Tooth acellular extrinsic fibre cementum incremental lines in humans are formed by parallel branched Sharpey's fibres and not by its mineral phase.

In humans, the growth pattern of the acellular extrinsic fibre cementum (AEFC) has been useful to estimate the age-at-death. However, the structural organization behind such a pattern remains poorly understood. In this study tooth cementum from seven individuals from a Mexican modern skeletal series were analyzed with the aim of unveiling the AEFC collagenous and mineral structure using multimodal imaging approaches. The organization of collagen fibres was first determined using: light microscopy, transmission electron microscopy (TEM), electron tomography, and plasma FIB scanning electron microscopy (PFIB-SEM) tomography. The mineral properties were then investigated using: synchrotron small-angle X-ray scattering (SAXS) for T-parameter (correlation length between mineral particles); synchrotron X-ray diffraction (XRD) for L-parameter (mineral crystalline domain size estimation), alignment parameter (crystals preferred orientation) and lattice parameters a and c; as well as synchrotron X-ray fluorescence for spatial distribution of calcium, phosphorus and zinc. Results show that Sharpey's fibres branched out fibres that cover and uncover other collagen bundles forming aligned arched structures that are joined by these same fibres but in a parallel fashion. The parallel fibres are not set as a continuum on the same plane and when they are superimposed project the AEFC incremental lines due to the collagen birefringence. The orientation of the apatite crystallites is subject to the arrangement of the collagen fibres, and the obtained parameter values along with the elemental distribution maps, revealed this mineral tissue as relatively homogeneous. Therefore, no intrinsic characteristics of the mineral phase could be associated with the alternating AEFC incremental pattern.

Deficiency of Trps1 in Cementoblasts Impairs Cementogenesis and Tooth Root Formation.

Cementum is the least studied of all mineralized tissues and little is known about mechanisms regulating its formation. Therefore, the goal of this study was to provide new insights into the transcriptional regulation of cementum formation by determining the consequences of the deficiency of the Trps1 transcription factor in cementoblasts. We used Trps1Col1a1 cKO (2.3Co1a1-CreERT2;Trps1fl/fl) mice, in which Trps1 is deleted in cementoblasts. Micro-computed tomography analyses of molars of 4-week-old males and females demonstrated significantly shorter roots with thinner mineralized tissues (root dentin and cementum) in Trps1Col1a1 cKO compared to WT mice. Semi-quantitative histological analyses revealed a significantly reduced area of cellular cementum and localized deficiencies of acellular cementum in Trps1Col1a1 cKO mice. Immunohistochemical analyses revealed clustering of cementoblasts at the apex of roots, and intermittent absence of cementoblasts on Trps1Col1a1 cKO cementum surfaces. Fewer Osterix-positive cells adjacent to cellular cementum were also detected in Trps1Col1a1 cKO compared to WT mice. Decreased levels of tissue-nonspecific alkaline phosphatase (TNAP), an enzyme required for proper cementogenesis, were apparent in cementum, periodontal ligament, and alveolar bone of Trps1Col1a1 cKO. There were no apparent differences in levels of bone sialoprotein (Bsp) in cementum. Quantitative analyses of picrosirius red-stained periodontal ligament revealed shorter and disorganized collagen fibers in Trps1Col1a1 cKO mice demonstrating impaired periodontal structure. In conclusion, this study has identified Trps1 transcription factor as one of the important regulators of cellular and acellular cementum formation. Furthermore, this study suggests that Trps1 supports the function of cementoblasts by upregulating expression of the major proteins required for cementogenesis, such as Osterix and TNAP.

Glycometabolic reprogramming in cementoblasts: A vital target for enhancing cell mineralization.

Cementum, a constituent part of periodontal tissues, has important adaptive and reparative functions. It serves to attach the tooth to alveolar bone and acts as a barrier delimit epithelial growth and bacteria evasion. A dynamic and highly responsive cementum is essential for maintaining occlusal relationships and the integrity of the root surface. It is a thin layer of mineralized tissue mainly produced by cementoblasts. Cementoblasts are osteoblast-like cells essential for the restoration of periodontal tissues. In recent years, glucose metabolism has been found to be critical in bone remodeling and osteoblast differentiation. However, the glucose metabolism of cementoblasts remains incompletely understood. First, immunohistochemistry staining and in vivo tracing with 18 F-fluorodeoxyglucose (18 F-FDG) revealed significantly higher glucose metabolism in cementum formation. To test the bioenergetic pathways of cementoblast differentiation, we compared the bioenergetic profiles of mineralized and unmineralized cementoblasts. As a result, we observed a significant increase in the consumption of glucose and production of lactate, coupled with the higher expression of glycolysis-related genes. However, the expression of oxidative phosphorylation-related genes was downregulated. The verified results were consistent with the RNA sequencing results. Likewise, targeted energy metabolomics shows that the levels of glycolytic metabolites were significantly higher in the mineralized cementoblasts. Seahorse assays identified an increase in glycolytic flux and reduced oxygen consumption during cementoblast mineralization. Apart from that, we also found that lactate dehydrogenase A (LDHA), a key glycolysis enzyme, positively regulates the mineralization of cementoblasts. In summary, cementoblasts mainly utilized glycolysis rather than oxidative phosphorylation during the mineralization process.

Runx2 heterozygosity alters homeostasis of the periodontal complex.

BACKGROUND AND OBJECTIVE: Haploinsufficiency of Runx2 (Runx2+/- ) causes dental anomalies. However, little is known about the involvement of Runx2 in the maintenance of dentin, cementum, and the periodontal ligament (PDL) during adulthood. This study aimed to observe the effects of Runx2+/- on homeostasis of the periodontal complex. MATERIALS AND METHODS: A total of 14 three-month-old Runx2+/- mice and their wild-type littermates were examined using micro-computed tomography, histology, and immunohistochemistry. Phenotypic alterations in the dentin, cementum, and PDL were characterized and quantified. RESULTS: Haploinsufficiency of Runx2 caused cellular changes in the PDL space including reduction of cell proliferation and apoptosis, and irregular attachment of the collagen fibers in the PDL space into the cementum. Absence of continuous thickness of cementum was also observed in Runx2+/- mice. CONCLUSION: Runx2 is critical for cementum integrity and attachment of periodontal fibers. Because of its importance to cementum homeostasis, Runx2 is essential for homeostasis of periodontal complex.

Variability in human tooth cementum thickness reflecting functional processes.

OBJECTIVE: The aim of this study was to investigate the thickness of acellular extrinsic fibre cementum (AEFC) at four root positions of anterior and posterior teeth with special focus on functional aspects. Furthermore, the correlations between cementum thickness and chronological age and sex are investigated. BACKGROUND: While numerous studies confirm continuous cementum apposition with age, masticatory forces as well as physiological and orthodontically induced tooth movements also have the potential to affect tooth cementum thickness. MATERIALS AND METHODS: Undecalcified teeth were embedded in resin and transverse-sectioned in the cervical third of the root. Two sections per root were selected, and digital images at four positions were obtained (mesial, distal, oral, and vestibular) using light microscopy. The AEFC thickness of 99 teeth (anterior = 66, posterior = 33, male = 54, female = 45) were measured in both sections. The differences in mean values between root positions and the association of root position variation with tooth type, age, sex, and subject as well as the overall effects of age and sex were analysed using a mixed model. RESULTS: First incisors and canines showed the greatest mean AFEC thickness, in contrast to premolars which had the lowest values. Differences were found across the four root positions, with a pattern varying considerably between anterior and posterior teeth and between maxilla and mandible in the anterior teeth. An interaction between root position and subject pointed to the existence of an individual component in the variation of AEFC thickness across the four root positions. There was an age trend with an almost linear increase in cementum thickness of 1 µm per year. Overall, females tended to exhibit a significantly lesser AEFC thickness compared to males. CONCLUSIONS: Distinct differences in the pattern of thickness values across the four root positions in anterior and posterior teeth support the assumption that the AEFC is strongly affected by functional processes. In addition to sex-specific differences and age-related trends, the root position variation of AEFC thickness varies from individual to individual.

4-META/MMA-TBB resin containing nano hydroxyapatite induces the healing of periodontal tissue repair in perforations at the pulp chamber floor.

We aimed to evaluate the materials based on 4-methacryloxyethyl trimellitate anhydride/methyl methacrylate tri-n-butylborane (Super-bond [SB]) and nano hydroxyapatite (naHAp) for the repair of perforation at pulp chamber floor (PPF) in vitro and in vivo models. SB and naHAp were mixed in the mass ratio of 10% or 30% to produce naHAp/SB. Human periodontal ligament stem cells (HPDLSCs) were cultured on resin discs of SB or naHAp/SB to analyze the effects of naHAp/SB on cell adhesion, proliferation, and cementoblastic differentiation. A rat PPF model was treated with SB or naHAp/SB to examine the effects of naHAp/SB on the healing of defected cementum and periodontal ligament (PDL) at the site of PPF. HPDLSCs were spindle-shaped and adhered to all resin discs. Changing the resin from SB to naHAp/SB did not significantly alter cell proliferation. Both 10% and 30% naHAp/SB were more effective than SB in promoting cementoblastic differentiation of HPDLSCs. In the rat PPF model, 30% naHAp/SB was more effective than SB in promoting the formation Sharpey's fiber-like structures with expression of the PDL-related marker and cementum-like structures with expression of cementum-related markers. In conclusion, 30% naHAp/SB can be the new restorative material for PPF because it exhibited the abilities of adhering to dentin and healing of defected periodontal tissue.

High-frequency low-intensity semiconductor laser irradiation enhances osteogenic differentiation of human cementoblast lineage cells.

PURPOSE: Laser irradiation activates a range of cellular processes in the periodontal components and promotes tissue repair. However, its effect on osteogenic differentiation of human cementoblast lineage cells remains unclear. This study aimed to examine the effects of high-frequency semiconductor laser irradiation on the osteogenic differentiation of human cementoblast lineage (HCEM) cells. METHODS: HCEM cells were cultured to reach 80% confluence and irradiated with a gallium-aluminum-arsenide (Ga-Al-As) semiconductor laser with a pulse width of 200 ns and wavelength of 910 at a dose of 0-2.0 J/cm2. The outcomes were assessed by analyzing the mRNA levels of alkaline phosphatase (ALP), runt-related transcription factor 2 (RUNX2), and type I collagen (COLL1) using real-time polymerase chain reaction (PCR) analysis 24 h after laser irradiation. Cell mineralization was evaluated using ALP activity, calcium deposition, and Alizarin Red staining. RESULTS: The laser-irradiated HCEM cells showed significantly enhanced gene expression levels of ALP, RUNX2, and COLL1 as well as ALP activity and calcium concentration in the culture medium compared with the non-irradiated cells. In addition, enhanced calcification deposits were confirmed in the laser-irradiated group compared with the non-irradiated group at 21 and 28 days after the induction of osteogenic differentiation. CONCLUSION: High-frequency semiconductor laser irradiation enhances the osteogenic differentiation potential of cultured HCEM cells, underscoring its potential utility for periodontal tissue regeneration.

Cementum is key to periodontal tissue regeneration: A review on apatite microstructures for creation of novel cementum-based dental implants.

The cementum is the outermost layer of hard tissue covering the dentin within the root portion of the teeth. It is the only hard tissue with a specialized structure and function that forms a part of both the teeth and periodontal tissue. As such, cementum is believed to be critical for periodontal tissue regeneration. In this review, we discuss the function and histological structure of the cementum to promote crystal engineering with a biochemical approach in cementum regenerative medicine. We review the microstructure of enamel and bone while discussing the mechanism underlying apatite crystal formation to infer the morphology of cementum apatite crystals and their complex structure with collagen fibers. Finally, the limitations of the current dental implant treatments in clinical practice are explored from the perspective of periodontal tissue regeneration. We anticipate the possibility of advancing periodontal tissue regenerative medicine via cementum regeneration using a combination of material science and biochemical methods.

Inactivation of Sufu in cementoblasts accelerates external tooth root resorption.

Cementum has been empirically regarded as an antiresorptive barrier against tooth roots. However, little is known about the factors of homeostasis and resistant mechanisms of tooth roots against resorption. Here, we investigated cementum factors and their interaction against resorption using transgenic mice exhibiting external cervical root resorption (ECRR). Ectopically thickened cervical cementum caused by functional inactivation of ectonucleotide pyrophosphotase/phosphodiesterase 1 (Enpp1) was susceptible to ECRR with aging. In addition, the inactivation of the suppressor of fused (Sufu), a Hedgehog signaling inhibitor, in cementoblasts led to ECRR. Interestingly, concurrent inactivation of Sufu and Enpp1 in cementoblasts remarkably exacerbated ECRR with higher Rankl expression. Cellular and molecular analyses using cementoblasts and bone marrow-derived macrophages indicated that Dickkopf-related protein 1 (Dkk1) induced by the inactivation of Sufu in cementoblasts has roles in the acceleration of ECRR triggered by Enpp1 inactivation. Using compound mutant mice for concurrent Wntless and Enpp1 inactivation, this synergistic cooperation of Dkk1 and Npp1 for resorption found in double mutant Sufu and Enpp1 mice was confirmed by the reproduction of amplified ECRR. On the basis of these findings, we conclude that proper Npp1 function and sustained Wnt activity in the cervical cementum are essential for the homeostasis of tooth roots against resorption in a physiological state.

Autophagy mediates cementoblast mineralization under compression through periostin/ß-catenin axis.

Repair of orthodontic external root resorption and periodontal tissue dysfunction induced by mechanical force remains a clinical challenge. Cementoblasts are vital in cementum mineralization, a process important for restoring damaged cementum. Despite autophagy plays a role in mineralization under various environmental stimuli, the underlying mechanism of autophagy in mediating cementoblast mineralization remains unclear. Here we verified that murine cementoblasts exhibit compromised mineralization under compressive force. Autophagy was indispensable for cementoblast mineralization, and autophagic activation markedly reversed cementoblast mineralization and prevented cementum damage in mice during tooth movement. Subsequently, messenger RNA sequencing analyses identified periostin (Postn) as a mediator of autophagy and mineralization in cementoblasts. Cementoblast mineralization was significantly inhibited following the knockdown of Postn. Furthermore, Postn silencing suppressed Wnt signaling by modulating the stability of ß-catenin. Together our results highlight the role of autophagy in cementoblast mineralization via Postn/ß-catenin signaling under compressive force and may provide a new strategy for the remineralization of cementum and regeneration of periodontal tissue.

Life history in primate teeth is revealed by changes in major and minor element concentrations measured via field-emission SEM-EDS analysis.

Overcoming the non-specificity of histological accentuated growth lines in hard tissues is an ongoing challenge. Identifying season at death and reproductive events has profound implications for evolutionary, ecological and conservation studies. Dental cementum is a mineralized tissue with yearly periodicity that continues deposition from tooth formation until death, maintaining a record spanning almost the entire life of an individual. Recent work has successfully employed elemental analysis of calcified incremental tissues to detect changes in extrinsic conditions such as diet and climate and to identify two important life-history milestones: weaning and sexual maturity. Here, we employ field-emission scanning electron microscopy and energy-dispersive X-ray analysis to measure the relative concentrations of calcium, phosphorous, oxygen, magnesium and sodium in the cementum of 34 teeth from seven male and female rhesus macaques with known medical and life-history information. We find that changes in relative magnesium concentrations correspond with reproductive events in females and breastfeeding in infants. Additionally, we observe seasonal calcium patterns in 77.3% of the samples.

Solution NMR structure of cementum protein 1 derived peptide (CEMP1-p1) and its role in the mineralization process.

We report the characterization of the three-dimensional structure of the CEMP1-p1 peptide [MGTSSTDSQQAQHRRCSTSN: corresponding to residues 1-20 of the N-terminus of cementum protein 1 (CEMP1)]. This peptide imitates the capacity of CEMP1 to stimulate hydroxyapatite (HA) crystal nucleation and growth, and promotes the differentiation of periodontal ligament cells into a cementoblastic phenotype. Additionally, in experimental models of critical-sized calvarial defects in Wistar rats, CEMP1-p1 has shown osteogenic properties that enhanced the physiological deposition and maturation of newly formed bone. In this work, studies of CEMP1-p1 by circular dichroism (CD) and nuclear magnetic resonance (NMR) were performed in trifluoroethanol D2 (TFED2) and aqueous solution to determine the 3D structure of the peptide. Using the 3D model, experimental data from HA crystals formation and calcium fluorescence emission, we explain the biological mechanisms involved in CEMP1-p1 activity to promote calcium recruitment and its affinity to HA crystals. This information is valuable because it proposes, for the first time, a plausible molecular mechanism during the mineralization process, from a specific cementum protein-derived peptide.

Loss of ß-catenin causes cementum hypoplasia by hampering cementogenic differentiation of Axin2-expressing cells.

BACKGROUND AND OBJECTIVE: Although cementum plays an essential role in tooth attachment and adaptation to occlusal force, the regulatory mechanisms of cementogenesis remain largely unknown. We have previously reported that Axin2-expressing (Axin2+ ) mesenchymal cells in periodontal ligament (PDL) are the main cell source for cementum growth, and constitutive activation of Wnt/ß-catenin signaling in Axin2+ cells results in hypercementosis. Therefore, the aim of the present study was to further evaluate the effects of ß-catenin deletion in Axin2+ cells on cementogenesis. MATERIALS AND METHODS: We generated triple transgenic mice to conditionally delete ß-catenin in Axin2-lineage cells by crossing Axin2CreERT2/+ ; R26RtdTomato/+ mice with ß-cateninflox/flox mice. Multiple approaches, including X-ray analysis, micro-CT, histological stainings, and immunostaining assays, were used to analyze cementum phenotypes and molecular mechanisms. RESULTS: Our data revealed that loss of ß-catenin in Axin2+ cells led to a cementum hypoplasia phenotype characterized by a sharp reduction in the formation of both acellular and cellular cementum. Mechanistically, we found that conditional removal of ß-catenin in Axin2+ cells severely impaired the secretion of cementum matrix proteins, for example, bone sialoprotein (BSP), dentin matrix protein 1 (DMP1) and osteopontin (OPN), and markedly inhibited the differentiation of Axin2+ mesenchymal cells into osterix+ cementoblasts. CONCLUSIONS: Our findings confirm the vital role of Axin2+ mesenchymal PDL cells in cementum growth and demonstrate that Wnt/ß-catenin signaling shows a positive correlation with cementogenic differentiation of Axin2+ cells.

Cementum protein 1 gene-modified adipose-derived mesenchymal stem cell sheets enhance periodontal regeneration in osteoporosis rat.

BACKGROUND AND OBJECTIVES: Osteoporosis (OP) and periodontitis are both diseases with excessive bone resorption, and the number of patients who suffer from these diseases is expected to increase. OP has been identified as a risk factor that accelerates the pathological process of periodontitis. Achieving effective and safe periodontal regeneration in OP patients is a meaningful challenge. This study aimed to assess the efficacy and biosecurity of human cementum protein 1 (hCEMP1) gene-modified cell sheets for periodontal fenestration defect regeneration in an OP rat model. MATERIALS AND METHODS: Rat adipose-derived mesenchymal stem cells (rADSCs) were isolated from Sprague-Dawley rats. After primary culture, rADSCs were subjected to cell surface analysis and multi-differentiation assay. And rADSCs were transduced with hCEMP1 by lentiviral vector, and hCEMP1 gene-modified cell sheets were generated. The expression of hCEMP1 was evaluated by reverse transcription polymerase chain reaction and immunocytochemistry staining, and transduced cell proliferation was evaluated by Cell Counting Kit-8. The hCEMP1 gene-modified cell sheet structure was detected by histological analysis and scanning electron microscopy. Osteogenic and cementogenic-associated gene expression was evaluated by real-time quantitative polymerase chain reaction. In addition, an OP rat periodontal fenestration defect model was used to evaluate the regeneration effect of hCEMP1 gene-modified rADSC sheets. The efficacy was assessed with microcomputed tomography and histology, and the biosecurity of gene-modified cell sheets was evaluated by histological analysis of the spleen, liver, kidney and lung. RESULTS: The rADSCs showed a phenotype of mesenchymal stem cells and possessed multi-differentiation capacity. The gene and protein expression of hCEMP1 through lentiviral transduction was confirmed, and there was no significant effect on rADSC proliferation. Overexpression of hCEMP1 upregulated osteogenic and cementogenic-related genes such as runt-related transcription factor 2, bone morphogenetic protein 2, secreted phosphoprotein 1 and cementum attachment protein in the gene-modified cell sheets. The fenestration lesions in OP rats treated with hCEMP1 gene-modified cell sheets exhibited complete bone bridging, cementum and periodontal ligament formation. Furthermore, histological sections of the spleen, liver, kidney and lung showed no evident pathological damage. CONCLUSION: This pilot study demonstrates that hCEMP1 gene-modified rADSC sheets have a marked ability to enhance periodontal regeneration in OP rats. Thus, this approach may represent an effective and safe strategy for periodontal disease patients with OP.

Screening of functionalized collagen membranes with a porcine periodontal regeneration model.

OBJECTIVES: Current methods for periodontal regeneration do not promote collagen fiber insertions into new bone and cementum. We used a pig wound model to screen different functionalized collagen membranes in promoting periodontal reattachment to root surfaces. METHODS: Treatment groups included (1) control with no membranes, (2) collagen-coated membranes, (3) membranes with insulin-like growth factor-1 (IGF-1), (4) membranes with amelotin, or (5) membranes attached with calcium phosphate cement (CPC), or with CPC combined with IGF-1. Flap procedures were performed on mandibular and maxillary premolars of each pig. RESULTS: Histomorphometric, micro-CT, and clinical measurements obtained at 4 and 12 weeks after surgery showed cementum formation on denuded roots and reformation of alveolar bone, indicating that the pig model can model healing responses in periodontal regeneration. Calcium phosphate cement simplified procedures by eliminating the need for sutures and improved regeneration of alveolar bone (p < 0.05) compared with other treatments. There was a reduction (p < 0.05) of PD only for the IGF group. Large observed variances between treatment groups indicated that a priori power analyses should be conducted to optimize statistical analysis. CONCLUSIONS: Pigs can model discrete elements of periodontal healing using collagen-based, functionalized membranes. Screening indicates that membrane anchorage with calcium phosphate cements improve regeneration of alveolar bone.

Activation of Wnt signaling in Axin2+ cells leads to osteodentin formation and cementum overgrowth.

OBJECTIVES: In this study, we used the mouse incisor model to investigate the regulatory mechanisms of Wnt/ß-catenin signaling on Axin2+ cells in tooth development. MATERIALS AND METHODS: Axin2lacZ/+ reporter mice were used to define the expression pattern of Axin2 in mouse incisors. We traced the fate of Axin2+ cells from postnatal Day 21 (P21) to P56 using Axin2CreERT2/+ and R26RtdTomato/+ reporter mice. For constitutive activation of Wnt signaling, Axin2CreERT2/+ , ß-cateninflox(Ex3)/+ , and R26RtdTomato/+ (CA-ß-cat) mice were generated to investigate the gain of function (GOF) of ß-catenin in mouse incisor growth. RESULTS: The X-gal staining of Axin2lacZ/+ reporter mice and lineage tracing showed that Axin2 was widely expressed in dental mesenchyme of mouse incisors, and Axin2+ cells were essential cell sources for odontoblasts, pulp cells, and periodontal ligament cells. The constitutive activation of Wnt signaling in Axin2+ cells resulted in the formation of osteodentin featured with increased DMP1 and dispersed DSP expression and overgrowth of cementum. CONCLUSION: Wnt signaling plays a key role in the differentiation and maturation of Axin2+ cells in mouse incisors.

Glycosaminoglycans accelerate biomimetic collagen mineralization in a tissue-based in vitro model.

Mammalian teeth are attached to the jawbone through an exquisitely controlled mineralization process: unmineralized collagen fibers of the periodontal ligament anchor directly into the outer layer of adjoining mineralized tissues (cementum and bone). The sharp interface between mineralized and nonmineralized collagenous tissues makes this an excellent model to study the mechanisms by which extracellular matrix macromolecules control collagen mineralization. While acidic phosphoproteins, localized in the mineralized tissues, play key roles in control of mineralization, the role of glycosaminoglycans (GAGs) is less clear. As several proteoglycans are found only in the periodontal ligament, it has been hypothesized that these inhibit mineralization of collagen in this tissue. Here we used an in vitro model based on remineralization of mouse dental tissues to determine the role of matrix GAGs in control of mineralization. GAGs were selectively removed from demineralized mouse periodontal sections via enzymatic digestion. Proteomic analysis confirmed that enzymatic GAG removal does not significantly alter protein content. Analysis of remineralized tissue sections by transmission electron microscopy (TEM) shows that GAG removal reduced the rate of remineralization in mineralized tissues compared to the untreated control, while the ligament remained unmineralized. Protein removal with trypsin also reduced the rate of mineralization, but to a lesser extent than GAG removal, despite a much larger effect on protein content. These results indicate that GAGs promote mineralization in mineralized dental tissues rather than inhibiting mineral formation in the ligament, which may have broader implications for understanding control of collagen mineralization in connective tissues.

In vitro effects of photobiomodulation on cell migration and gene expression of ALP, COL-1, RUNX-2, and osterix in cementoblasts.

The aim of this study was to evaluate the effects of photobiomodulation (PBM) on cell migration and alkaline phosphatase (ALP), type I collagen (Col-1), runt-related transcription factor 2 (RUNX-2), and Osterix (OSX) gene expression in a cementoblast culture (OCCM-30), in a microenvironment mimicking an injury on the cementoblast layer, such as it occurs during root resorption. For this, OCCM-30 cells were cultured in 6-well plates and the following parameters were assayed: (1) migration by scratch assay and ALP, Col-1, Runx2, and Osx by real-time PCR. PBM was performed in two protocols using a LED device emitting light at 660 nm (± 30 nm). OCCM-30 cementoblasts were grown and divided into four groups: (1) negative control; (2) positive control (scratch); (3) scratch + PBM with a total energy of 36 J and energy density 1.6 J/cm2; and (4) scratch + PBM with a total energy of 72 J and energy density of 3.2 J/cm2. Data were statistically analyzed, with the level of significance set at 5%. Cementoblasts migrated from the edge of the scratch toward the center, and the wound closed after 24 h, with the PBM3.2J/cm2 group showing the higher cell migration compared with the other groups at 2 h, 6 h, 8 h, and 13 h (p < 0.05). The control and PBM1.6J/cm2 groups showed similar levels of cell migration, with no significant differences (p > 0.05). PBM3.2J/cm2 group exhibited greater ALP, Col-1, OSX, and RUNX2 in comparison with the other experimental groups (p < 0.05). Similar levels of all genes evaluated were observed between the PBM1.6J/cm2 group and the positive control group (p > 0.05). In conclusion, our findings support the effectiveness of photobiomodulation on cementoblast migration and gene expression, which may contribute to the formation of a new cementum layer.

Development, Establishment, and Validation of a Model for the Mineralization of Periodontium Remodelling Cells: Cementoblasts.

Chronic kidney disease (CKD) patients undergoing dialysis are at high risk of bone fractures. CKD-induced mineral and bone disorder is extended to periodontal disease due to changes in the ionic composition of saliva in CKD patients, dysregulating mineralization, hindering regeneration and thereby promoting the progression of dental complications. Despite the importance of cementum for overall oral health, the mechanisms that regulate its development and regeneration are not well comprehended, and a lack of sufficient in vitro experimental models has hindered research progress. In this study, the impact of experimental conditions on the calcification of cementoblasts was systematically investigated, aimed at establishing a standardized and validated model for the calcification of cementoblasts. The effects of phosphate, calcium, ascorbic acid, ß-glycerolphosphate, dexamethasone, and fetal calf serum on the calcification process of cementoblasts were analyzed over a wide range of concentrations and time points by investigating calcium content, cell viability, gene expression and kinase activity. Cementoblasts calcified in a concentration- and time-dependent manner with higher concentrations of supplements cause a higher degree of calcification but decreased cell viability. Phosphate and calcium have a significantly stronger effect on cementoblast calcification processes compared to osteogenic supplements: ascorbic acid, ß-glycerolphosphate, and dexamethasone induce calcification over a wide range of osteogenic signalling pathways, with osteopontin being a central target of gene regulation. Conversely, treatment with ascorbic acid, ß-glycerolphosphate, and dexamethasone leads to activating only selected pathways, especially promoting bone sialoprotein expression. The developed and validated cementoblast calcification protocol, incubating up to 60% confluent cementoblasts with 1.9 mmol L-1 of phosphate supplementation for a reasonable, multi-pathway calcification induction and 10 mmol L-1 ß-glycerolphosphate, 75 µmol L-1 ascorbic acid and 10 nmol L-1 dexamethasone for a reasonable osteogenic differentiation-based calcification induction, provides standard in vitro experimental models for better understanding cementoblast function and regeneration.

Between a rock and a hard place: Regulation of mineralization in the periodontium.

The periodontium supports and attaches teeth via mineralized and nonmineralized tissues. It consists of two, unique mineralized tissues, cementum and alveolar bone. In between these tissues, lies an unmineralized, fibrous periodontal ligament (PDL), which distributes occlusal forces, nourishes and invests teeth, and harbors progenitor cells for dentoalveolar repair. Many unanswered questions remain regarding periodontal biology. This review will focus on recent research providing insights into one enduring mystery: the precise regulation of the hard-soft tissue borders in the periodontium which define the interfaces of the cementum-PDL-alveolar bone structure. We will focus on advances in understanding the molecular mechanisms that maintain the unmineralized PDL "between a rock and a hard place" by regulating the mineralization of cementum and alveolar bone.