Mineralization Front Research Articles

Hemocyte-mediated shell mineralization in the eastern oyster.

The growth of molluscan shell crystals is usually thought to be initiated from solution by extracellular organic matrix. We report a class of granulocytic hemocytes that may be directly involved in shell crystal production for oysters. On the basis of scanning electron microscopy (SEM) and x-ray microanalysis, these granulocytes contain calcium carbonate crystals, and they increase in abundance relative to other hemocytes following experimentally induced shell regeneration. Hemocytes are observed at the mineralization front using vital fluorescent staining and SEM. Some cells are observed releasing crystals that are subsequently remodeled, thereby at least augmenting matrix-mediated crystal-forming processes in this system.

Tooth enamel biomineralization in extant horses: implications for isotopic microsampling

Isotopic analyses of tooth enamel from fossil equids are increasingly being used to reconstruct paleoclimatic and paleoenvironmental conditions. However, the accuracy of these reconstructions is currently limited, partly because the precise timing and spatial patterns of enamel mineralization in equids have not been documented. We used radiographic analyses of mandibles collected from modern juvenile and adult domestic horses ( Equus caballus) to document the timing of enamel mineralization in equid cheek teeth (premolars and molars). Optical and radiographic analyses of thin sections of mature and developing teeth were used to document the spatial pattern of enamel mineralization in each tooth. We found that the enamel layers in equine cheek teeth took longer to mineralize than had been previously assumed, largely because the enamel continued to mineralize for approximately 6 to 12 months after each tooth had begun to erupt. Total enamel mineralization times for individual teeth ranged from ∼1.5 to ∼2.8 years. Examination of thin sections revealed that Retzius' striae extend outward from the enamel–dentin junction at an angle of ∼5° to ∼10° and run near-parallel to the nonocclusal surface of the tooth. Daily cross-striations average ∼5 μm in width, suggesting that, during the initial phase of enamel mineralization (matrix formation), new material is added at a rate of ∼5 μm/day. Radiographic analyses demonstrate that the secondary mineralization front (enamel maturation front) is orientated approximately parallel to the Retzius' striae, but complete maturation (i.e., full mineralization) lags behind matrix formation by several weeks to months.

Nucleobindin--a Ca2+-binding protein present in the cells and mineralized tissues of the tooth.

Nucleobindin, a Ca2+-binding protein, has been previously identified within the nucleus and endoplasmic reticulum, and in association with the Golgi membrane. In addition, nucleobindin has been shown to be a minor constituent of bone extracellular matrix and has been postulated to play a role in mineralization. In the current investigation, we report the expression and localization of nucleobindin within odontoblasts and the dentin matrix. Nucleobindin mRNA transcripts were detected in the tooth, and in situ hybridization analysis substantiated the findings, showing nucleobindin expression within mature odontoblasts and within the cells of surrounding developing alveolar bone. Western blot analysis of tooth protein extracts demonstrated the presence of a 63 kDa protein, which showed immunologic affinity for a rat nucleobindin peptide antibody. The distribution of the protein was shown in mature odontoblasts by using immunohistochemistry. Moreover, immunogold labeling of nucleobindin and subsequent ultrastructural analysis demonstrated a similar pattern of distribution. Nucleobindin was identified within odontoblast cellular compartments: the nucleus, endoplasmic reticulum, and mitochondria. Of interest, nucleobindin localization was observed within the surrounding dentin extracellular matrix, and immunogold labeling was shown to accumulate with tissue development toward the cusp. The study clearly demonstrated the presence of nucleobindin within dental tissues. In consideration of the known functional properties of nucleobindin, it may be postulated that nucleobindin may contribute to the accumulation and transport of Ca2+ ions to the mineralization front prior to hydroxyapatite deposition.

Hoechst 33342 is a useful cell tracer for a long-term investigation of articular cartilage repair.

The repair process of a full-thickness osteochondral defect was observed in a rat model using Hoechst 33342 as a cell tracer. The osteochondral defect was created at the medial femoral condyle of the right knee joints of twelve 11 week old male rats. Three weeks after the surgery, Hoechst 33342 was injected into the same knee joints. Calcein, a marker of the mineralization front, was then injected subcutaneousely twice at seven days and one day before harvesting of the tissue. At six, ten, and fourteen weeks and one year after the surgery, femoral condyles were obtained from the operated knee joints, fixed by alcohol, and embedded in polymethylmethacrylate. The sections were examined by fluorescent and then light microscopy. In the lateral femoral condyle cartilage, Hoechst 33342 labeling of chondrocyte nuclei was observed in all layers of the intact cartilage, and the dye never infiltrated beneath the subchondral bone plate. At 6 weeks after the surgery, Hoechst 33342-positive cells were observed not only in the regenerated fibrous cartilage, but also in the newly formed mineralized tissue in the medial femoral condyle. Interestingly, Hoechst 33342 labeling remained undiminished even one year after the intra-articular injection. The findings of the present study suggest that intra-articular injection of Hoechst 33342 is a useful tracer for long-term investigations of chondrocyte differentiation in vivo.

Immunodetection of osteoadherin in murine tooth extracellular matrices.

An antiserum was generated from synthetic peptides highly conserved between different mammalian species to immunolocalise the small leucine-rich proteoglycan osteoadherin (OSAD) in murine teeth. In 19-day-old embryos of rats and mice, a positive staining was found in incisor predentin and alveolar bone surrounding developing incisors and molars. In newborns, OSAD was detected at the tip of the first molar cusp where it accumulated in predentin concomitantly with odontoblast differentiation. In 2-day-old rats and mice, in the first molar, immunostaining revealed positive predentin, enamel matrix close to the apical pole of ameloblasts and a strong signal in dentin. At this stage, OSAD was detected in predentin in the second molar. Ultrastructural immunocytochemistry showed gold particles associated with collagen fibres in predentin and in foci at the dentin mineralisation front. Gold particles were also detected near the secretory pole of ameloblasts where enamel crystallites elongate. No staining was detected in pulp tissue and dental follicle. Restriction of OSAD expression to the extracellular matrix of bone, dentin and enamel suggests a role of this proteoglycan in the organisation of mineralised tissues.

Defective bone remodelling in osteoprotegerin-deficient mice.

Previous studies have reported enhanced osteoclastogenesis, increased bone resorption and osteoporosis in osteoprotegerin (OPG)-deficient mice. In the present study, we show that the tibial epiphyses contain abundant, thin trabeculae lined with numerous osteoclasts and cuboidal osteoblasts. The increase in osteoblasts and osteoclasts was associated with a dramatic increase in calcein labelling of the mineralization fronts and replacement of much of the intertrabecular marrow with numerous alkaline phosphatase-positive preosteoblasts. Furthermore, the discrete, linear cement lines seen in wild-type mice were replaced by a randomly oriented meshwork of cement lines that were stained intensely for tartrate-resistant acid phosphatase and osteopontin in the OPG-/- mice. These indices of accelerated bone remodelling in mutant bone were associated with irregular trabecular surfaces, a disorganized collagen matrix interspersed with amorphous ground substance and numerous fissures between old and new bone. In total, these observations indicate that enhanced osteoclastic activity in OPG-/- epiphyses led to a coupled increase in osteoblast differentiation and activity and an increase in bone remodelling. The high bone turnover, disorganized matrix and impaired attachment of new to old bone in the cement lines in OPG-/- mice appear to cause bone fragility.

MRNA expression and protein localization of dentin matrix protein 1 during dental root formation

Dentin matrix protein 1 (DMP1) is an acidic phosphoprotein. DMP1 was initially detected in dentin and later in other mineralized tissues including cementum and bone, but the DMP1 expression pattern in tooth is still controversial. To determine the precise localization of DMP1 messenger RNA (mRNA) and the protein in the tooth, we performed in situ hybridization and immunohistochemical analyses using rat molars and incisors during various stages of root formation. During root dentin formation of molars, DMP1 mRNA was detected in root odontoblasts in parallel with mineralization of the dentin. However, the level of DMP1 mRNA expression in root odontoblasts decreased near the coronal part and was absent in coronal odontoblasts. DMP1 protein was localized along dentinal tubules and their branches in mineralized root dentin, and the distribution of DMP1 shifted from the end of dentinal tubules to the base of the tubules as dentin formation progressed. During the formation of the acellular cementum, DMP1 mRNA was detected in cementoblasts lining the acellular cementum where its protein was localized. During the formation of the cellular cementum, DMP1 mRNA was detected in cementocytes embedded in the cellular cementum but not in cementoblasts, and its protein was localized in the pericellular cementum of cementocytes including their processes. During dentin formation of incisors, DMP1 mRNA was detected in odontoblasts on the cementum-related dentin, where its protein was localized along dentinal tubules near the mineralization front. The localization of DMP1 mRNA and protein in dentin and cementum was related to their mineralization, suggesting that one of the functions of DMP1 may be involved in the mineralization of dentin and cementum during root formation.

Abnormalities in bone mineral density and bone histology in thalassemia.

This study demonstrated that there was extensive iron staining on trabecular surface and marked reduction in trabecular bone volume without significant alteration in bone formation and bone resorption rates as well as significant reduction in bone mineral density in 18 thalassemic patients. Serum IGF-I was reduced and may modulate the reduction of bone mass. Bone histomorphometric studies in thalassemia to show alterations in bone histology and their relationship to biochemical parameters are very limited. Therefore, this study was systematically conducted to determine the alterations in thalassemia patients. Serum biochemical parameters, trans-iliac crest bone biopsy, and determination of bone mineral density of femur and lumbar spine were done in 18 thalassemic patients (10 females and 8 males). Serum osteocalcin, carboxy terminal teleopeptide fragment of type I collagen, and parathyroid hormone levels were within normal limits, but serum 25(OH) vitamin D (19.3 +/- 1.6 ng/ml) and 1,25(OH)2 vitamin D (33.77 +/- 1.51 pg/ml) levels were decreased. Serum insulin-like growth factor I (IGF-I; 145.2 +/- 20 ng/ml) was suppressed, whereas serum ferritin (1366.6 +/- 253.9 ng/ml) was markedly elevated. Reduced bone mineral density was found in all studied areas. Trabecular bone volume was significantly decreased (16.65 +/- 1.12%), whereas bone formation rate, eroded surface, and other bone histomorphometric parameters were within normal limits. The trabecular bone volume varied significantly with bone mineral density of total femur (r = 0.48, p = 0.04). There was an extensive stainable iron surface on the mineral front (9-60%). Significant correlation between serum IGF-I, serum ferritin, stainable iron surface, and bone mineral density, lumbar spine, and total femur were found. Serum IGF-I correlated with trabecular bone volume (r = 0.6, p = 0.03), inversely with both serum ferritin level (r = -0.6, p < 0.01), and inversely with stainable iron surface (r = -0.53, p = 0.02). Multiple regression analysis demonstrated that IGF-I was the only independent variable that determined bone mineral density of lumbar spine and total femur. Low bone mineral density and reduced trabecular bone volume with extensive iron deposition are the predominant findings in thalassemic patients. There was no evidence of increased bone resorption or mineralization defect. A reduction in circulatory IGF-I may modulate the reduction of bone mass.

Bone histomorphometry in children and adolescents with beta-thalassemia disease: iron-associated focal osteomalacia.

Thalassemia/hemoglobinopathy is a hereditary disease that causes chronic anemia and increased erythropoiesis. Consequently, an expansion of bone marrow spaces may contribute to osteopenia/osteoporosis. However, the pathogenesis of bone changes is not yet known. We, therefore, carried out the study on bone histomorphometry and biochemical and hormonal profiles in children and adolescents with suboptimally treated beta-thalassemia disease with the hope of gaining some new insight into the cellular and structural alterations of thalassemic bone. Seventeen patients underwent iliac crest bone biopsy for histomorphometric analyses. Bone mineral density (BMD) measurements were performed by dual energy x-ray absorptiometry. Most patients had growth retardation and delayed bone age. BMD was low especially at the lumbar spine. Serum IGF-I levels were almost always low. Bone histomorphometry revealed increased osteoid thickness, osteoid maturation time, and mineralization lag time, which indicate impaired bone matrix maturation and defective mineralization. In addition, iron deposits appeared along mineralization fronts and osteoid surfaces. Moreover, focal thickened osteoid seams were found together with focal iron deposits. Dynamic bone formation study revealed reduced bone formation rate. These findings indicate that delayed bone maturation and focal osteomalacia are the pathogenesis of bone disease in suboptimally blood-transfused thalassemics with iron overload. Iron deposits in bone and low circulating IGF-I levels may partly contribute to the above findings.

Dentin Sialophosphoprotein Knockout Mouse Teeth Display Widened Predentin Zone and Develop Defective Dentin Mineralization Similar to Human Dentinogenesis Imperfecta Type III

Dentin sialophosphoprotein (Dspp) is mainly expressed in teeth by the odontoblasts and preameloblasts. The Dspp mRNA is translated into a single protein, Dspp, and cleaved into two peptides, dentin sialoprotein and dentin phosphoprotein, that are localized within the dentin matrix. Recently, mutations in this gene were identified in human dentinogenesis imperfecta II (Online Mendelian Inheritance in Man (OMIM) accession number 125490) and in dentin dysplasia II (OMIM accession number 125420) syndromes. Herein, we report the generation of Dspp-null mice that develop tooth defects similar to human dentinogenesis imperfecta III with enlarged pulp chambers, increased width of predentin zone, hypomineralization, and pulp exposure. Electron microscopy revealed an irregular mineralization front and a lack of calcospherites coalescence in the dentin. Interestingly, the levels of biglycan and decorin, small leucine-rich proteoglycans, were increased in the widened predentin zone and in void spaces among the calcospherites in the dentin of null teeth. These enhanced levels correlate well with the defective regions in mineralization and further indicate that these molecules may adversely affect the dentin mineralization process by interfering with coalescence of calcospherites. Overall, our results identify a crucial role for Dspp in orchestrating the events essential during dentin mineralization, including potential regulation of proteoglycan levels.

Induction of apoptosis in skeletal tissues: phosphate-mediated chick chondrocyte apoptosis is calcium dependent.

In an earlier study, we have shown that Pi induced apoptosis of terminally differentiated hypertrophic chondrocytes. To ascertain whether Ca2+ modulates Pi-induced cell death, we asked the following two questions: First, can we prevent Pi-induced apoptosis by removing Ca2+ from the culture medium; alternatively, can we potentiate cell death by increasing the Ca2+ concentration? Second, can we inhibit chondrocyte apoptosis by blocking Pi transport? We also explored the mechanism of apoptosis by evaluating mitochondrial activity and reactive oxygen species (ROS) generation in cells treated with the ion pair. We noted that EDTA and EGTA blocked Pi-induced apoptosis in a dose-dependent manner. While high levels of Ca2+ alone had little effect on chondrocyte viability, the cation enhanced Pi-dependent cell death and greatly increased Pi uptake. When Pi transport was blocked, there was complete inhibition of cell killing. The process of cell death was characterized by mitochondrial hyperpolarization; two hours following apoptogen treatment, there was a significant decrease in the mitochondrial membrane potential. Coincident with the changes in mitochondrial function, there was an increase in intracellular Ca2+ that was maintained throughout the experimental period. A raised Ca2+ signal was observed in blebs at the cell membrane. Finally, we noted that, 75 minutes after treatment with the ion pair, there was a six-fold elevation in ROS levels. This increase declined to baseline values after three hours. Based on these observations, we suggest that, at the cartilage mineralization front, an elevation in local environmental Ca2+ and Pi concentrations modulates oxidative metabolism, and triggers apoptosis of terminally differentiated chondrocytes.

Mineralized microstructure of calcified avian tendons: a scanning small angle X-ray scattering study.

The micrometer level spatial distribution of the size, shape, and orientation of mineral crystallites in the calcifying matrix of tendons near the edge of the mineralizing front was investigated by scanning small angle X-ray scattering using synchrotron X-ray radiation. Using a special microbeam arrangement enabling 20 microm beam resolution and short measurement times, linear diffraction scans were made on sections from the normally calcifying tendons (tibialis cranialis) from the domestic turkey, which calcify in the distal to proximal direction. A change in shape and arrangement of mineral crystals was observed within the first 200 microm of the mineralization front, and the mineral crystal distribution was highly anisotropic with crystals aligned parallel to the fiber axis. In a cross-section of the tendon cut at right angles to the fiber axis, the orientation distribution of crystals was not azimuthally symmetric, and showed a small but nonzero anisotropy and a continuous change in mean orientation angle across the width of the tendon cross-section.

From serum to the mineral phase. The role of the odontoblast in calcium transport and mineral formation.

Dentin may be considered as a calcified connective tissue and is in its composition as well as in its mode of formation closely related to bone. Dentin is formed by two simultaneous processes in which the odontoblasts are instrumental: the formation of the proteinaceous dentin matrix, and mineral crystal formation in this matrix. As part of this, the odontoblasts actively transport Ca2+ ions towards the site of mineral formation. The cells maintain a delicate intracellular Ca2+ ion balance by the concerted action of transmembraneous transport mechanisms, including Ca-ATPase, Na+/Ca2+ exchangers and calcium channels of the L-type, and possibly intracellular Ca(2+)-binding proteins. The net effect of this is a maintenance of a cytoplasmic sub-micromolar Ca2+ activity and an extracellular accumulation of Ca2+ ions at the mineralization front. In addition to the major matrix constituent, collagen, non-collagenous macromolecules, such as dentin phosphoprotein (phosphophoryn), dentin sialoprotein, and proteoglycan, are synthesized by the odontoblasts and deposited in the matrix. Such polyanionic macromolecules are presumably responsible for the extracellular induction of hydroxyapatite crystals, but may also function to inhibit mineral growth and to regulate crystal size. Accordingly, it can be concluded that dentinogenesis comprises an interplay between several factors in the tissue, cellular as well as extracellular.

Dentin Extracellular Matrix (ECM) Proteins: Comparison to Bone ECM and Contribution to Dynamics of Dentinogenesis

Dentinogenesis involves the initial odontoblastic synthesis of a collagen-rich extracellular matrix (ECM) and predentin that is converted to dentin when the collagen fibrils become mineralized. Since the width of predentin is rather uniform, we postulate that extracellular events regulate dentinogenesis. Similarly, osteogenesis involves an initial unmineralized osteoid that is mineralized and converted to bone. To gain insights into these two processes, we compared ECM proteins in bone with those in dentin, focusing upon the sialic acid (SA)-rich proteins. We observed qualitative similarities between the SA-rich proteins, but distinct differences in the amounts of osteopontin (OPN) and dentin sialoprotein (DSP). OPN, a predominant protein in bone, was found in much smaller amounts in dentin. Conversely, DSP was abundant in dentin ECM, but found sparingly in bone. Molecular cloning experiments indicate that coding sequences for DSP and dentin phosphoprotein (DPP) are found on the same mRNA. We believe that the initial form of the precursor protein DSPP is inactive in influencing the mineralization process and that it must be activated by cleavage of peptide bonds in conserved regions. Thus, unknown proteinases would act on DSPP, possibly at the mineralization front, and liberate active DPP, which plays an initiation and regulatory role in the formation of apatite crystals. This post-translational processing reaction would represent an important control point in dentinogenesis. Recently, we identified uncleaved DSPP in dentin extracts, which should allow us to test portions of our hypothesis.

Dentin Extracellular Matrix (ECM) Proteins: Comparison to Bone ECM and Contribution to Dynamics of Dentinogenesis

Dentinogenesis involves the initial odontoblastic synthesis of a collagen-rich extracellular matrix (ECM) and predentin that is converted to dentin when the collagen fibrils become mineralized. Since the width of predentin is rather uniform, we postulate that extracellular events regulate dentinogenesis. Similarly, osteogenesis involves an initial unmineralized osteoid that is mineralized and converted to bone. To gain insights into these two processes, we compared ECM proteins in bone with those in dentin, focusing upon the sialic acid (SA)-rich proteins. We observed qualitative similarities between the SA-rich proteins, but distinct differences in the amounts of osteopontin (OPN) and dentin sialoprotein (DSP). OPN, a predominant protein in bone, was found in much smaller amounts in dentin. Conversely, DSP was abundant in dentin ECM, but found sparingly in bone. Molecular cloning experiments indicate that coding sequences for DSP and dentin phosphoprotein (DPP) are found on the same mRNA. We believe that the initial form of the precursor protein DSPP is inactive in influencing the mineralization process and that it must be activated by cleavage of peptide bonds in conserved regions. Thus, unknown proteinases would act on DSPP, possibly at the mineralization front, and liberate active DPP, which plays an initiation and regulatory role in the formation of apatite crystals. This post-translational processing reaction would represent an important control point in dentinogenesis. Recently, we identified uncleaved DSPP in dentin extracts, which should allow us to test portions of our hypothesis.

Rapid new bone tissue remodeling during distraction osteogenesis is associated with apoptosis

During the process of distraction osteogenesis new bone forms and undergoes rapid remodeling. Apoptosis may be one of the regulatory mechanisms governing the removal of the redundant callus during distraction osteogenesis. A rabbit tibial lengthening model was used and lengthened at 0.7 mm/day for 3 weeks. The regenerating tissues from the distraction gap were examined for apoptotic changes by transmission electron microscopy (TEM) and the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) method. Osteoclastic bone resorption activities were demonstrated by tartrate resistant acid phosphatase (TRAP) staining. The apoptotic cells were mainly present in the transitional regions between the fibrous tissue and the new bone in the mineralization front, and close to or on the new bone surfaces near the center of the regenerate. The TUNEL labeling was greatly reduced in the mature bone near the osteotomied bone ends. TEM examination confirmed the presence of cells with apoptotic changes at various regions of the regenerate. TRAP staining revealed that osteoclastic bone resorption activities in the regenerate were in a similar pattern of distribution to those of the TUNEL labeling. The localization of apoptotic cells at the different regions of the regenerate, accompanied by the osteoclast activities, suggest that apoptosis is closely related to bone formation and remodeling during distraction osteogenesis.

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.

Aluminium and bone disease in chronic renal failure.

Aluminium is absorbed by the intestines and is rapidly transported into bone, where it disrupts mineralization and bone cell growth and activity. Its toxicities result in or exacerbate painful forms of renal osteodystrophy, most notably adynamic bone disease and osteomalacia, but also other forms of the disease. Because aluminium is sequestered in bone for long periods, its toxic effects are cumulative. As a result, even intermittent or low-dose use of aluminium-based phosphate binders adds to the total load of this toxin in the bone; thus, aluminium use is inadvisable, even for a 'rescue indication'. Aluminium blood levels are not a reliable marker of aluminium absorption or organ load in dialysis patients: only stainable aluminium at the mineralization front reflects the histopathological changes observed in bone. Therefore, bone biopsies remain the only approach for definitive diagnosis of aluminium-related bone disease. Most importantly, lack of correlation between overall organ concentrations of a toxin, such as aluminium, and pathological changes does not rule out toxicity. Thus, the specific localization of the toxin is more important than overall organ concentration. What has been observed with aluminium during 25 years of research might be reproduced with other metals that are absorbed, transported and accumulated in bone. What we have learned about the toxicity of aluminium should inform our interpretation of data from studies of other metal-based therapeutics for renal patients. This calls for careful evaluation of any newly introduced therapeutic agents for bone disease in patients lacking excretory kidney function.

Expression, Structure, and Function of Enamel Proteinases

Proteinases serve two important functions during dental enamel formation: They (a) process and (b) degrade enamel proteins. Different enzymes carry out these functions. Enamelysin (MMP-20) is the foremost enamel matrix-processing enzyme. Its expression initiates prior to the onset of dentin mineralization and continues throughout the secretory stage of amelogenesis. In vitro, enamelysin catalyzes all of the amelogenin cleavages that are known to occur during the secretory stage in vivo, and it is probably the enzyme responsible for the processing of all enamel proteins. There is evidence suggesting that enamelysin activity is critical for proper enamel formation. Uncleaved and processed enamel proteins often segregate into different compartments within the developing enamel layer, suggesting that they may have different functions. Intact ameloblastin and its C-terminal cleavage products localize in the superficial rod and interrod enamel, while its N-terminal cleavage products congregate in the sheath space. Intact enamelin is only present at the mineralization front within a micrometer of the enamel surface, while its cleavage products concentrate in the rod and interrod enamel. Processed enamel proteins accumulate during the secretory stage, but disappear early in the maturation stage. Enamel matrix serine proteinase 1 (EMSP1), now officially designated kallikrein 4 (KLK4), is believed to be the predominant degradative enzyme that clears enamel proteins from the matrix during maturation. KLK4 expression initiates during the transition stage and continues throughout maturation. KLK4 concentrates at the enamel surface when the enamel matrix disappears, and aggressively degrades amelogenin in vitro. During tooth development, proteinases are secreted by ameloblasts into the extracellular space, where they cleave enamel proteins by catalyzing the hydrolysis of peptide bonds. Enamel proteinases are present in low abundance and are not likely to participate directly in the mineralization process. Two major enamel proteinases have been identified: enamelysin (MMP20) and kallikrein 4 (KLK4). These proteinases are expressed at different times and have different functions. Their roles are to modify and/or to eliminate enamel matrix proteins, which affects the way enamel proteins interact with each other and with the developing enamel crystallites. A brief review of dental enamel formation is presented, followed by a more detailed analysis of enamelysin and KLK4 expression, structure, and function.

Fourier transform infrared microspectroscopic investigation of the maturation of nonstoichiometric apatites in mineralized tissues: a horse dentin study

Fourier transform infrared microspectroscopy (FTIRM) was used to study carbonated apatite/collagen interactions and maturation in horse secondary dentin. Unlike human dentin, this model contains no peritubular material around the odontoblastic processes and is thus quite similar to bone in composition, but not subject to tissue turnover. Crystals close to the mineralization front were very immature, showing high HPO 4 and very low CO 3 levels. Carbonate ions were located essentially in very labile, reactive environments, probably on the crystal surface. Removal of some of the HPO 4 ions from crystals during maturation was linked to an increase in total carbonate content. The CO 3 ions in labile environments decreased, probably after incorporation into more organized regions of the lattice. However, this increase of total carbonate content was associated with greater mineral crystallinity, confirming findings in other studies of synthetic apatite maturation in vitro. The good correlation between these results and those of in vitro experiments suggests that crystal maturation is essentially due to physicochemical processes and that the organic matrix controls only crystal size, multiplication, and/or organization.