SIRT6 overexpression inhibits cementogenesis by suppressing glucose transporter 1.

Cementum, which shares common features with bone in terms of biochemical composition, is important for the homeostasis of periodontium during periodontitis and orthodontic treatment. Sirtuin 6 (SIRT6), as a member of the sirtuin family, plays key roles in the osteogenic differentiation of bone marrow mesenchymal stem cells. However, the involvement of SIRT6 in cementoblast differentiation and mineralization and the underlying mechanisms remain unknown. In this study, we observed that the expression of SIRT6 increased during cementoblast differentiation initially. Analysis of the gain- and loss-of-function indicated that overexpressing SIRT6 in OCCM-30 cells suppresses cementoblast differentiation and mineralization and downregulating SIRT6 promotes cementogenesis. GLUT1, a glucose transporter necessary in cementogenesis, was inhibited by SIRT6. Overexpressing GLUT1 in SIRT6-overexpressed OCCM-30 cells rescued the inhibitory effect of SIRT6 on cementoblast differentiation and mineralization. Moreover, AMPK was activated after overexpressing SIRT6 and inhibited cementoblast differentiation and mineralization. Downregulating the expression of SIRT6 inhibited AMPK activity. Meanwhile, GLUT1 overexpression significantly decreased AMPK activity. Overall, on one hand, SIRT6 inhibited cementoblast differentiation and mineralization by suppressing GLUT1. On the other hand, SIRT6 inhibited cementoblast differentiation and mineralization by activating the AMPK pathway. GLUT1 overexpression also rescued the increased AMPK pathway activated by SIRT6.

Intermittent parathyroid hormone (PTH) promotes cementogenesis and alleviates the catabolic effects of mechanical strain in cementoblasts.

BACKGROUND: External root resorption, commonly starting from cementum, is a severe side effect of orthodontic treatment. In this pathological process and repairing course followed, cementoblasts play a significant role. Previous studies implicated that parathyroid hormone (PTH) could act on committed osteoblast precursors to promote differentiation, and inhibit apoptosis. But little was known about the role of PTH in cementoblasts. The purpose of this study was to investigate the effects of intermittent PTH on cementoblasts and its influence after mechanical strain treatment. RESULTS: Higher levels of cementogenesis- and differentiation-related biomarkers (bone sialoprotein (BSP), osteocalcin (OCN), Collagen type I (COL1) and Osterix (Osx)) were shown in 1-3 cycles of intermittent PTH treated groups than the control group. Additionally, intermittent PTH increased alkaline phosphatase (ALP) activity and mineralized nodules formation, as measured by ALP staining, quantitative ALP assay, Alizarin red S staining and quantitative calcium assay. The morphology of OCCM-30 cells changed after mechanical strain exertion. Expression of BSP, ALP, OCN, osteopontin (OPN) and Osx was restrained after 18 h mechanical strain. Furthermore, intermittent PTH significantly increased the expression of cementogenesis- and differentiation-related biomarkers in mechanical strain treated OCCM-30 cells. CONCLUSIONS: Taken together, these data suggested that intermittent PTH promoted cementum formation through activating cementogenesis- and differentiation-related biomarkers, and attenuated the catabolic effects of mechanical strain in immortalized cementoblasts OCCM-30.

Metabotropic glutamate receptor 1 promotes cementoblast proliferation via MAP kinase signaling pathways.

PURPOSE/AIM: Glutamate is one of the signaling molecules responsible for transmission in the central nervous system. Periodontal ligament (PDL) cells were recently reported to express metabotropic glutamate receptors (mGluRs). However, the functions of mGluR signaling in PDL cells or PDL-related cells remain largely unknown. The aim of this study was to investigate the expression and function of mGluRs in PDL-related cells. MATERIALS AND METHODS: OCCM-30 cells, immortalized murine cementoblasts, were stimulated with l-glutamate or mGluRs antagonists. The cells' proliferative response was evaluated using a colorimetric assay and gene expression was assessed using real-time polymerase chain reaction. The nuclear translocation of cyclin D1 was evaluated by immunohistochemistry. RESULTS: l-Glutamate promoted the proliferation of OCCM-30 cells, which expressed mGluR1, but not mGluR5. Dihydroxyphenylglycine (DHPG), an agonist of group I mGluRs (mGluR1 and mGluR5), also promoted cell proliferation, and this was inhibited by LY456236, an mGluR1 antagonist. DHPG increased the expression of cyclin D1, a key regulator of cell proliferation, and its nuclear translocation. DHPG also increased the expression of Bcl2A1, an antiapoptotic oncogene and simultaneously reduced the expression of Bax, a pro-apoptotic marker. Furthermore, the DHPG-induced proliferation of OCCM-30 cells was reduced by pretreatment with SB203580, SP600125, and PD98059, inhibitors of p38, JNK, and ERK1/2, respectively. CONCLUSIONS: These findings indicate that activation of mGluR1 expressed by OCCM-30 cells induces cell proliferation in a manner that is dependent on mitogen-activated protein kinase pathways and that cyclin D1 and Bcl2A1/Bax may be involved. Our results provide useful information for elucidating the mechanisms underlying cementum homeostasis and regeneration.

Effects of TNF-α on Cementoblast Differentiation, Mineralization, and Apoptosis.

Tumor necrosis factor-α (TNF-α) is involved in various inflammatory processes, including periodontitis. Although the influences of TNF-α on periodontal ligament fibroblasts and osteoblasts have been widely documented, its effects on cementoblasts, the cells responsible for cementum production, remain largely unknown. In this study, we found that TNF-α suppressed the mineralization ability of cementoblasts by inhibiting differentiation and inducing apoptosis. Various signaling pathways, such as p53, PP2AC, p38, Erk1/2, JNK, PI3K-Akt, and NF-κB, were activated during this process. The use of a specific inhibitor and siRNA transfection confirmed that the effects of TNF-α on differentiation and apoptosis in cementoblasts were partially abrogated by inhibiting p53 activity. By contrast, the effects of TNF-α were even exacerbated by the inhibition of the p38, Erk1/2, JNK, PI3K-Akt, and NF-κB pathways. Moreover, p53 activity was further enhanced by blocking the p38, Erk1/2, JNK, and PI3K-Akt signaling pathways. Taken together, these results suggested that the differentiation inhibition and apoptosis in cementoblasts induced by TNF-α were partially dependent on p53 activity. The p38, Erk1/2, JNK, PI3K-Akt, and NF-κB pathways were also activated but acted as balancing players to limit rather than conduct the negative effects of TNF-α. These balancing effects were dependent, or at least partially dependent, on p53, except for the NF-κB pathway.

TNF-α stimulates MMP-3 production via PGE2 signalling through the NF-kB and p38 MAPK pathway in a murine cementoblast cell line.

BACKGROUND: Cementoblasts are considered to play an important role in the homeostasis of periodontal tissues under both physiologic and pathologic conditions. Matrix metalloproteinases (MMPs) is the key family of enzymes participating in extracellular matrix remodelling. In the present study, the effects and regulatory mechanisms of tumour necrosis factor (TNF)-α on the expression of MMPs and their inhibitors (tissue inhibitor of metalloproteinases; TIMPs) were investigated. MATERIALS AND METHODS: OCCM-30, an immortalised murine cementoblast cell line, was stimulated with TNF-α at 1 and 10ng/ml for 24h. The expression of Mmp-2, Mmp-3, Mmp-13, Mmp-14, Timp-1, and Timp-2 as well as PGE2 was determined. Inhibitors of MAPKs, PI3K/Akt, NF-kB and Cox-2 were employed to reveal possible TNF-α induced regulatory signalling pathway(s). The mRNA and protein expression were analysed by (semi)quantitative real-time PCR and enzyme-linked immunosorbent assay (ELISA), respectively. RESULTS: TNF-α dose-dependently stimulated MMP-3 expression by cementoblasts. This was found for mRNA as well as protein expression. No significant differences were found in the mRNA expression of Mmp-2, Mmp-13, Mmp-14, Timp-1, and Timp-2 upon TNF-α stimulation. The level of PGE2, however, was significantly increased along with MMP-3. Treatment with a selective Cox-2 inhibitor resulted in partial suppression of TNF-α-induced Mmp-3 mRNA expression. Addition of PGE2 enhanced Mmp-3 mRNA in a dose dependent manner, suggesting an inductive effect of TNF-α partly via PGE2. The up-regulation of Mmp-3 by TNF-α was completely suppressed by a combination of NF-kB and p38 MAPK inhibitors, while partial suppression was found with each inhibitor. The effect of PGE2 on Mmp-3 expression was abolished by treating cells with an NF-kB inhibitor; a p38 MAPK inhibitor had only a small effect. CONCLUSIONS: The present study indicates that cementoblasts respond to TNF-α by increasing MMP-3 production partially via PGE2 and signalling through the NF-kB and p38 MAPK pathway. MMP-3 may participate in periodontal tissue degradation/remodelling.

Strontium promotes cementoblasts differentiation through inhibiting sclerostin expression in vitro.

Cementogenesis, performed by cementoblasts, is important for the repair of root resorption caused by orthodontic treatment. Based on recent studies, strontium has been applied for osteoporosis treatment due to its positive effect on osteoblasts. Although promising, the effect of strontium on cementoblasts is still unclear. So the aim of this research was to clarify and investigate the effect of strontium on cementogenesis via employing cementoblasts as model. A series of experiments including MTT, alkaline phosphatase activity, gene analysis, alizarin red staining, and western blot were carried out to evaluate the proliferation and differentiation of cementoblasts. In addition, expression of sclerostin was checked to analyze the possible mechanism. Our results show that strontium inhibits the proliferation of cementoblasts with a dose dependent manner; however, it can promote the differentiation of cementoblasts via downregulating sclerostin expression. Taking together, strontium may facilitate cementogenesis and benefit the treatment of root resorption at a low dose.

Age-related adaptation of bone-PDL-tooth complex: Rattus-Norvegicus as a model system.

Functional loads on an organ induce tissue adaptations by converting mechanical energy into chemical energy at a cell-level. The transducing capacity of cells alters physico-chemical properties of tissues, developing a positive feedback commonly recognized as the form-function relationship. In this study, organ and tissue adaptations were mapped in the bone-tooth complex by identifying and correlating biomolecular expressions to physico-chemical properties in rats from 1.5 to 15 months. However, future research using hard and soft chow over relevant age groups would decouple the function related effects from aging affects. Progressive curvature in the distal root with increased root resorption was observed using micro X-ray computed tomography. Resorption was correlated to the increased activity of multinucleated osteoclasts on the distal side of the molars until 6 months using tartrate resistant acid phosphatase (TRAP). Interestingly, mononucleated TRAP positive cells within PDL vasculature were observed in older rats. Higher levels of glycosaminoglycans were identified at PDL-bone and PDL-cementum entheses using alcian blue stain. Decreasing biochemical gradients from coronal to apical zones, specifically biomolecules that can induce osteogenic (biglycan) and fibrogenic (fibromodulin, decorin) phenotypes, and PDL-specific negative regulator of mineralization (asporin) were observed using immunohistochemistry. Heterogeneous distribution of Ca and P in alveolar bone, and relatively lower contents at the entheses, were observed using energy dispersive X-ray analysis. No correlation between age and microhardness of alveolar bone (0.7 ± 0.1 to 0.9 ± 0.2 GPa) and cementum (0.6 ± 0.1 to 0.8 ± 0.3 GPa) was observed using a microindenter. However, hardness of cementum and alveolar bone at any given age were significantly different (P<0.05). These observations should be taken into account as baseline parameters, during development (1.5 to 4 months), growth (4 to 10 months), followed by a senescent phase (10 to 15 months), from which deviations due to experimentally induced perturbations can be effectively investigated.

Correction of hypophosphatasia-associated mineralization deficiencies in vitro by phosphate/pyrophosphate modulation in periodontal ligament cells.

BACKGROUND: Mutations in the liver/bone/kidney alkaline phosphatase (ALPL) gene in hypophosphatasia (HPP) reduce the function of tissue non-specific alkaline phosphatase (ALP), resulting in increased pyrophosphate (PP(i)) and a severe deficiency in acellular cementum. We hypothesize that exogenous phosphate (P(i)) would rescue the in vitro mineralization capacity of periodontal ligament (PDL) cells harvested from HPP-diagnosed patients, by correcting the P(i)/PP(i) ratio and modulating expression of genes involved with P(i)/PP(i) metabolism. METHODS: Ex vivo and in vitro analyses were used to identify mechanisms involved in HPP-associated PDL/tooth root deficiencies. Constitutive expression of PP(i)-associated genes was contrasted in PDL versus pulp tissues obtained from healthy individuals. Primary PDL cell cultures from patients with HPP (monozygotic twin males) were established to assay ALP activity, in vitro mineralization, and gene expression. Exogenous P(i) was provided to correct the P(i)/PP(i) ratio. RESULTS: PDL tissues obtained from healthy individuals featured higher basal expression of key PP(i) regulators, genes ALPL, progressive ankylosis protein (ANKH), and ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), versus paired pulp tissues. A novel ALPL mutation was identified in the twin patients with HPP enrolled in this study. Compared to controls, HPP-PDL cells exhibited significantly reduced ALP and mineralizing capacity, which were rescued by addition of 1 mM P(i). Dysregulated expression of PP(i) regulatory genes ALPL, ANKH, and ENPP1 was also corrected by adding P(i), although other matrix markers evaluated in our study remained downregulated. CONCLUSION: These findings underscore the importance of controlling the P(i)/PP(i) ratio toward development of a functional periodontal apparatus and support P(i)/PP(i) imbalance as the etiology of HPP-associated cementum defects.

Isolation of protein-tyrosine phosphatase-like member-a variant from cementum.

Cementum has been shown to contain unique polypeptides that participate in cell recruitment and differentiation during cementum formation. We report the isolation of a cDNA variant for protein-tyrosine phosphatase-like (proline instead of catalytic arginine) member-a (PTPLA) from cementum. A cementifying fibroma-derived λ-ZAP expression library was screened by panning with a monoclonal antibody to cementum attachment protein (CAP), and 1435 bp cDNA (gb AC093525.3) was isolated. This cDNA encodes a 140-amino-acid polypeptide, and its N-terminal 125 amino acids are identical to those of PTPLA. This isoform, designated as PTPLA-CAP, results from a read-through of the PTPLA exon 2 splice donor site, truncating after the second putative transmembrane domain. It contains 15 amino acids encoded within the intron between PTPLA exons 2 and 3, which replace the active site for PTPLA phosphatase activity. The recombinant protein, rhPTPLA-CAP, has Mr 19 kDa and cross-reacts with anti-CAP antibody. Anti-rhPTPLA-CAP antibody immunostained cementum cells, cementum, heart, and liver. Quantitative RT-PCR showed that PTPLA was expressed in all periodontal cells; however, PTPLA-CAP expression was limited to cementum cells. The rhPTPLA-CAP promoted gingival fibroblast attachment. We conclude that PTPLA-CAP is a splice variant of PTPLA, and that, in the periodontium, cementum and cementum cells express this variant.

Expression of alkaline phosphatase in immortalized murine cementoblasts in response to compression-force.

OBJECTIVE: To determine the effect of compression-force on the expression of alkaline phosphatase (ALP), and ALP activity in cementoblasts. METHODS: We performed this study in the State Key Laboratory of Oral Diseases, West China Stomatology Hospital, Sichuan University, Chengdu, Sichuan, China from October to December 2010. We exposed murine immortalized cementoblasts (OCCM-30) to 2000-ustrain compression-force at a frequency of 0.5 Hz for 1, 3, 6, 12, and 24 hours. We assayed the cellular ALP activity after the treatments. We used real-time polymerase chain reaction (RT-PCR) and western blot to examine the gene and protein expression of ALP in the OCCM-30 cells at each time point. RESULTS: Two-thousand ustrain compressive force significantly up-regulated the mRNA expression of ALP in OCCM-30 cells, which reached a peak at 12 hours loading, and the protein expression change of ALP in response to compression-force was consistent with the variation of gene level. We also noted marked enhancement of ALP activity in OCCM-30 cells during the application of mechanical stress. CONCLUSION: The compression-force increased the expression of ALP in OCCM-30 cells, suggesting that mechanical stimulation may affect the cellular function of cementoblasts by regulating ALP expression, which may participate in cementum metabolism during orthodontic tooth movement.

The MMP activity in developing rat molar roots and incisors demonstrated by in situ zymography.

Matrix metalloproteinases (MMPs) have been expressed during root development and periodontal tissue formation, whereas it is not known if these MMP molecules are enzymatically active to degrade the extracellular matrices (ECMs). The present study was designed to investigate the gelatinolytic and collagenolytic activity in rat molar root and incisor development. Three-week old rat mandibles were frozen and cut without fixation or decalcification and processed for in situ zymography using substrates gelatin and collagen. The enzymatic activity was assessed according to the intensity of fluorescence due to the lysis of the substrates. Odontoblasts, predentin, cementum, bone and the enamel matrix showed the high activity. The present study demonstrated MMP activity in calcified tissues using in situ zymography for the first time and the possible involvement of the MMP activity in molar root and incisor development and periodontal tissue formation.

Brain-derived neurotrophic factor enhances periodontal tissue regeneration.

To address whether brain-derived neurotrophic factor (BDNF) could be involved in periodontal tissue regeneration, we examined the effects of BDNF on proliferation and the expression of bone (cementum)- related proteins (osteopontin, bone morphogenetic protein [BMP]-2, type I collagen, alkaline phosphatase [ALPase], and osteocalcin) in cultures of human periodontal ligament (HPL) cells, which are thought to be prerequisite for periodontal tissue regeneration, and on proliferation and angiogenesis in human endothelial cells. Furthermore, we examined the effect of BDNF on the regeneration of periodontal tissues in experimentally induced periodontal defects in dogs. BDNF elevated the expression of ALPase and osteocalcin mRNAs and increased the synthesis of osteopontin, BMP-2, and type I collagen DNA in HPL cells. BDNF stimulated mRNA expression of vascular endothelial growth factor-B and tenascin-X, and proliferation and angiogenesis in human endothelial cells. In vivo studies showed that BDNF stimulated the formation of new alveolar bone cementum and connective new fibers, which were inserted into the newly formed cementum and bone. BDNF also stimulated blood capillary formation. These findings suggest that the regulation of functioning of periodontal ligament cells and endothelial cells by BDNF results in the promotion of periodontal tissue regeneration.

[Isolation, culture and identification of bovine cementum-derived cells].

OBJECTIVE: To isolate and culture bovine cementum-derived cells and investigate their biological properties. METHODS: Culture of primary bovine cementoblast (CB) were established from new-born bovine teeth. Cementum was manually dissected, fragmented, and digested twice with collagenase. Following a thorough wash to remove liberated cells, the remaining cementum fragments were plated and cultured in RPMI1640 containing 10% FCS and 1.5% Hepes. The cells in culture were identified using immunocytochemistry by expression of cementum attachment protein (CAP), osteocalcin (OCN) and alkaline phosphatase (ALP). ALP activity was measured by modified Gomori staining. RESULTS: (1) The cells in culture possessed the typical morphology of CB and there were no obvious change of the cell morphology up to 5th passage; (2) Cells in culture exhibited alkaline phosphatase activity and were also positive to CAP, OPN and ALP immunohistochemistry staining. CONCLUSION: The cells isolated and cultured in this experiment possess the properties of CB. This enables us to further observe its biological characteristics during cementogenesis and periodontal regeneration.

Bone morphogenetic protein 2 induces dental follicle cells to differentiate toward a cementoblast/osteoblast phenotype.

When triggered appropriately, dental follicle cells are considered to be able to differentiate toward a cementoblast/osteoblast phenotype. However, factors and mechanisms regulating follicle cell differentiation remain undefined. This study focused on determining the ability of bone morphogenetic protein (BMP) 2 to promote the differentiation of follicle cells and periodontal ligament (PDL) cells along a cementoblast/ osteoblast pathway. Follicle cells and PDL cells were isolated from the first molar region of CD-1 mice and immortalized with SV40. Both cell types expressed BMP-4 and BMP receptors (BMPR) IA and II, but only follicle cells expressed BMP-2 mRNA. Cells were exposed to recombinant human BMP (rhBMP)-2 (0-100 ng/ml) and Northern blots were used to determine the expression of mineral-associated markers. BMP-2, in a dose- and time-dependent manner, induced cementoblast/osteoblast differentiation of follicle cells, as reflected by enhanced core binding factor alpha (Cbfal), bone sialoprotein (BSP), and osteocalcin (OCN) mRNA expression and enhanced mineral formation. U0126, a specific inhibitor of MEK-1/2 members of the MAPK family, abolished BMP-2-mediated expression of BSP and OCN. In contrast, exposure of PDL cells to BMP-2 resulted in modest expression of OCN and minimal promotion of mineralization. These results suggest that BMP-2 triggers follicle cells to differentiate toward a cementoblast/osteoblast phenotype and that the MAPK pathway is involved.

On the origin of intrinsic matrix of acellular extrinsic fiber cementum: studies on growing cementum pearls of normal and bisphosphonate-affected guinea pig molars.

Cementum pearls (CPs) belong to a type of acellular extrinsic fiber cementum (AEFC) that form on the maturing enamel of guinea pig molars. This study aimed to elucidate the forming process of intrinsic matrix of AEFC using the CPs of normal and bisphosphonate-affected guinea pig molars as experimental models. A group of guinea pigs were subjected to continuous administration of 1-hydroxyethylidene-1,1-bisphosphonate (HEBP) for 2 wk to inhibit mineralization of growing CPs. Fenestration of the enamel organ and migration of periodontal cells on to the exposed surface of maturing enamel appeared to be unaffected by HEBP, whereas de novo formation as well as growth of pre-existing CPs did not proceed under the same conditions. Immunoreactions for osteopontin were located exclusively on the mineralized matrix of preformed CPs, implying the absence of additional deposition or accumulation of putative intrinsic cementum matrix on the affected CPs, where the propagation of mineral phase had been arrested. In both normal and HEBP-treated groups, distinct enzymatic reactions for alkaline phosphatase appeared on the cells of the periodontal ligament associated closely with the sites of CP formation, and along the mineralization front of CPs. These observations suggest that the mineralization process per se plays a central role in the deposition of AEFC matrix and that alkaline phosphatase of periodontal cells penetrating through the enamel organ to the maturing enamel surface plays a key role in the mineralization process of CPs.

Role of MAP kinases p42erk-2/p44erk-1 in cementum-derived attachment-protein-mediated cell attachment.

Cementum-derived attachment protein (CAP) is a collagenous protein which promotes the attachment and spreading of periodontal cell types. We examined the role of the MEK/MAPK pathway in CAP-mediated fibroblast attachment. Human gingival fibroblasts were labeled with 35S-methionine, and the effect of MAP kinase pathway inhibitor PD98059 on attachment and spreading on CAP-coated dishes was examined. Effect on cell proliferation on CAP-coated plates was determined by [3H]-thymidine uptake. Attachment of human gingival fibroblasts to CAP-containing surfaces activated extracellular-signal-regulated kinases (ERK) ERK-2 and ERK-1. In the absence of serum, the ERKs were activated 15 min after attachment, reaching peak levels after 3 hours, and the activity was sustained for at least 12 hours. The enzyme levels were inhibited in cells treated with PD98059. The PD98059 did not significantly affect the kinetics of fibroblast attachment or the number of cells attaching to CAP-coated plates. However, cell spreading was retarded. DNA synthesis as indicated by [3H]-thymidine uptake was not significantly affected. In contrast to PD98059, attachment, spreading, and [3H]-thymidine uptake were inhibited by the protein tyrosine kinase inhibitor genestein. Our results indicate that the MEK/MAPK pathway participates in CAP-mediated fibroblast spreading, but cell attachment and proliferation do not appear to require ERK-2.

In situ hybridization for matrix metalloproteinase-1 and cathepsin K in rat root-resorbing tissue induced by tooth movement.

The movement of teeth during orthodontic treatment occasionally induces undesirable root resorption. Although high collagenolytic activity has been detected in resorbing tissue of deciduous teeth, the cellular origin of collagenolytic enzymes in root-resorbing tissue caused by tooth movement has not been identified. Here, rats were subject to 7 days of experimental tooth movement to induce root resorption. In situ hybridization with digoxigenin-labelled RNA probes was performed on sections of the maxillary bone to detect the mRNAs that encode matrix metalloproteinase-1 (MMP-1) and cathepsin K in root-resorbing tissue. MMP-1 mRNA was detected in fibroblastic cells, cementoblasts and osteoblasts, but not in odontoclasts nor osteoclasts. Moreover, MMP-1 mRNA was highly expressed in some cementocytes located near odontoclasts and in many osteocytes. In contrast, cathepsin K mRNA was expressed only in odontoclasts and osteoclasts. These results suggest that MMP-1 and cathepsin K are important in root resorption during tooth movement in a mode similar to bone resorption.

Should cementoblasts express alkaline phosphatase activity? Preliminary study of rat cementoblasts in vitro.

BACKGROUND: A well-characterized cell culture model for cementoblasts is essential to understand the mechanisms of periodontal ligament (PDL) reattachment and regeneration. Whether cementoblasts express alkaline phosphatase (ALP) activity in vivo and in vitro remains to be determined. METHODS: Using a 2-step method of enzyme digestion/explant culture, osteoblasts, gingival/PDL fibroblasts, and cementoblasts were obtained from alveolar bone, gingiva, and the root surface of rat first molars and cultured. Initially, bone sialoprotein (BSP) was immunolocalized on tissue sections of periodontium and on cultured cells to distinguish mineral-forming cells from fibroblasts. Proteins were extracted from these cells to assess ALP activity by using an enzyme assay. RNA was extracted from the same cell source to detect ALP mRNA by reverse transcriptase polymerase chain reaction (RT-PCR). RESULTS: Cultured PDL/gingival fibroblasts were spindle shaped. Osteoblasts were irregularly shaped, and cell clusters/nodules were observed as they approached confluence. The cementoblasts manifested a polygonal shape and had two morphotypes: osteoblast-like and cuboidal or stratified. BSP was localized within the mineralized tissues and in osteoblasts and cementoblasts in culture and in tissue sections. The highest level of ALP activity was found in osteoblasts, a moderate level in PDL fibroblasts, and the lowest level in gingival fibroblasts. The cementoblasts lacked ALP activity, and this was reflected by a very weak signal (or no signal at all) for ALP mRNA in the cementoblasts. CONCLUSIONS: These studies indicate that cells consistent with a cementoblast-like phenotype may be successfully cultured, and that they lack ALP activity.

Root development in mice lacking functional tissue non-specific alkaline phosphatase gene: inhibition of acellular cementum formation.

Tissue non-specific alkaline phosphatase (TNAP) is richly present in developing teeth including the cells of the periodontal ligament. Here, we investigated tooth and root development in mice lacking the TNAP gene. Heterozygous mutants were obtained from The Jackson Laboratory, Animal Resources (Bar Harbor, ME, USA) and bred. TNAP-deficient mice and their littermates were killed from 6 to 25 days after birth and their molar blocks processed for light and electron microscopy. It was observed that the eruption of the incisors into the oral cavity was delayed for 2 to 3 days. Also, the onset of mineralization of the mantle dentin in the roots of the developing molars was delayed for 2 to 3 days. Yet, dentin and enamel formation in the homozygous mutants showed a more or less normal pattern, with the exception of localized enamel hypoplasias. The most conspicuous finding was the defective formation of acellular cementum along the molar roots. Instead of a continuous layer, the cementum was deposited as very thin and irregularly shaped patches around the bases of the periodontal ligament fibers. Sharpey's fibers were short and poorly developed. In contrast, the development of the alveolar bone, the periodontal ligament, and the cellular cementum was seemingly unaffected. It is concluded that TNAP represents an essential factor in mantle dentin mineralization and in the formation of acellular cementum.

Alkaline phosphatase activity in human periodontal ligament: age effect and relation to cementum growth rate.

Recently, a relationship was demonstrated between the thickness of the cementum layer in rat molars and the activity of alkaline phosphatase (ALP) in the adjoining periodontal ligament (PDL). It was the aim of the present study to investigate whether such a relationship also exists in the periodontium of man. Healthy deciduous and permanent teeth free from periodontitis were obtained from 74 patients, varying in age from 3 to 78 yr, and their PDL dissected from the middle one-third of the roots. ALP activity was measured in PDL extracts and expressed per hydroxyproline content. It was shown that ALP activity was relatively high in children. After puberty its concentration decreased to level off at about half the concentration found in the younger age groups. The activity of the enzyme in the PDL correlated positively with the yearly cementum thickness increment as calculated from data published previously.