Periodontology 2000, Vol. 41, 2006, 196–217 Copyright Blackwell Munksgaard 2006 Printed in Singapore. All rights reserved PERIODONTOLOGY 2000

Regeneration of periodontal tissues: cementogenesis revisited

MARGARITA ZEICHNER-DAVID

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

196 Regeneration of periodontal tissues: cementogenesis revisited

Fig. 1. Root development and periodontium formation. continues, and there is formation of the periodontium Histological sections of 7-day postnatal mouse mandibu- with cementum, periodontal ligament and bone. Am, lar molars showing the initial development of the root by ameloblasts; C, cementum; D, dentin; Ds, dental sac; formation of the Hertwig’s epithelial root sheath. At the HERS, Hertwig’s epithelial root sheath; Od, odontoblasts; 14-day postnatal time-point, apical migration of the roots PDL, periodontal ligament. of odontoblasts to form the root dentin (183, 182, 222, chymal transformation to become functional ce- 243, 251), or to differentiate dental sac cells into mentoblasts in charge of producing the acellular cementoblasts (181). The current notion states that cementum (251, 275). Hertwig’s epithelial root sheath cells produce the The gingival tissues appear to be derived from both basement membrane containing chemotactic pro- the oral mucosa and the developing tooth germ (135). teins, which serve to direct the migration of prece- It has been suggested that the dental follicle (con- mentoblast cells (140, 141, 182, 235, 251) and to nective tissue surrounding the developing teeth) induce cementoblast differentiation (191, 232, 234). gives rise to the fibroblasts forming the periodontal Amongst the basement membrane molecules are ligament as well as to the alveolar bone and several extracellular matrix proteins, growth factors, cementoblasts (45, 136, 186, 243), all of which have enamel proteins and adhesion molecules, such as a a common neural crest origin (34). Therefore, it is collagenous-like protein, known as cementum postulated that there are different types of attachment protein (CAP), which has chemotactic cementoblasts: those originating from the Hertwig’s potential capable of recruiting putative cementoblast epithelial root sheath via epithelial–mesenchymal precursors (11, 149, 156, 196, 275). In the second transformation and which form the acellular stage of cementogenesis (when the tooth reaches cementum; and those derived from the dental follicle, occlusion and cellular cementum is formed), the which form the cellular cementum (9, 19, 105, 251, proliferation of cells of the Hertwig’s epithelial root 275). It is also believed that progenitors for perio- sheath is considerably reduced, and some cells are dontal ligament, osteoblast and cementoblast cells entrapped in the newly formed mineral where they adopt a paravascular location in the periodontal may influence phenotypic changes in the dental sac ligament, and these cells, which exhibit some fea- cells (252). It is also suggested that Hertwig’s epi- tures of stem cells, can regenerate functional tissues thelial root sheath cells undergo epithelial–mesen- when the need arises (150–153, 195). Periodontal

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

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

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

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

201 Zeichner-David gingival fibroblast proliferation and synthesizing biological activity (7, 6, 65). The potential use of gene activity, has been implicated in periodontal inflam- therapy in vivo to stimulate periodontal tissue mation and may also be responsible for delayed regeneration has been studied in large tooth-associ- wound healing following periodontal therapy (12). ated alveolar bony defects in rats. The results showed In vivo studies using the beagle dog (natural perio- that the direct gene transfer of platelet-derived dontal disease) and the nonhuman primate (ligature- growth factor-B stimulates the regeneration of induced attachment loss) models showed that the alveolar bone and cementum (104). application of platelet-derived growth factor/insulin- As stated above, some members of the BMPs are like growth factor-1 resulted in significant amounts of normally expressed during the development of the new bone and cementum formation (138, 210). periodontium, such as BMP-3 and BMP-7/OP-1, Treatment with insulin-like growth factor-1 alone did which have been localized immunologically in not significantly alter healing compared with controls, alveolar bone, cementum, and periodontal ligament, while treatment with platelet-derived growth factor whereas BMP-2 was only localized in the alveolar alone showed significant regeneration of attachment. bone (249, 266). Although the exact role of BMPs in Although there are differences in the response to the development of the periodontium has not yet platelet-derived growth factor/insulin-like growth been determined, these proteins are good candidates factor-1, depending on which animal model is used for stimulating periodontal regeneration because of (the osseous response in dogs appears to be greater their ability to promote not only osteogenesis but than that of the nonhuman primate, while new also cementogenesis. The expected role of BMPs in attachment formation appears to be greater in the stimulating intramembranous bone formation with- nonhuman primate than in the dog), there is consis- out an endochondral intermediate may provide tency in promoting periodontal regeneration (63, 64). greater osteogenic potential than autogenous bone or Rutherford et al. (211) showed that platelet-derived other bone substitutes (121, 118, 119, 170, 205, 240). growth factor and dexamethasone, combined with a Studies indicate that recombinant BMP-2 exerts no collagen carrier matrix, induced regeneration of the effect on the growth and differentiation of human periodontium in monkeys. It has also been shown that periodontal ligament cells in vitro; however, BMP-2 the combination of platelet-derived growth factor and stimulates alkaline phosphatase activity and para- guided tissue regeneration work better than either of thyroid hormone-dependent 3¢,5¢-cyclic adenosine the two modalities alone (36, 188). monophosphate (cAMP) accumulation, which are Clinical trials in humans using platelet-derived early markers of osteoblast differentiation. Never- growth factor/insulin-like growth factor to treat per- theless, BMP-2 produced no mature osteoblasts, as iodontal osseous defects showed that only high doses measured by expression of osteocalcin, and also of these factors gave rise to a statistically significant inhibited 1,25(OH)2D3-induced osteocalcin synthesis increase in alveolar bone formation (92). When in these cells (123). In vitro studies using mouse-de- platelet-derived growth factor was used in combina- rived dental follicle and periodontal ligament cells tion with bone allografts to treat Class II furcations suggest that BMP-2 induced dental follicle cells to and interproximal intrabony defects, histological differentiate towards a cementoblast/osteoblast phe- evaluation showed regeneration of new alveolar notype but had no effect on periodontal ligament cells bone, cementum, and periodontal ligament (30, 171). (278). Paradoxally, BMP-2 was found to inhibit ce- Platelet-rich plasma is a fraction of plasma that mentoblast cell mineralization in vitro by decreasing contains platelet-derived growth factor and trans- the expression of BSP and collagen type 1 (279). In forming growth factor-b (180). An alternative to the studies of BMP-2 on early wound healing in a rat use of recombinant growth factors is the use of a model of periodontal regeneration, the connective platelet gel in combination with demineralized tissue attachment was found to be similar in animals freeze-dried bone allografts (5, 43). receiving BMP-2 and in controls. However, BMP-2 The limitations of topical protein delivery to peri- induced bone formation at some distance from the odontal osseous defects include transient biological defect, which indicates the need for a suitable delivery activity and bioavailability of platelet-derived growth system to maintain the BMP-2 at the site of implan- factor at the wound site. To overcome these limita- tation (120). Other studies suggest that the effects of tions, studies have used genetic engineering to BMPs may be influenced by certain factors, such as transduce cells derived from the periodontium, using root surface conditioning, delivery systems, mastica- adenovirus carrying the platelet-derived growth fac- tory forces, etc., and that BMP-2 stimulates the pro- tor gene to promote sustained release and ensure liferation and migration of cells from the adjacent

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

203 Zeichner-David 204 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. ND (178) No effect on bone phenotype Hu (primary) ND ND ND Increase matrix (versican, biglycan, ND (73) decorin, hyaluronan 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 ND (273) and OCN, increase BMP2 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 Yes- more (268) and MMPS Mo (primary) ND ND Yes – increase ND ND (101) Mo (primary) ND ND ND Increase Col, IL-6 and PGHS-2; ND (102) no effect on OCN and IGF-1 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) 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, ND (73) decorin, hyaluronan 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) revisited cementogenesis tissues: periodontal of Regeneration Cementoblasts Mo (SV40) ND ND Yes – increase Decrease Ocn Inhibits (254) Mo (OCCM-30)* ND ND ND Decrease BSP Inhibits (258) Mo (OCCM-30)NDà 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; 205 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. àMouse leucine rich amelogenin peptide (LRAP). Zeichner-David epithelium onto dental root surfaces, a process that that BMPs are the molecules responsible for enamel frequently interferes with the formation of new con- matrix derivative activity. Although these studies nective tissue attachments (79, 78). suggest that the action of Emdogain is a result of The majority of available in vitro studies have the presence of contaminating growth factors, other analyzed the effect of enamel matrix derivative on studies have shown that pure recombinant enamel gene expression and differentiation, and most of proteins indeed have activity as inducers. The results these studies found either an increased or a de- obtained in our laboratory indicate that mouse creased expression of certain transcription and recombinant amelogenin can increase attachment growth factors, extracellular matrix proteins or min- and proliferation of mouse periodontal ligament cells eralization-associated proteins in the cells tested. in vitro (272, 273). Furthermore, a post-translational Where mineralization was measured, it was found modified recombinant ameloblastin, another enamel- that enamel matrix derivative induced mineralization associated protein, had an effect similar to that of of periodontal ligament cells (166), increased miner- amelogenin on periodontal ligament cells. Both alization of osteoblasts (268) and gingival fibroblasts recombinant amelogenin and ameloblastin can (115), decreased mineralization of cementoblast cells change the phenotype expressed by periodontal (254) and inhibited the mineralization of dental fol- ligament cells by inhibiting the expression of colla- licle cells (74). Differences in results amongst studies gen type I and inducing de novo expression of can be explained by differences in sources and con- osteocalcin. Amelogenin also induced the expression centrations of enamel matrix derivative and in the of bone sialoprotein and BMP-2, while ameloblastin cell preparations used. Most studies employed pri- induced the de novo expression of BMP-3 (273). mary cell cultures derived from different patients, These results indicate that both enamel-associated which probably contained mixed populations of a proteins have a modulatory effect on the expression variety of cells present in the periodontium. Never- of BMPs, suggesting that perhaps these proteins exert theless, taken together, these studies suggest that their signaling effect by means of BMPs. Recombin- enamel matrix derivative can act as a multipurpose ant mouse amelogenin improved osteoblast adhe- growth factor capable of stimulating the proliferation sion (90), and increased the expression of bone of mesenchymal cells while inhibiting the cell divi- sialoprotein and decreased the formation of miner- sion of epithelial cells, and can stimulate attachment alized nodules in cementoblasts (258). A leucine-rich and phenotypical changes in some cells, while amelogenin peptide, which exhibited no effect on inhibiting matrix production in others. cell proliferation, down-regulated osteocalcin and Given the widespread use of Emdogain, and the up-regulated osteopontin in a dose- and time- fact that it is made from an extract of enamel pro- dependent manner, and inhibited the capacity to teins, it is important to identify the actual protein form mineral nodules (17). Taken together, these responsible for its function. Studies by Maycock et al. reports point towards a growth factor activity for (148) found that, in addition to amelogenin, Emdo- enamel proteins that may be of importance in gain contains metalloproteases and serine pro- periodontal tissue regeneration. teases. Studies by Kawase et al. (114) demonstrated Several clinical trials have shown an increase in that porcine enamel matrix derivative contains periodontal attachment and bone formation in indi- transforming growth factor-b1 (or a transforming viduals treated with Emdogain (54, 85, 87, 154, 179, growth factor-b-like substance), and that the action 200, 217, 216, 218, 219, 277). However, in many of of enamel matrix derivative is mediated by the smad- these studies, the results were no better than those 2 signaling pathway. In addition, a neutralizing obtained with other previously established treat- anti-transforming growth factor-b immunoglobulin ments, such as guided tissue regeneration, which blocked the action of enamel matrix derivative on yields better outcomes in the management of deep epithelial cells, although it failed to block completely intrabony periodontal defects (84, 187, 218, 221, 231). enamel matrix derivative-induced fibroblastic prolif- Histological studies revealed that treatment with eration, suggesting the presence of more than one Emdogain is unpredictable, resulting in the forma- growth factor. Iwata et al. (98) isolated the inductive tion of cellular cementum rather than acellular activity of enamel matrix derivative by using chro- cementum, and this cementum was only partially matography and characterized it as being BMP-2 and attached to the root surface, similar to the cementum BMP-4 using specific antibodies. Furthermore, in the formed with the use of guided tissue regeneration. presence of noggin (an inhibitor of BMPs), enamel Furthermore, more bone regeneration occurred by matrix derivative lost its inductive activity, indicating using a guided tissue regeneration procedure than

206 Regeneration of periodontal tissues: cementogenesis revisited

Emdogain (216, 219, 218). Other studies showed no tum and promotes attachment of the supporting evidence of improvement in radiographic bone level, periodontal tissues, human histological studies have and surgical re-entry found new tissue with a rubbery questioned both the consistency of the histological consistency and that was not mineralized (189, 190). outcomes and the ability of enamel matrix derivative Experiments in rats, using a wounded rat periodon- to predictably stimulate the formation of acellular tium model followed by immunohistochemical cementum (107). It appears that following treatment analysis, showed that Emdogain does not affect the with enamel matrix derivative, a bone-like tissue expression of differentiation markers or bone matrix resembling cellular intrinsic fibrous cementum is protein synthesis in the repopulation response of formed (22). wounded rat molar periodontium (35). Despite the mixed results obtained from both Systematic studies, using literature reviews and in vitro and in vivo studies, new applications of meta-analysis, suggest that treatment with enamel Emdogain are continuously being reported. Some matrix derivative results in significant variations in studies suggest that it has the ability to induce the clinical outcomes (107). Although Emdogain is able formation of reparative dentin in pulpotomized teeth to significantly improve probing attachment levels (94, 96, 168, 169). It is being used to coat titanium and pocket depth reduction, some studies found no implants with mixed results; one study suggests that evidence of clinically important differences between there is enhanced formation of trabecular bone (229) guided tissue regeneration and Emdogain (47, 62) while the other found no effect (53). It has also been and reported that guided tissue regeneration is more suggested that enamel matrix derivative can combat predictable for cementum and bone regeneration bacteria in postsurgical periodontal wounds, which (257). Although animal histological studies with sur- otherwise could hamper wound healing and reduce gically created defects suggest that enamel matrix the outcome of regenerative procedures (8, 172, 220, derivative induces the formation of acellular cemen- 237). More recently, an acceleration of skin wound

PLF PL-7 DPM ControlHERS-CM Control HERS-CM Control HERS-CM

14 days

21 days

28 days

35 days

Fig. 2. Effect of Hertwig’s epithelial root sheath-condi- mentoblasts (PL-7) and dental papillae mesenchyme tioned media (HERS-CM) on periodontium-associated cell fibroblasts (DPM) were prepared from Immortomouse mineralization. HERS-CM was prepared by growing the (275). Cells were grown in differentiation conditions cells in Dulbecco’s modified Eagle’s minimal essential (DMEM supplemented with 10% FCS, 100 U/ml of peni- medium (DMEM) supplemented with 10% fetal calf serum cillin/streptomycin, 50 mg/ml of ascorbic acid and 2 mM (FCS) and 100 U/ml of penicillin/streptomycin. Cells were sodium b-glycerophosphate), with or without (controls) incubated at 39.5C in a humidified atmosphere of 95% 100 lg of HERS-CM proteins. At different time-points of air and 5% CO2 for 7 days, after which the media were culture, cells were fixed with 70% methanol and 30% collected, the protein concentration determined and then acetic acid and stained with Von Kossa to determine lyophilized. Periodontal ligament fibroblasts (PLF), ce- mineralization.

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

208 Regeneration of periodontal tissues: cementogenesis revisited

(Fig. 2). This effect is specific for periodontal liga- References ment cells because other types of fibroblasts, such as those derived from the dental pulp, do not produce a 1. Adams DF. Diagnosis and treatment of refractory perio- mineralized extracellular matrix, even in the presence dontitis. Curr Opin Dent 1992: 2: 33–38. of HERS-CM. When cementoblast cells, capable of 2. Akizuki T, Oda S, Komaki M, Tsuchioka H, Kawakatsu N, producing a mineralized extracellular matrix, were Kikuchi A, Yamato MJ, Okano T, Ishikawa I. Application of periodontal ligament cell sheet for periodontal regener- grown in the presence of HERS-CM, an acceleration ation: a pilot study in beagle dogs. J Periodontal Res 2005: in the formation of mineral was detected. Analysis of 40: 245–251. the phenotype at the molecular level indicated a 3. Alvarez-Perez MA, Narayanan S, Zeichner-David M, de novo induction of the expression of bone sialo- Rodriguez Carmona B, Arzate H. 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