate into periodontal ligament fibroblasts, osteoblasts and cementoblasts, and to contribute to periodontal regeneration (131). Regeneration of cementum, periodontal ligament and alveolar bone has also been observed using auto-transplantation of bone marrow mesenchymal stem cells into periodontal osseous defects in dogs (111). Similar results have been observed after the application of periodontal ligament cell sheets (2). The ability of cementoblasts and dental follicle cells to promote periodontal regeneration in a rodent periodontal fenestration model was analyzed recently (280). The results indicated that cementoblast-treated and carrier alone-treated defects showed complete bone bridging and periodontal ligament formation; however, no new cementum was formed along the root surface in either group. Puzzling, however, was the fact that no repair, or even osteogenesis, was seen within dental follicle cell-treated defects, even though these cells are believed to be precursors of cementoblasts and to be responsible for alveolar bone formation. As our laboratory has established immortal cell lines for the Hertwig s epithelial root sheath (275) and the Epithelial Rest of Malassez cells, we are exploring the ability of these cells, or their secreted products, to induce periodontal ligament cells to differentiate into cementoblasts in vitro. When periodontal ligament cells, which do not produce a mineralized extracellular matrix, are grown in the presence of Hertwig s epithelial root sheath conditioned media (HERS-CM), these cells produce a mineralized extracellular matrix, as determined by a positive Von-Kossa staining BSP OCN OSN OPN AP BMP4 Col1 Actin Effect of HERS-CM on PLF cell differentiation P 21d 21d + HERS Fig. 3. Effect of Hertwig s epithelial root sheath-conditioned media (HERS-CM) on the phenotype of periodontal ligament cells. HERS-CM was prepared as previously described. Periodontal ligament cells were grown under proliferation (P) conditions (in the presence of interferonc at 33 C) or differentiation conditions [Dulbecco s modified Eagle s minimal essential medium (DMEM) supplemented with 10% fetal calf serum (FCS), 100 U/ml of penicillin/streptomycin, 50 mg/ml of ascorbic acid and 2 mm sodium b-glycerophosphate] with or without (controls) 100 lg of HERS-CM proteins. Cells were collected after 21 days in culture (media were changed every other day), the media were removed, cells were rinsed in phosphate-buffered saline (PBS) and total RNA was extracted for determination of phenotype by using reverse transcription polymerase chain reaction (RT PCR). AP, alkaline phosphatase; BMP-4, bone morphogenetic protein-4; BSP, bone sialoprotein; Col1, collagen type I; OCN, osteocalcin; OPN, osteopontin; OSN, osteonectin. 208