Tooth root dentin mineralization defects in a mouse model of hypophosphatasia.

Tissue-nonspecific alkaline phosphatase (TNAP) is expressed in mineralizing tissues and functions to reduce pyrophosphate (PP(i) ), a potent inhibitor of mineralization. Loss of TNAP function causes hypophosphatasia (HPP), a heritable disorder marked by increased PP(i) , resulting in rickets and osteomalacia. Tooth root cementum defects are well described in both HPP patients and in Alpl(-/-) mice, a model for infantile HPP. In Alpl(-/-) mice, dentin mineralization is specifically delayed in the root; however, reports from human HPP patients are variable and inconsistent regarding dentin defects. In the current study, we aimed to define the molecular basis for changes in dentinogenesis observed in Alpl(-/-) mice. TNAP was found to be highly expressed by mature odontoblasts, and Alpl(-/-) molar and incisor roots featured defective dentin mineralization, ranging from a mild delay to severely disturbed root dentinogenesis. Lack of mantle dentin mineralization was associated with disordered and dysmorphic odontoblasts having disrupted expression of marker genes osteocalcin and dentin sialophosphoprotein. The formation of, initiation of mineralization within, and rupture of matrix vesicles in Alpl(-/-) dentin matrix was not affected. Osteopontin (OPN), an inhibitor of mineralization that contributes to the skeletal pathology in Alpl(-/-) mice, was present in the generally unmineralized Alpl(-/-) mantle dentin at ruptured mineralizing matrix vesicles, as detected by immunohistochemistry and by immunogold labeling. However, ablating the OPN-encoding Spp1 gene in Alpl(-/-) mice was insufficient to rescue the dentin mineralization defect. Administration of bioengineered mineral-targeting human TNAP (ENB-0040) to Alpl(-/-) mice corrected defective dentin mineralization in the molar roots. These studies reveal that TNAP participates in root dentin formation and confirm that reduction of PP(i) during dentinogenesis is necessary for odontoblast differentiation, dentin matrix secretion, and mineralization. Furthermore, these results elucidate developmental mechanisms underlying dentin pathology in HPP patients, and begin to explain the reported variability in the dentin/pulp complex pathology in these patients.

Activity of matrix metalloproteinases in bovine versus human dentine.

Metalloproteinases (MMPs) have been implicated with metabolism of collagen in physiological and pathological processes in human dentine. As bovine teeth have been used as a substitute for human teeth in laboratory analysis, this study evaluated the activity of MMP-2 and -9 in bovine versus human dentine. Bovine and human dentine fragments, from crowns and roots, were powderized. Protein extraction was performed by two protocols: a neutral extraction with guanidine-HCl/EDTA (pH 7.4) and an acidic extraction with citric acid (pH 2.3). Gelatinolytic activities of extracts were revealed by zymography. MMP-2 and -9 were detected in crown and root dentine from bovine and human teeth. Total activities of MMP-2 were 11.4 ± 2.2, 14.6 ± 2.0, 9.7 ± 1.2 and 12.4 ± 0.9 ng/ml for bovine root, human root, bovine crown and human crown dentine, respectively. Corresponding activities for MMP-9 were 14.9 ± 2.0, 15.3 ± 1.3, 15.4 ± 1.3 and 15.5 ± 1.3 ng/ml, respectively. Bovine dentine was found to be a reliable substrate for studies involving the activity of MMP-2 and -9.

Differential expression of matrix metalloproteinase-2 in human coronal and radicular sound and carious dentine.

OBJECTIVE: To examine the differential expression of matrix metalloproteinase-2 (MMP-2) in human coronal and radicular sound and carious dentine using combined trichrome staining technique and immunofluorescence approach. METHODS: Freshly extracted human premolars were fixed with formaldehyde, demineralised with 10% EDTA (pH 7.4), dehydrated and sectioned for light and immunofluorescence microscopy. Half of the sections were stained with Masson's trichrome and examined with light microscopy to identify regions in the coronal and radicular parts of the teeth that contained sound, caries-affected and caries-infected dentine. The rest of the sections were hybridized with anti-mouse MMP-2 primary antibody and FITC-conjugated secondary antibody. Immunofluorescence of the FITC that was indicative of the distribution of the MMP-2 in coronal and radicular dentine was analysed by fluorescence light microscopy. RESULTS: Trichrome staining revealed a green zone of unaffected sound dentin, red irregular regions of caries-infected dentine and pink regions of caries-affected dentine. Immunofluorescence signals that were indicative of MMP expression were the lowest in sound dentine and most intense in the caries-infected dentine. Caries-affected dentine showed intermediate immunoreactivity. The variations in the intensities of immunofluorescence corresponded well with the distribution of caries-infected and caries-affected dentine in the trichrome-stained sections, for both coronal and radicular dentine. CONCLUSION: Caries stimulates MMP-2 expression, resulting in the differential expression of this protease in sound, caries-affected and caries-infected dentine. The more intense MMP-2 expression in caries-affected dentine compared with sound dentine may imply more rapid hybrid layer degradation when caries-affected dentine is employed as the substrate for bonded restorations.

Determination of matrix metalloproteinases in human radicular dentin.

Matrix metalloproteinases (MMPs) are present in sound coronal dentin and may play a role in collagen network degradation in bonded restorations. We investigated whether these enzymes can also be detected in root dentin. Crown and root sections of human teeth were powderized, and dentin proteins were extracted by using guanidine-HCl and EDTA. Extracts were analyzed by zymography and Western blotting for matrix metalloproteinases detection. Zymography revealed gelatinolytic activities in both crown and root dentin samples, corresponding to MMP-2 and MMP-9. MMP-2 was more evident in demineralized root dentin matrix, whereas MMP-9 was mostly extracted from the mineralized compartment of dentin and presented overall lower levels. Western blot analysis detected MMP-8 equally distributed in crown and root dentin. Because MMPs are also present in radicular dentin, their contribution to the degradation of resin-dentin bonds should be addressed in the development of restorative strategies for the root substrate.

Chlorhexidine Inhibits the Proteolytic Activity of Root and Coronal Carious Dentin in vitro.

The purpose of this study was to evaluate the effect of chlorhexidine on the proteolytic activity of carious coronal and root dentin collected from patients. Sound dentin from freshly extracted human teeth was used as a control. Dentin fragments were mixed with a synthetic substrate for proteolytic enzymes (N-benzoyl-DL-arginine-naphthylamide--BANA) and the suspensions mixed with either 0.12% chlorhexidine digluconate or distilled water. These mixtures were incubated for 18 h at 37 degrees C, color was developed by the addition of 0.1% Fast Garnet and their optical density was recorded spectrophotometrically. BANA hydrolysis measured by the optical density of incubated specimens was detected in all tested groups, but was significantly higher for carious than for sound dentin (p < 0.05). The proteolytic activity was reduced for carious coronal and root dentin by chlorhexidine (p < 0.05; 50 and 30%, respectively). Chlorhexidine also reduced the proteolytic activity in sound root dentin (p < 0.05; 20%). Conversely, changes in the proteolytic activity of sound coronal dentin were not observed in the presence of chlorhexidine. The reduction in proteolytic activity by chlorhexidine was significantly higher in carious coronal dentin than in carious root dentin (p < 0.05). In conclusion, part of the effect of chlorhexidine in controlling caries progression in humans may be due to a decrease in the proteolytic activity of carious coronal and root dentin. Because of the prolonged incubation time in the present study, similar results may be obtained clinically with prolonged dentin exposure to chlorhexidine, e.g. chlorhexidine-containing varnishes.

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.

[The effect of matrix metalloproteinase-1 on root surface dentin matrix: a scanning electron microscope observation].

OBJECTIVE: To observe the effect of matrix metalloproteinase-1 (MMP-1) from human host on degradation of dentin organic matrix of root dentin. METHODS: The freshly extracted caries-free impacted teeth were selected. Teeth were cut transversely under the enamel-cementum junction into dentin sections with a thickness of about 5 mm. Then all sections with removal of cementum, pulp and predentin were randomly divided into four groups. In the first group, dentin sections were demineralized with acid solution for 21 days, and then incubated with MMP-1 solution for 7 days; the second group were only treated with acid solution for 21 days; the third group were only attacked by MMP-1 solution for 7 days; and the fourth group were untreated as a control. Then all sections were dehydrated in ascending strength of alcohol, critically dried, coated with platinum, and then observed under scanning electron microscope(SEM). RESULTS: The dentin sections of root surface attacked by acid and MMP-1 showed that demineralization of dentin mineral and degradation of dentin matrix fibrae synchronously happened. The dentin matrix fibrae wasn't degradated in the groups treated with acid or MMP-1. CONCLUSION: The proteinases from human host may play an important role in the development of root surface caries. MMP-1 may distinctly degradate the organic matrix of demineralized dentin.

Inhibition of molar eruption and root elongation in MT1-MMP-deficient mice.

To study whether eruption of teeth and root growth require remodeling of collagen in the peridental tissues, we studied molar development in mice deficient in MT1-MMP, an enzyme essential for remodeling of soft tissue-hard tissue interfaces. The lower jaws of deficient mice and their wildtype littermates were subjected to stereologic analysis. It was shown that in deficient animals, eruption and root elongation were severely inhibited, signifying a role of the enzyme in these developmental processes.

The localization of matrix metalloproteinase-20 (MMP-20, enamelysin) in mature human teeth.

MMP-20 (enamelysin), the matrix metalloproteinase family member discovered in the enamel organ, has also been detected in odontoblasts during dentin formation. We studied the presence and localization of MMP-20 in mature human teeth in health and disease. In immunohistochemistry, MMP-20-positive staining was observed most intensively in the radicular odontoblastic layer and also in dilated dentinal tubuli of caries lesions. By Western blotting, MMP-20 was detected in odontoblasts and pulp tissue of both sound and carious teeth, in dentinal fluid and dentin of sound teeth, but not in soft carious dentin. We conclude that MMP-20 produced during primary dentinogenesis is incorporated into dentin and may be released during caries progression. The main cellular source of MMP-20 in the dentin-pulp complex is the odontoblasts, which secrete MMP-20 into the dentinal fluid.

On the role of MT1-MMP, a matrix metalloproteinase essential to collagen remodeling, in murine molar eruption and root growth.

Although the connective tissues of the periodontium are subject to a high turnover rate, no conclusive evidence has yet emerged that periodontal collagen turnover is essential for the eruption of teeth or for root elongation. These processes were studied in mice deficient in MT1-MMP, a membrane type matrix metalloproteinase essential for remodeling of soft tissue-hard tissue interfaces. Mandibular first molars of deficient mice and their wild-type littermates were subjected to stereological analysis in order to assess root length, eruption and the volume density of phagocytosed collagen in periodontal ligament fibroblasts. The data showed that both eruption and root elongation were severely inhibited in animals lacking the enzyme. We also found, in periodontal ligament fibroblasts from MT1-MMP-deficient mice, a massive age-related accumulation (up to 60-fold over controls) of collagen fibril-containing phagosomes. Phagolysosomes, which represent the next downstream step in collagen fibril degradation by the lysosomal pathway, did not accumulate. These observations indicate that MT1-MMP plays a central role in periodontal remodeling. The stunted root growth and the failure to erupt indicate the important role of the enzyme in tooth development.

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.

Effects of collagenase on root demineralization.

The role of proteolytic enzymes in the root caries process remains unclear. The aim of this study was to investigate collagenase activity during tooth root demineralization and remineralization in an in vitro demineralization/remineralization pH-cycling model, Human tooth roots were subjected to pH cycling (alternating demineralization and remineralization) in one of two different time cycles for five days. Collagenase at 90, 180, or 360 micrograms per root was placed into either the demineralizing solution or the remineralizing solution in the pH-cycling system. The effects of additional exposure to collagenase before or after pH cycling were also studied. After the exposure, thin sections of the roots were examined histologically by polarized light microscopy. Changes of calcium and phosphate in the solutions were analyzed chemically. Surface erosion occurred only in the groups where collagenase was contained in the remineralizing solution and in which the root samples were exposed to severe demineralization. However, no differences among the control and experimental groups were found in calcium and phosphate changes in the pH-cycling solutions. These findings suggest that collagenase works during the remineralizing phase and predominantly attacks the organic matrix of the root after demineralization. Additional exposure to collagenase before or after pH cycling did not increase surface erosion except for exposure to collagenase in the absence of phosphate following pH cycling.

Periodontal tissue-vitality after different etching modalities.

The purpose of the present study was to investigate possible immediate necrotizing effects on periodontal tissues of etching agents operating either at low or neutral pH. Phosphoric and citric acids, both of which operate at low pH, exerted immediate (within 20 s) necrotizing effects on both mucosal flaps and periodontal tissues. The penetration depth increased with time up to 1/4 of the circumference of the root after 3 min. This was in sharp contrast to EDTA which operates at neutral pH. EDTA failed to induce any detectable necrosis during the experimental periods. Whether or not the immediate necrotizing effects of low pH etching agents also impair periodontal healing in the long-term perspective, needs to be evaluated with controlled experimental studies in vivo as well as clinically in order to determine whether etching at neutral pH is preferable.

Identification of tissue inhibitor of metalloproteinases-1 (TIMP-1) in human teeth and its distribution in cementum and dentine.

An inhibitory activity toward matrix metalloproteinases such as interstitial collagenase, 72-kDa gelatinase/type IV collagenase, and stromelysin-1 was detected in an EDTA extract of pulverized roots of human teeth, and identified as TIMP-1 by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and immunoblotting. Distribution of TIMP-1 in human cementum and dentine was investigated by a sandwich enzyme immunoassay in combination with an abrasive microsampling technique. TIMP-1 could not be detected in cementum from some teeth but in others decreased from a fairly low value at the surface towards the cementodentinal junction. TIMP-1 concentrations in the dentine increased consistently from the cementodentinal junction toward the predentine. The average TIMP-1 concentration in the dentine (54.1 +/- 18.5 pg/mg +/- SE) was significantly (P < 0.05) higher than that (9.6 +/- 6.0 pg/mg +/- SE) in the cementum.

The presence of an inhibitor of human skin collagenase in the roots of healthy and periodontally diseased teeth: changes that occur with age.

The amount of anti-collagenase in the root has been reported in a group 18 to 35 years of age. Healthy roots had more than the diseased; and apical more than the cervical. The purpose of the present study is to determine the effect of age on these values. In a group 50 to 92 years of age, all values were markedly higher than in the younger group. In addition, all relationships between values were preserved. These include location and presence of disease. The higher levels might be due to an aging process or a protective defense against collagenase in the gingival crevice.

Susceptibility of the collagenous matrix from bovine incisor roots to proteolysis after in vitro lesion formation.

The susceptibility of the organic matrix from permanent bovine incisor roots to proteolytic breakdown after in vitro lesion formation was investigated. Root surfaces were exposed to 0.1 M acetic acid, pH 4.0, to produce erosive lesions or to 0.1 M lactic acid, 0.2 mM methane hydroxy diphosphonate, pH 5.0, to produce subsurface lesions. After demineralization, the roots were treated with a bacterial collagenase. The quantity of enzyme-degradable collagen in the root tissue was found to be proportional to the calcium released during demineralization, until a plateau value was reached at calcium concentrations in solution of 3.3 mM at pH 4.0 and 2.7 mM at pH 5.0. The degradability of collagen was found to be substantially less in subsurface lesions than in erosive lesions. The presence of cementum-free areas did not affect the results. These findings suggest that the mineral component of the roots is composed of several fractions which differ in their solubility properties in weak acids.

Adenine nucleotide contents and ATPases activities in porcine deciduous dental pulp during the root formation, in fully formed root and during root resorption phases.

Nucleotide content and activity of certain enzymes were compared in pigs of various ages in order to study the energetic metabolism of deciduous dental pulps in the three phases of the cycle of tooth ontogeny, namely, root formation, fully formed root and root resorption phases. The frozen pulps were removed with the help of a screw vise and analysed for ATP, ADP and AMP contents and Ca2+ and Mg2+-ATPases activities. The highest ATP content in the first deciduous molar pulp was found when the tooth was still in an intrabony position. The calculated energy charge, although low for all groups, at this stage of development, indicated an activation of the consuming processes. In the root resorption phase, lowest ATP content and higher Ca2+ and Mg2+-ATPases activities were observed.

Replantation of enzymatically treated teeth in monkeys. Part I.

Chemical alterations in cementum of avulsed teeth may be implied as a causative factor in their eventual resorption when replanted. Root surface decalcification, with subsequent enzymatic deletion of glycoproteins and cross linking of cementum collagen, results in little or no root resorption for up to 3 months after replantation.