Null mutation of chloride channel 7 (Clcn7) impairs dental root formation but does not affect enamel mineralization.

ClC-7, located in late endosomes and lysosomes, is critical for the function of osteoclasts. Secretion of Cl(-) by the ruffled border of osteoclasts enables H(+) secretion by v-H(+)-ATPases to dissolve bone mineral. Mice lacking ClC-7 show altered lysosomal function that leads to severe lysosomal storage. Maturation ameloblasts are epithelial cells with a ruffled border that secrete Cl(-) as well as endocytose and digest large quantities of enamel matrix proteins during formation of dental enamel. We tested the hypothesis that ClC-7 in maturation ameloblasts is required for intracellular digestion of matrix fragments to complete enamel mineralization. Craniofacial bones and developing teeth in Clcn7(-/-) mice were examined by micro-CT, immunohistochemistry, quantified histomorphometry and electron microscopy. Osteoclasts and ameloblasts in wild-type mice stained intensely with anti-ClC-7 antibody but not in Clcn7(-/-) mice. Craniofacial bones in Clcn7(-/-) mice were severely osteopetrotic and contained 1.4- to 1.6-fold more bone volume, which was less mineralized than the wild-type littermates. In Clcn7(-/-) mice maturation ameloblasts and osteoclasts highly expressed Ae2 as in wild-type mice. However, teeth failed to erupt, incisors were much shorter and roots were disfigured. Molars formed a normal dental crown. In compacted teeth, dentin was slightly less mineralized, enamel did not retain a matrix and mineralized fairly normal. We concluded that ClC-7 is essential for osteoclasts to resorb craniofacial bones to enable tooth eruption and root development. Disruption of Clcn7 reduces bone and dentin mineral density but does not affect enamel mineralization.

Buffering of protons released by mineral formation during amelogenesis in mice.

Regulation of pH by ameloblasts during amelogenesis is critical for enamel mineralization. We examined the effects of reduced bicarbonate secretion and the presence or absence of amelogenins on ameloblast modulation and enamel mineralization. To that end, the composition of fluorotic and non-fluorotic enamel of several different mouse mutants, including enamel of cystic fibrosis transmembrane conductance regulator-deficient (Cftr null), anion exchanger-2-deficient (Ae2a,b null), and amelogenin-deficient (Amelx null) mice, was determined by quantitative X-ray microanalysis. Correlation analysis was carried out to compare the effects of changes in the levels of sulfated-matrix (S) and chlorine (Cl; for bicarbonate secretion) on mineralization and modulation. The chloride (Cl- ) levels in forming enamel determined the ability of ameloblasts to modulate, remove matrix, and mineralize enamel. In general, the lower the Cl- content, the stronger the negative effects. In Amelx-null mice, modulation was essentially normal and the calcium content was reduced least. Retention of amelogenins in enamel of kallikrein-4-deficient (Klk4-null) mice resulted in decreased mineralization and reduced the length of the first acid modulation band without changing the total length of all acidic bands. These data suggest that buffering by bicarbonates is critical for modulation, matrix removal and enamel mineralization. Amelogenins also act as a buffer but are not critical for modulation.

Composition of mineralizing incisor enamel in cystic fibrosis transmembrane conductance regulator-deficient mice.

Formation of crystals in the enamel space releases protons that need to be buffered to sustain mineral accretion. We hypothesized that apical cystic fibrosis transmembrane conductance regulator (CFTR) in maturation ameloblasts transduces chloride into forming enamel as a critical step to secrete bicarbonates. We tested this by determining the calcium, chloride, and fluoride levels in developing enamel of Cftr-null mice by quantitative electron probe microanalysis. Maturation-stage enamel from Cftr-null mice contained less chloride and calcium than did wild-type enamel, was more acidic when stained with pH dyes ex vivo, and formed no fluorescent modulation bands after in vivo injection of the mice with calcein. To acidify the enamel further we exposed Cftr-null mice to fluoride in drinking water to stimulate proton release during formation of hypermineralized lines. In Cftr-deficient mice, fluoride further lowered enamel calcium without further reducing chloride levels. The data support the view that apical CFTR in maturation ameloblasts tranduces chloride into developing enamel as part of the machinery to buffer protons released during mineral accretion.

Parenteral monofluorophosphate (MFP) is a more potent inducer of enamel fluorotic defects in neonatal hamster molars than sodium fluoride.

Supra-optimal intake of sodium fluoride (NaF) during early childhood results in formation of irreversible enamel defects. Monofluorophosphate (MFP) was considered as less toxic than NaF but equally cariostatic. We compared the potency of MFP and NaF to induce pre-eruptive sub-ameloblastic cysts and post-eruptive white spots and pits in developing hamster enamel. Hamster pups were injected subcutaneously with either NaF or MFP in equimolar doses of either 9 mg or 18 mg F/kg body weight. At 9 mg F/kg, MFP induced more but smaller sub-ameloblastic cysts with a collective cyst volume twice as large as that induced by NaF. Eight days after F injection, all F-injected groups had formed 4-6 white spots per molar, with an additional 2 pits per molar in the low MFP group. Twenty-eight days after injection, most white spots had turned into pits (5-6 per molar) and only the high MFP group still contained 2 white spots per molar. We conclude that parenterally applied MFP is more potent in inducing enamel defects than NaF. Most white spots formed turn into pits by functional use of the dentition. The higher potency of parenteral MFP may be associated with sustained elevated F levels in the enamel organ by enzymatic hydrolysis of MFP by alkaline phosphatase activity.

Enhanced periodontal tissue regeneration by periodontal cell implantation.

AIM: Due to a lack of regenerative potential, current treatments for periodontal defects do not always provide satisfactory clinical results. Previously, the implantation of a biomaterial scaffold-cell construct has been suggested as a clinically achievable approach. In this study, it was aimed to investigate the contribution of implanted periodontal ligament (PDL) cells to periodontal tissue regeneration. MATERIALS & METHODS: Gelatin sponges were seeded with green fluorescent protein (GFP) transfected PDL or gingival fibroblasts (GF) cells, and implanted into a surgically created rat intrabony periodontal defect model. After six weeks, decalcified maxillae were used for histomorphometrical and immunohistochemical analyses. RESULTS: After six weeks, animals that had received the PDL cells exhibited significantly more functional bone and ligament. Furthermore, there were remarkable differences in the distribution of the transplanted cells. Periodontal ligament cells were always located directly lining the newly regenerated areas. In contrast, GF cells dispersed over the whole defect area, and did not provide a favourable effect on the regeneration of the periodontal tissues. CONCLUSION: We concluded that PDL cells transplanted into a periodontal defect survive and favour regeneration of periodontium, possibly in a paracrine manner.

Regeneration of the periodontium using enamel matrix derivative in combination with an injectable bone cement.

OBJECTIVES: Enamel matrix derivative (EMD) has proven to enhance periodontal regeneration; however, its effect is mainly restricted to the soft periodontal tissues. Therefore, to stimulate not only the soft tissues, but also the hard tissues, in this study EMD is combined with an injectable calcium phosphate cement (CaP; bone graft material). The aim was to evaluate histologically the healing of a macroporous CaP in combination with EMD. MATERIALS AND METHODS: Intrabony, three-wall periodontal defects (2 × 2 × 1.7 mm) were created mesial of the first upper molar in 15 rats (30 defects). Defects were randomly treated according to one of the three following strategies: EMD, calcium phosphate cement and EMD, or left empty. The animals were killed after 12 weeks, and retrieved samples were processed for histology and histomorphometry. RESULTS: Empty defects showed a reparative type of healing without periodontal ligament or bone regeneration. As measured with on a histological grading scale for periodontal regeneration, the experimental groups (EMD and CaP/EMD) scored equally, both threefold higher compared with empty defects. However, most bone formation was measured in the CaP/EMD group; addition of CAP to EMD significantly enhanced bone formation with 50 % compared with EMD alone. CONCLUSIONS: Within the limits of this animal study, the adjunctive use of EMD in combination with an injectable cement, although it did not affect epithelial downgrowth, appeared to be a promising treatment modality for regeneration of bone and ligament tissues in the periodontium. CLINICAL RELEVANCE: The adjunctive use of EMD in combination with an injectable cement appears to be a promising treatment modality for regeneration of the bone and ligament tissues in the periodontium.

Na+ and K+ transport and maturation stage ameloblast modulation.

Introduction: Enamel mineralization requires calcium transport into the extracellular matrix for the synthesis of hydroxyapatite (HA) crystals. Formation of HA releases protons into the matrix, which are then neutralized when ameloblasts modulate from cells with apical invaginations, the so-called ruffle-ended ameloblasts (RE), to smooth-ended ameloblasts (SE). Ameloblast modulation is associated with the translocation of the calcium exchanger Nckx4 to the apical border of RE, to remove Na+ from the enamel matrix in exchange for Ca2+ and K+. As enamel matures, Na+ and K+ in the matrix progressively decrease. However, the transporter to remove K+ from mineralizing enamel has not been identified. Methods: Expression of K+ exchangers and channels in secretory and maturation stage of enamel organs were compared following an RNA-seq analysis. Kcnj15, which encodes the Kir4.2 inwardly rectifying K+ channel, was found to be the most upregulated internalizing K+ transporter in maturation stage of enamel organs. Kir4.2 was immunolocalized in wt, Nckx4-/-, Wdr72-/-, and fluorosed ameloblasts. Regulation of Wdr72 expression by pH was characterized in vitro and in vivo. Results: Kir4.2 immunolocalized to the apical border of wild type (wt) mouse RE and cytosol of SE, a spatial distribution pattern shared by NCKX4. In Nckx4-/- ameloblasts, Kir4.2 also localized to the apical surface of RE and cytosol of SE. However, in fluorosed and Wdr72-/- ameloblasts, in which vesicle trafficking is disrupted, Kir4.2 remained in the cytosol. In vitro, Wdr72 was upregulated in LS8 cells cultured in medium with a pH 6.2, which is the pH of the enamel matrix underlying RE, as compared to pH 7.2 under SE. Conclusion: Taken together these results suggest that Kir4.2 participates in K+ uptake by maturation ameloblasts, and that K+ and Na+ uptake by Kir4.2 and Nckx4, respectively, may be regulated by pH through WDR72-mediated endocytosis and membrane trafficking.

Tooth Formation as Experimental Model to Study Chemotherapy on Tissue Development: Effect of a Specific Dose of Temozolomide/Veliparib.

BACKGROUND: Chemotherapy treatment of cancer in children can influence formation of normal tissues, leading to irreversible changes in their structure and function. Tooth formation is susceptible to several types of chemotherapy that induce irreversible changes in the structure of enamel, dentin and dental root morphology. These changes can make the teeth more prone to fracture or to caries when they have erupted. Recent studies report successful treatment of brain tumors with the alkylating drug temozolomide (TMZ) in combination with veliparib (VLP) in a glioblastoma in vivo mouse model. Whether these drugs also affect tooth formation is unknown. AIM: In this study the effect of TMZ/VLP on incisor formation was investigated in tissue sections of jaws from mice and compared with mice not treated with these drugs. MATERIALS AND METHOD: The following aspects were studied using immunohistochemistry of specific protein markers including: (1) proliferation (by protein expression of proliferation marker Ki67) (2) a protein involved in paracellular ion transport (expression of tight junction (TJ) protein claudin-1) and (3) in transcellular passage of ions across the dental epithelium (expression of Na+, K+ 2Cl- cotransporter/NKCC1). RESULTS: Chemotherapy with TMZ/VLP strongly reduced immunostaining for claudin-1 in distal parts of maturation ameloblasts. No gross changes were found in the treated mice, either in cell proliferation in the dental epithelium at the cervical loop or in the immunostaining pattern for NKCC1 in (non-ameloblastic) dental epithelium. The salivary glands in the treated mice contained strongly reduced immunostaining for NKCC1 in the basolateral membranes of acinar cells. DISCUSSION/CONCLUSIONS: Based on the reduction of claudin-1 immunostaining in ameloblasts, TMZ/VLP may potentially influence forming enamel by changes in the structure of TJs structures in maturation ameloblasts, structures that are crucial for the selective passage of ions through the intercellular space between neighboring ameloblasts. The strongly reduced basolateral NKCC1 staining seen in fully-grown salivary glands of TMZ/VLP-treated mice suggests that TMZ/VLF could also influence ion transport in adult saliva by the salivary gland epithelium. This may cause treated children to be more susceptible to caries.

Mechanical loading stimulates BMP7, but not BMP2, production by osteocytes.

Bone mechanical adaptation is a cellular process that allows bones to adapt their mass and structure to mechanical loading. This process is governed by the osteocytes, which in response to mechanical loading produce signaling molecules that affect osteoblasts and osteoclasts. Bone morphogenic proteins (BMPs) are excellent candidates as signaling molecules, but it is unknown whether mechanically stimulated osteocytes affect bone adaptation through BMP production. Therefore, the aim of this study was to assess whether osteocytes produce BMPs in response to mechanical loading. In addition, since BMP7 has a vitamin D receptor (VDR) response element in the promoter region, we also investigated whether VDR is involved in the BMP7 response to mechanical loading. Human or VDR(-/-) mouse primary bone cells were submitted in vitro to 1 h pulsating fluid flow (PFF) and postincubated without PFF (PI) for 1-24 h, and gene and protein expression of BMP2 and BMP7 were quantified. In human bone cells, PFF did not change BMP2 gene expression, but it upregulated BMP7 gene expression by 4.4- to 5.6-fold at 1-3 h PI and stimulated BMP7 protein expression by 2.4-fold at 6 h PI. PFF did not stimulate BMP7 gene expression in VDR(-/-) mouse bone cells. These results show for the first time that mechanical loading upregulates BMP7, likely via the VDR, but not BMP2, gene and protein expression in osteocytes in vitro. Since BMP7 plays a major role in bone development and remodeling, these data might contribute to a better understanding of the mechanism leading to the mechanical adaptation of bone.

Developmental expression of solute carrier family 26A member 4 (SLC26A4/pendrin) during amelogenesis in developing rodent teeth.

Ameloblasts need to regulate pH during the formation of enamel crystals, a process that generates protons. Solute carrier family 26A member 4 (SLC26A4, or pendrin) is an anion exchanger for chloride, bicarbonate, iodine, and formate. It is expressed in apical membranes of ion-transporting epithelia in kidney, inner ear, and thyroid where it regulates luminal pH and fluid transport. We hypothesized that maturation ameloblasts express SLC26A4 to neutralize acidification of enamel fluid in forming enamel. In rodents, secretory and maturation ameloblasts were immunopositive for SLC26A4. Staining was particularly strong in apical membranes of maturation ameloblasts facing forming enamel. RT-PCR confirmed the presence of mRNA transcripts for Slc26a4 in enamel organs. SLC26A4 immunostaining was also found in mineralizing connective tissues, including odontoblasts, osteoblasts, osteocytes, osteoclasts, bone lining cells, cellular cementoblasts, and cementocytes. However, Slc26a4-null mutant mice had no overt dental phenotype. The presence of SLC26A4 in apical plasma membranes of maturation ameloblasts is consistent with a potential function as a pH regulator. SLC26A4 does not appear to be critical for ameloblast function and is probably compensated by other pH regulators.

Fluoride inhibits the response of bone cells to mechanical loading.

The response of bone cells to mechanical loading is mediated by the cytoskeleton. Since the bone anabolic agent fluoride disrupts the cytoskeleton, we investigated whether fluoride affects the response of bone cells to mechanical loading, and whether this is cytoskeleton mediated. The mechano-response of osteoblasts was assessed in vitro by measuring pulsating fluid flow-induced nitric oxide (NO) production. Osteocyte shape was determined in hamster mandibles in vivo as parameter of osteocyte mechanosensitivity. Pulsating fluid flow (0.7 ± 0.3 Pa, 5 Hz) stimulated NO production by 8-fold within 5 min. NaF (10-50 µM) inhibited pulsating fluid flow-stimulated NO production after 10 min, and decreased F-actin content by ~3-fold. Fluid flow-induced NO response was also inhibited after F-actin disruption by cytochalasin B. NaF treatment resulted in more elongated, smaller osteocytes in interdental bone in vivo. Our results suggest that fluoride inhibits the mechano-response of bone cells, which might occur via cytoskeletal changes. Since decreased mechanosensitivity reduces bone mass, the reported anabolic effect of fluoride on bone mass in vivo is likely mediated by other factors than changed bone cell mechanosensitivity.

Ae2(a,b)-deficient mice exhibit osteopetrosis of long bones but not of calvaria.

Extracellular acidification by osteoclasts is essential to bone resorption. During proton pumping, intracellular pH (pH(i)) is thought to be kept at a near-neutral level by chloride/bicarbonate exchange. Here we show that the Na(+)-independent chloride/bicarbonate anion exchanger 2 (Ae2) is relevant for this process in the osteoclasts from the long bones of Ae2(a,b)(-/-) mice (deficient in the main isoforms Ae2a, Ae2b(1), and Ae2b(2)). Although the long bones of these mice had normal numbers of multinucleated osteoclasts, these cells lacked a ruffled border and displayed impaired bone resorption activity, resulting in an osteopetrotic phenotype of long bones. Moreover, in vitro osteoclastogenesis assays using long-bone marrow cells from Ae2(a,b)(-/-) mice suggested a role for Ae2 in osteoclast formation, as fusion of preosteoclasts for the generation of active multinucleated osteoclasts was found to be slightly delayed. In contrast to the abnormalities observed in the long bones, the skull of Ae2(a,b)(-/-) mice showed no alterations, indicating that calvaria osteoclasts may display normal resorptive activity. Microfluorimetric analysis of osteoclasts from normal mice showed that, in addition to Ae2 activity, calvaria osteoclasts--but not long-bone osteoclasts--possess a sodium-dependent bicarbonate transporting activity. Possibly, this might compensate for the absence of Ae2 in calvaria osteoclasts of Ae2(a,b)(-/-) mice.

Inorganic phosphate stimulates DMP1 expression in human periodontal ligament fibroblasts embedded in three-dimensional collagen gels.

Stable integration of collagenous tissue-engineered constructs to surrounding solid devices can be accomplished by coating the solid surfaces with exogenous alkaline phosphatase (ALP). We showed previously that coating of culture well surfaces with the enzyme in combination with the presence of its substrate beta-glycerophosphate (beta-GP) induces mineral deposition at the interface of matrix and surface, thereby preventing matrix detachment. In this study the effect of such mineral-inducing conditions on differentiation of human periodontal ligament (PDL) fibroblasts into osteoblasts/cementoblasts was analyzed in three-dimensional collagen gels. Mineral-inducing conditions decreased collagen type I gene expression and induced dentin matrix protein 1 (DMP1; a marker of late osteoblasts/cementoblasts) gene expression by fibroblasts. DMP1 protein was detected in some fibroblasts only in mineralizing gels. Exogenous ALP released high levels of inorganic phosphate from beta-GP. Addition of inorganic phosphate alone induced DMP1 gene expression, which could be prevented by blocking phosphate entry into fibroblasts by foscarnet. We concluded that mineralizing conditions induced by exogenous ALP affect the phenotype of PDL fibroblasts. The fibroblasts are stimulated to express the late osteoblast/osteocyte marker protein DMP1, which is mediated by uptake of inorganic phosphate into the cells. The enzyme-mediated mineral deposition may thus facilitate enhanced integration of collagenous tissue-engineered constructs to devices or implants in vitro.

Localization and function of the anion exchanger Ae2 in developing teeth and orofacial bone in rodents.

To explore the functions of the anion exchanger 2 (Ae2) in the development of bones and teeth we examined the distribution of Ae2 in cells involved in the formation of teeth and surrounding bone in young hamsters, mice and rats. In all three species strongest immunostaining for Ae2 was obtained in basolateral membranes of maturation ameloblasts and in osteoclasts resorbing bone. In hamsters a weaker staining was also seen in the Golgi apparatus of secretory ameloblasts, young osteoblasts and osteocytes, odontoblasts and fibroblasts of the forming periodontal ligament. In adult Ae2(a,b) (-/-) mice, in which Ae2-targeted disruption precluded the expression of Ae2a, Ae2b1 and Ae2b2 isoforms, the immunostaining for Ae2 in ameloblasts and osteoclasts was totally abolished. The enamel formation was abnormal but teeth erupted, osteoclasts in jaw bone were functional and structure of dentin and bone was normal. In another mouse model, Ae2(-/-) mice in which the expression of all five Ae2 isoforms was disrupted, teeth failed to erupt and the alveolar bone proved poorly formed with giant but apparently functional osteoclasts. Our data indicate that basolaterally located Ae2a, Ae2b1 or Ae2b2 (or a combination of these) is present in maturation ameloblasts critical for the cells' normal functioning. Although isoforms of Ae2 were also present in basolateral membranes of osteoclasts, they proved to be not critical to osteoclast resorption of orofacial bone. Poorly formed bone and the failure of teeth to erupt seen in the Ae2(-/-) mice with gene disruption affecting all isoforms may result from secondary (systemic) changes that are different from Ae2(a,b) (-/-) mice.

The Importance of Connexin 43 in Enamel Development and Mineralization.

During enamel development, formation of hydroxyapatite crystals and regulation of pH in the enamel matrix require massive transport of ions. Both ameloblasts and adjacent dental epithelial cells in the stellate reticulum co-express several transmembrane cotransporters/ion-exchangers for transport of ions across plasma membranes. Gap junctions (GJs) enable intercellular exchanges of ions between neighboring cells. This suggests that the ameloblasts and other cell layers of the enamel organ, form a functional unit. During the bell stage of tooth formation, the non-ameloblast dental epithelium highly expresses the Na-K-Cl cotransporter (Nkcc1). Nkcc1-null mice are associated with enamel hypomineralization and increased expression of GJ protein connexin 43 (Cx43), suggesting that reduced ion transport in the Nkcc1-null mouse is in part compensated by increased intercellular ion transport through GJs. To understand the role of GJs in ion transport and its effect on pH regulation, we examined in a mouse strain in which Cx43 was ablated selectively in DMP1 expressing cells (Cx43flox/flox mice crossed with DMP1-8kb-Cre mice), including ameloblasts. Micro-CT analysis showed that the mineral density at late maturation stage incisal enamel of the Cx43-null mice was 10% less than in controls, whereas that in dentin was unchanged. Maturation stage ameloblasts of mice lacking the pH regulating sodium/bicarbonate transporter NBCe1 (Nbce1-null), or chloride channel Cftr (Cftr-null) were found to have increased Cx43-immunostaining. These results support the possibility that GJs in the ameloblast-papillary complex at the maturation stage contribute to ion transport by enabling passage of ions directly from cells of the papillary layer into ameloblast layer. Increasing the number of GJs may partly compensate the reduction of ion-cotransporters and ion exchangers in dental epithelium.

Immunolocalization of sibling and RUNX2 proteins during vertical distraction osteogenesis in the human mandible.

We tested the hypothesis that mechanical loading of human bone increases expression of the transcription factor RUNX2 and bone matrix proteins osteopontin (OPN), bone sialoprotein (BSP), dentin matrix protein-1 (DMP1), and matrix extracellular phosphoglycoprotein (MEPE). We examined this in tissue sections of atrophic mandibular bone taken from edentulous patients who had undergone distraction osteogenesis. In undistracted bone, weak to moderate staining for OPN and BSP was found in osteoblasts and bone matrix of immature woven bone. RUNX2 was also detectable in osteoblasts and in cells of the periosteum. In woven bone, but not in lamellar bone, a small number of osteocytes stained for all proteins tested. After distraction, staining intensity had increased in the existing old bone and staining was seen in more bone cells than before distraction. We also found a high expression of DMP1 and MEPE in many osteocytes embedded in woven bone and in some osteocytes of lamellar bone not seen before distraction. New bone trabeculae were forming in the fibrous tissue of the distraction gap containing all stages of intramembranous bone formation. Moderate to strong staining was seen for all five proteins tested in osteocytes located in woven bone of these trabeculae and for RUNX2, OPN, and BSP in osteoblasts lining the trabecular surfaces. We conclude that loading of atrophic human jawbone by distraction activates matrix synthesis of bone cells in and around existing bone. Increased staining of DMP1 and MEPE in osteocytes after loading is in line with the concept that these proteins may be involved in signaling the effector cells to adapt the bone structure to its mechanical demands.

Alkaline phosphatase-induced mineral deposition to anchor collagen fibrils to a solid surface.

Reconstruction of tendon and ligament tissues requires proper attachment of the tissue-engineered construct to surrounding tissues. A problem of reconstructing collagen-rich tissues is that an in vitro engineered collagenous network containing fibroblasts will contract and detach from a solid surface. In vivo anchorage of soft connective tissues to mineralized tissues like bones and teeth is accomplished by embedding collagen fibrils into mineralized layers. Mineralization is partially the result of local activity of the enzyme alkaline phosphatase (ALP). In this study, we tested whether ALP-induced mineral deposition at the interface between a collagen gel and a polystyrene or polyetheretherketone (PEEK) surface could prevent gel detachment from the surface. Coating of culture wells with intestinal ALP prevented detachment of gels harbored with human periodontal ligament (PDL) fibroblasts in the presence of its substrate beta-glycerophosphate. Mineral deposition was observed predominantly at the interface of collagen gel and well surface. The contractile properties of fibroblasts were not influenced by either ALP, beta-glycerophosphate or both. The presence of ALP on a solid surface and providing its substrate to allow mineral deposition can prevent detachment of collagen matrices. Our findings provide a tool to induce attachment of fibrillar collagen to a solid surface; an approach that seems useful for reconstruction of load-bearing tissues and attachment of ligaments to implants.

Mineralization-defects are comparable in fluorotic impacted human teeth and fluorotic mouse incisors.

OBJECTIVE: Fluoride excess of 0.05-0.07mgF/kgbw/day in water or food additives like salt is the principal cause of endemic dental fluorosis. How fluoride causes these defects is not clear yet. Recent studies in rodents suggest that development of enamel fluorosis is associated with insufficient neutralization of protons released during the formation of hypermineralized lines. DESIGN: Here we examined whether hypermineralization could also be assessed by MicroCT in developing molar enamel of humans exposed to fluoride. RESULT: Micro-CT analysis of hypomineralized enamel from human fluorotic molars graded by the Thylstrup-Fejerskov (TF) Index as III-IV showed weak hypermineralized lines and hypermineralized patches not seen in TF-I/II grade enamel. The mesio-distal sides of these molar teeth were significantly smaller (∼18%, p=0.02) than in TF-I/II teeth. CONCLUSION: The patterns of changes observed in human fluorotic teeth were similar to those in fluorotic rodent incisors. The data are consistent with the hypothesis that also in developing human teeth fluoride-stimulated local acidification of enamel could be a mechanism for developing fluorotic enamel.

No Change in Bicarbonate Transport but Tight-Junction Formation Is Delayed by Fluoride in a Novel Ameloblast Model.

We have recently developed a novel in vitro model using HAT-7 rat ameloblast cells to functionally study epithelial ion transport during amelogenesis. Our present aims were to identify key transporters of bicarbonate in HAT-7 cells and also to examine the effects of fluoride exposure on vectorial bicarbonate transport, cell viability, and the development of transepithelial resistance. To obtain monolayers, the HAT-7 cells were cultured on Transwell permeable filters. We monitored transepithelial resistance (TER) as an indicator of tight junction formation and polarization. We evaluated intracellular pH changes by microfluorometry using the fluorescent indicator BCECF. Activities of ion transporters were tested by withdrawal of various ions from the bathing medium, by using transporter specific inhibitors, and by activation of transporters with forskolin and ATP. Cell survival was estimated by alamarBlue assay. Changes in gene expression were monitored by qPCR. We identified the activity of several ion transporters, NBCe1, NHE1, NKCC1, and AE2, which are involved in intracellular pH regulation and vectorial bicarbonate and chloride transport. Bicarbonate secretion by HAT-7 cells was not affected by acute fluoride exposure over a wide range of concentrations. However, tight-junction formation was inhibited by 1 mM fluoride, a concentration which did not substantially reduce cell viability, suggesting an effect of fluoride on paracellular permeability and tight-junction formation. Cell viability was only reduced by prolonged exposure to fluoride concentrations greater than 1 mM. In conclusion, cultured HAT-7 cells are functionally polarized and are able to transport bicarbonate ions from the basolateral to the apical fluid spaces. Exposure to 1 mM fluoride has little effect on bicarbonate secretion or cell viability but delays tight-junction formation, suggesting a novel mechanism that may contribute to dental fluorosis.

Collagen type V enhances matrix contraction by human periodontal ligament fibroblasts seeded in three-dimensional collagen gels.

Extracellular matrix components play an important role in modulating cellular activity. To study such capacities of the matrix, fibroblasts are frequently cultured in a three-dimensional gel and contraction is assessed as a measure of cellular activity. Since a connective tissue contains several types of collagen, we investigated the effect of gels composed of collagen I alone or in combination with 10% collagen III and/or 5% collagen V on contraction by human periodontal ligament fibroblasts. Gels containing collagen V contracted much faster than those without this type of collagen. Blocking of the integrin beta1-subunit with an activity-blocking antibody delayed (gels with collagen V) or almost completely blocked (gels without collagen V) contraction. Use of an antibody directed against integrin alpha2beta1 resulted in delay of gel contraction for gels both with and without collagen V. Anti-integrin alpha v beta3 or RGD peptides partially blocked contraction of gels containing collagen V, but had no effect on gels consisting of collagen I alone. The beta1-containing integrins are involved in the basal contraction by fibroblasts that bind to collagens I and III. The enhanced contraction, stimulated by collagen V, appears to be mediated by integrin alpha v beta3. We conclude that collagen V may play an important modulating role in connective tissue contraction. Such a modulation may occur during the initial stages of wound healing and/or tissue regeneration.