Higinio Arzate

Role of Hertwig's Epithelial Root Sheath Cells in Tooth Root Development

During tooth development, after the completion of crown formation, the apical mesenchyme forms the developing periodontium while the inner and outer enamel epithelia fuse below the level of the crown cervical margin to produce a bilayered epithelial sheath termed Hertwigs epithelial root sheath (HERS). The role of HERS cells in root formation is widely accepted; however, the precise function of these cells remains controversial. Functions suggested have ranged from structural (subdivide the dental ectomesenchymal tissues into dental papilla and dental follicle), regulators of timing of root development, inducers of mesenchymal cell differentiation into odontoblasts and cementoblasts, to cementoblast cell precursors. The characterization of the HERS phenotype has been hindered by the small amount of tissue present at a given time during root formation. In this study, we report the establishment of an immortal HERS‐derived cell line that can be maintained in culture and then induced to differentiate in vitro. Characterization of the HERS phenotype using reverse transcriptase‐polymerase chain reaction and Western blot immunostaining suggests that HERS cells initially synthesize and secrete some enamel‐related proteins such as ameloblastin, and then these cells appear to change their morphology and produce a mineralized extracellular matrix resembling acellular cementum. These studies suggest that the acellular and cellular cementum are synthesized by two different types of cells, the first one by HERS‐derived cementoblasts and the later by neural crest‐derived cementoblasts. Developmental Dynamics 228:651–663, 2003.

High glucose concentrations alter the biomineralization process in human osteoblastic cells

Diabetes mellitus (DM) may alter bone remodeling, as osteopenia and osteoporosis are among the complications. Moreover, DM increases the risk and severity of chronic inflammatory periodontal disease, in which bone resorption occurs. Broad evidence suggests that chronic inflammation can contribute to the development of DM and its complications. Hyperglycemia is a hallmark of DM that may contribute to sustained inflammation by increasing proinflammatory cytokines, which are known to cause insulin resistance, via toll-like receptor (TLR)-4-mediated mechanisms. However, the mechanisms by which bone-related complications develop in DM are still unknown. Studies done on the effect of high glucose concentrations on osteoblast functions are contradictory because some suggest increases (although others suggest reductions) in the biomineralization process. Therefore, we evaluated the effect of high glucose levels on biomineralization and inflammation markers in a human osteoblastic cell line. Cells were treated with either physiological 5.5 mM or increasing concentrations of glucose up to 24 mM, and we determined the following: i) the quantity and quality of calcium-deposit crystals in culture and ii) the expression of the following: a) proteins associated with the process of biomineralization, b) the receptor activator of nuclear factor kappa-B ligand (RANKL) and osteoprotegerin (OPG), c) cytokines IL1, IL6, IL8, IL10, MCP-1 and TNF alpha, and d) TLR-2, -3, -4 and -9. Our results show that high glucose concentrations (12 mM and particularly 24 mM) alter the biomineralization process in osteoblastic cells and provoke the following: i) a rise in mineralization, ii) an increase in the mRNA expression of RANKL and a decrease of OPG, iii) an increase in the mRNA expression of osteocalcin, bone sialoprotein and the transcription factor Runx2, iv) a diminished quality of the mineral, and v) an increase in the expression of IL1beta, IL6, IL8, MCP-1 and IL10 mRNAs. In addition we found that both high glucose levels and hyperosmotic conditions provoked TLR-2, -3, -4 and -9 overexpression in osteoblastic cells, suggesting that they are susceptible to osmotic stress.

Cementum protein 1 (CEMP1) induces a cementoblastic phenotype and reduces osteoblastic differentiation in periodontal ligament cells

Cementum is a calcified tissue covering the tooth root surface, which functions as rigid tooth‐anchoring structure. Periodontal ligament is a unique non‐mineralized connective tissue, and is a source of mineralized tissue forming cells such as cementoblasts and osteoblasts. The CEMP1 is a novel cementum component the presence of which appears to be limited to cementoblasts and their progenitors. In order to understand the function of CEMP1, we investigated CEMP1 expression during the differentiation of human periodontal ligament cells. Immunomagnetically enriched alkaline phosphatase (ALP)‐positive periodontal ligament cells preferentially expressed CEMP1. CEMP1 expression was reduced when periodontal ligament cells differentiated to osteoblasts in vitro. Over‐expression of CEMP1 in periodontal ligament cells enhanced cementoblast differentiation and attenuated periodontal and osteoblastic phenotypes. Our data demonstrate for the first time that the CEMP1 is not only a marker protein for cementoblast‐related cells, but it also regulates cementoblast commitment in periodontal ligament cells. J. Cell. Physiol. 227: 649–657, 2012.

Calcium Hydroxide Promotes Cementogenesis and Induces Cementoblastic Differentiation of Mesenchymal Periodontal Ligament Cells in a CEMP1- and ERK-Dependent Manner

Periodontal tissue engineering is a complex process requiring the regeneration of bone, cementum, and periodontal ligament (PDL). Since cementum regeneration is poorly understood, we used a dog model of dental pulpal necrosis and in vitro cellular wounding and mineralization assays to determine the mechanism of action of calcium hydroxide, Ca(OH)2, in cementogenesis. Laser capture microdissection (LCM) followed by qRT-PCR were used to assay responses of periapical tissues to Ca(OH)2 treatment. Additionally, viability, proliferation, migration, and mineralization responses of human mesenchymal PDL cells to Ca(OH)2 were assayed. Finally, biochemical inhibitors and siRNA were used to investigate Ca(OH)2-mediated signaling in PDL cell differentiation. In vivo, Ca(OH)2-treated teeth formed a neocementum in a STRO-1- and cementum protein-1 (CEMP1)-positive cellular environment. LCM-harvested tissues adjacent to the neocementum exhibited higher mRNA levels for CEMP1, integrin-binding sialoprotein, and Runx2 than central PDL cells. In vitro, Ca(OH)2 and CEMP1 promoted STRO-1-positive cell proliferation, migration, and wound closure. Ca(OH)2 stimulated expression of the cementum-specific proteins CEMP1 and PTPLA/CAP in an ERK-dependent manner. Lastly, Ca(OH)2 stimulated mineralization by CEMP1-positive cells. Blocking CEMP1 and ERK function abolished Ca(OH)2-induced mineralization, confirming a role for CEMP1 and ERK in the process. Ca(OH)2 promotes cementogenesis and recruits STRO-1-positive mesenchymal PDL cells to undergo cementoblastic differentiation and mineralization via a CEMP1- and ERK-dependent pathway.

Immunolocalization of a Human Cementoblastoma-conditioned Medium-derived Protein

Little is known about the molecular mechanisms that regulate the cementogenesis process, because specific cementum markers are not yet available. To investigate whether a cementoblastoma-conditioned medium-derived protein (CP) could be useful as a cementum biological marker, we studied its expression and distribution in human periodontal tissues, human periodontal ligament, alveolar bone, and cementoblastoma-derived cells. In human periodontal tissues, immunoreactivity to anti-CP was observed throughout the cementoid phase of acellular and cellular cementum, cementoblasts, cementocytes, cells located in the endosteal spaces of human alveolar bone, and in cells in the periodontal ligament located near the blood vessels. Immunopurified CP promoted cell attachment on human periodontal ligament, alveolar bone-derived cells, and gingival fibroblasts. A monoclonal antibody against bovine cementum attachment protein (CAP) cross-reacted with CP. These findings indicate that CP identifies potential cementoblast progenitor cells, is immunologically related to CAP species, and serves as a biological marker for cementum.

Periodontal regeneration following implantation of cementum and periodontal ligament-derived cells.

BACKGROUND AND OBJECTIVE The periodontal regeneration of bone defects is often unsatisfactory and could be largely improved by cell therapy. Therefore, the purpose of this study was to evaluate the regenerative potential of implanting canine cementum-derived cells (CDCs) and canine periodontal ligament-derived cells (PDLDCs) in experimentally created periodontal intrabony defects in beagle dogs. MATERIAL AND METHODS Cells were obtained from premolars extracted from four beagle dogs. Three-wall intrabony periodontal defects, 3 mm wide and 4 mm deep, were surgically created in their second and fourth premolars and plaque was allowed to accumulate. Once the defects were surgically debrided, periodontal regeneration was attempted by random implantation of collagen sponges embedded with 750,000 CDCs, 750,000 PDLDCs or culture medium. After 3 mo of healing, specimens were obtained and periodontal regenerative outcomes were assessed histologically and histometrically. RESULTS The histological analysis showed that a minimal amount of new cementum was formed in the control group (1.56 ± 0.39 mm), whereas in both test groups, significantly higher amounts of new cementum were formed (3.98 ± 0.59 mm in the CDC group and 4.07 ± 0.97 mm in the PDLDC group). The test groups also demonstrated a larger dimension of new connective tissue, resulting in a significantly more coronal level of histological attachment. CONCLUSION This proof-of-principle study suggests that cellular therapy, in combination with a collagen sponge, promoted periodontal regeneration in experimental intrabony periodontal defects.

Human cementoblasts express enamel-associated molecules in vitro and in vivo

BACKGROUND AND OBJECTIVE Cementum is a mineralized tissue that facilitates the attachment of periodontal ligament to the root and surrounding alveolar bone and plays a key role in the regeneration of periodontal tissues. The molecular mechanisms that regulate the proliferation and differentiation of cementoblasts, however, have not been elucidated to date. Enamel molecules are believed to regulate cementoblast differentiation and to initiate the formation of acellular extrinsic fiber cementum. The purpose of this study was therefore to isolate and culture human root-derived cells (HRDC) in order to determine whether they are able to express both cementum and specific enamel proteins and subsequently to confirm these findings in vivo. MATERIAL AND METHODS Human root-derived cells were isolated and expanded in vitro. Cells were characterized using RT-PCR, immunostaining, western blotting and by examination of total mRNA to determine the expression of cementum and enamel markers. Human periodontal tissues were also examined for the expression of enamel-related proteins by immunostaining. RESULTS We showed that HRDC express mRNA corresponding to ameloblastin (AMBN), amelogenin (AMEL), enamelin (ENAM), tuftelin (TUFT) and cementum-associated molecules such as cementum protein 1 (CEMP1) and cementum attachment protein (CAP). Western blotting revealed that HRDC express both AMEL and AMBN gene products, as well as the cementum markers CEMP1 and CAP. In vivo, we have showed that AMBN and AMEL are expressed by cementoblasts lining cementum, paravascular cells and periodontal ligament cells. CONCLUSION These results suggest that enamel-associated and cementum-associated proteins could act synergistically in regulating cementoblast differentiation and cementum deposition and offer new approaches on how the cementogenesis process is regulated.

Anti-cementoblastoma-derived protein antibody partially inhibits mineralization on a cementoblastic cell line

The effect of human anti-cementoblastoma-derived protein antibody during cementogenesis in vitro was investigated by using human cementoblastoma-derived cells. Cultures treated with 5 microg/ml of CP antibody from day 1 to day 15 revealed a significant decrease in alkaline phosphatase activity (ALP) 40% (p < 0.005), 44% (p < 0.001), 49% (p < 0.1), and 45% (p < 0.02) at 9, 11, 13, and 15 days, respectively. Immunoexpression of osteopontin revealed that in cultures treated with anti-CP antibody, the positive number of cementoblastoma cells was reduced by 87, 83, 69, and 52% at 5, 7, 9, and 11 days, respectively. Bone sialoprotein immunoexpression showed a decrease in positive cells of 82, 51, 60, 80, 83, and 87% at 5, 7, 9, 11, 13, and 15 days, respectively, as compared to controls. The Ca/P ratio of the mineral-like tissue deposited in vitro by cementoblastoma cells revealed that control cultures had a Ca/P ratio of 1.45 and 1.61 at 5 and 15 days, whereas experimental cultures revealed a Ca/P ratio of 0.50 and 0.79 at 5 and 15 days, respectively. Electron diffraction patterns showed inner double rings representing D-spacing that were consistent with those of hydroxyapatite in both control and experimental cultures. Examination of the crystallinity with high resolution transmission electron microscopy showed homogeneous and preferential spatial arrangement of hydroxyapatite crystallites in control and experimental cultures at 15 days. Atomic force microscopy images of control cultures at 5 and 15 days revealed small granular particles and grain agglomeration that favored the formation of crystalline plaques with a lamellar-like pattern of the mineral-like tissue. Experimental cultures at 5 and 15 days showed tiny and homogeneous granular morphology. The agglomerates maintained spherical morphology without organization of needle-like crystals to form plaque-like structures. Based on these findings, it is hypothesized that cementoblastoma-derived protein may be associated to crystal growth, compositional and morphological features during the mineralization process of cementum in vitro.

Characterization of recombinant human cementum protein 1 (hrCEMP1): Primary role in biomineralization

Cementum protein 1 (CEMP1) has been recently cloned, and in vitro experiments have shown functions as regulator of cementoblast behavior and inducer of differentiation of non-osteogenic cells toward a cementoblastic/osteoblastic phenotype. In this study, we have produced a full-length human recombinant CEMP1 protein in a human gingival fibroblast cell line. The purified protein (hrCEMP1) has a M(r) 50,000. Characterization of hrCEMP1 indicates that its secondary structure is mainly composed of beta-sheet (55%), where random coil and alpha helix conformations correspond to 35% and 10%, respectively. It was found that hrCEMP1 is N-glycosylated, phosphorylated and possesses strong affinity for hydroxyapatite. Even more important, our results show that hrCEMP1 plays a role during the biomineralization process by promoting octacalcium phosphate (OCP) crystal nucleation. These features make CEMP1 a very good candidate for biotechnological applications in order to achieve cementum and/or bone regeneration.

Cementum proteins: role in cementogenesis, biomineralization, periodontium formation and regeneration

Destruction of the periodontium is normally associated with periodontal disease, although many other factors, such as trauma, aging, infections, orthodontic tooth movement and systemic and genetic diseases, can contribute to this process. Strategies (such as guided tissue regeneration) have been developed to guide and control regeneration using bioresorbable membranes and bone grafts. Although effective to a certain point, these strategies have the problem that they are not predictable and do not completely restore the architecture of the original periodontium. To achieve complete repair and regeneration it is necessary to recapitulate the developmental process with complete formation of cementum, bone and periodontal ligament fibers. Detailed knowledge of the biology of cementum is key for understanding how the periodontium functions, identifying pathological issues and for developing successful therapies for repair and regeneration of damaged periodontal tissue. It is the purpose of this review to focus on the role of cementum and its specific components in the formation, repair and regeneration of the periodontium. As cementum is a matrix rich in growth factors that could influence the activities of various periodontal cell types, this review will examine the characteristics of cementum, its composition and the role of cementum components, especially the cementum protein-1, during the process of cementogenesis, and their potential usefulness for regeneration of the periodontal structures in a predictable therapeutic manner.