Immunohistochemical Identification of Decorin and Biglycan in Human Dentin: A Correlative Field Emission Scanning Electron Microscopy/Transmission Electron Microscopy Study

Decorin and biglycan, two small leucine-rich proteoglycans, have been proposed to play important roles in matrix-mediated formation of mineralized tissues, and their three-dimensional arrangement in human dentin is still not completely understood. The aim of this study was to immunohistochemically analyze the distribution of decorin and biglycan in human predentin/dentin organic matrix under a high-resolution field emission in-lens scanning electron microscope (FEI-SEM) and a transmission electron microscope (TEM). Tooth dentin specimens were submitted to either a preembedding or a postembedding immunolabeling technique using primary antibodies antidecorin and antibiglycan and gold-conjugated secondary antibodies. Correlative FEI-SEM/TEM observations showed that the two antibodies yielded a similar labeling pattern over the processes of odontoblasts and the predentin. Decorin and biglycan were mainly associated with the collagen fibers within the predentin layer, revealing a moderate immunoreaction that was significantly higher compared to the one observed on dentin. Thus, a generally weak labeling for decorin was found in dentin, which, however, was significantly higher on odontoblast processes within dentinal tubules than in intertubular dentin. On the other hand, biglycan immunolocalization on dentin revealed few gold particles rather uniformly distributed, without showing significant differences between tubular and intertubular regions. In conclusion, this study reveals distinct distribution patterns of decorin and biglycan and their relation with collagen. Decorin’s and biglycan’s precise roles within prematrix and mineralized matrix in human teeth should be further clarified.     

Immunoelectron Microscopic Visualization of Pro- and Secreted Forms of Decorin and Biglycan in the Predentin and During Dentin Formation in the Rat Incisor

Using antibodies raised against the proform and fully processed (secreted) forms of the proteoglycans decorin and biglycan, combined with gold electron immunohistochemistry, we observed in the incisors of five Sprague-Dawley rats that the proforms were mostly located in the cell bodies of odontoblasts, with a presence reduced to one-third or one-fourth in the processes. Proforms, also present in the extracellular matrix, were uniformly distributed throughout predentin, with higher labeling for probiglycan than pro-decorin. Both were present in lesser amounts in metadentin and dentin. With respect to the secreted form, grain density was at a constant level for biglycan in predentin and dentin, whereas a gradient was detected for decorin, the grain density being increased three times in the distal predentin. Although decorin labeling was diminished in metadentin, staining in circumpulpal dentin was similar to that found in distal predentin. We have previously reported a reverse gradient for chondroitin sulfate/dermatan sulfate distribution. To reconcile these diverging data, our hypothesis is that enzymatic proteolytic cleavage may remove the glycosylated N-terminal-containing region, resulting in a non- proteoglycan form of the molecule. Although substantial differences in distribution were apparent between the two proteoglycans, increasing interactions between proteoglycans and collagen, facilitated by the cleavage and loss of the N-terminal glycosaminoglycan chain region in the distal predentin, may be a prerequisite for dentin mineralization.     

Deletion of Serotonin 2B Receptor Provokes Structural Alterations of Mouse Dental Tissues

Rampant caries and periodontal diseases occur in patients treated with antidepressants such as serotonin reuptake inhibitors (SRIs; e.g., Prozac) which target the serotonin transporter (SERT). As the serotonin 2B receptor (5HT_2BR) regulates SERT functionality and capacity to recognize SRIs, we investigated the potential role of 5HT_2BR on dental tissues by exploiting 5HT_2BR knockout (KO) mice. Compared to wild-type (WT) mice, several structural differences were identified in the teeth of KO mice. In the molar of KO mice, rod curvatures and twisting were altered compared to WT mice, suggesting involvement of 5HT_2BR at early stages of enamel formation. The volume of the KO enamel layer was also reduced, and larger porosities were observed in the prismatic enamel, with smaller crystallite thickness. Crystallite pattern disorganization and occlusal abrasion were enhanced in female KO mice, indicating a sexual dimorphism. In the incisor, no difference was detected in the width of the enamel layer between KO and WT mice; however, enamel maturation differed in absence of 5HT_2BR. Specifically, the outer aprismatic enamel border was 1.5- to 2-fold larger in KO compared to WT mice, together with a decreased etching pattern. Finally, although no noticeable difference was observed in dentin, the micro-CT three-dimensional pulp reconstruction evidenced a decrease in both length and width of dentin formation in the root canals of the KO versus WT mice. These data provide evidence that 5HT_2BR-mediated signaling pathways are involved in enamel formation and dentinogenesis.     

TGF-β/Extracellular Matrix Interactions in Dentin Matrix: A Role in Regulating Sequestration and Protection of Bioactivity

TGF-β isoforms sequestrated in dentin matrix potentially provide a reservoir of bioactive molecules that may influence cell behavior in the dentin–pulp complex following tissue injury. The association of these growth factors with dentin matrix and the influence of such associations on the bioactivity of growth factors are still unclear. We used surface plasmon resonance technology in the BIAcore^TM 3000 system to investigate the binding of TGF-β isoforms 1 and 3 to purified decorin, biglycan, and EDTA soluble dentin matrix components. TGF-β isoforms 1 and 3 were immobilized on sensorchips CM4 through amine coupling. For kinetic studies of protein binding, purified decorin and biglycan, isolated EDTA soluble dentin matrix, and dentin matrix immunodepleted of decorin and/or biglycan were injected over TGF-β isoforms and allowed to interact. Programmed kinetic analysis software provided sensorgrams for each concentration of proteoglycan or dentin matrix extract injected. Purified decorin and biglycan and dentin matrix extract bound to the TGF-β isoforms. However, the association with TGF-β3 was much weaker than that with TGF-β1. After immunoaffinity depletion of the dentin matrix extract, the level of interaction between the dentin matrix extract and TGF-β was significantly reduced. These results suggest isoform-specific interactions between decorin/biglycan and TGF-β isoforms 1 and 3, which may explain why TGF-β3 is not detected in the dentin matrix despite being expressed at higher levels than TGF-β1 in odontoblasts. These proteoglycans appear to play a significant role in TGF-β/extracellular matrix interactions and may be important in the sequestration of these growth factors in the dentin matrix.     

Ameloblastin Fusion Protein Enhances Pulpal Healing and Dentin Formation in Porcine Teeth

Ameloblastin (Ambn, also named “amelin” or “sheathlin”) is a protein participating in enamel formation and mesenchymal-ectodermal interaction during early dentin formation in developing teeth. Experiments have demonstrated an association between Ambn expression and healing of acute pulp wounds. The purpose of this study was to investigate if local application of recombinant fusion Ambn (rAmbn) could influence reparative dentin formation in pulpotomized teeth. In this randomized, double-blinded study, pulpotomy was performed in 28 lower central incisors in 17 adult miniature pigs. Following the surgical procedure, the exposed pulp tissue was covered either with rAmbn or with calcium hydroxide. After 2, 4, or 8 weeks, the teeth were extracted and examined by histomorphometry and immunohistochemistry using antibodies against porcine ameloblastin, collagen type I, and dentin sialoprotein (DSP). In rAmbn-treated teeth, a substantial amount of newly formed reparative dentin was observed at the application site, completely bridging the pulpal wound. Dentin formation was also observed in calcium hydroxide-treated teeth; however, the amount of reparative dentin was significantly smaller (P < 0.001) than after rAmbn treatment. Immunohistochemistry confirmed that the new hard tissue formed was similar to dentin. This is the first time a direct link between ameloblastin and dentin formation has been made in vivo. The results suggest potential for rAmbn as a biologically active pulp-dressing agent for enhanced pulpal wound healing and reparative dentin formation after pulpotomy procedures.     

Wnt Signaling Regulates Pulp Volume and Dentin Thickness

Odontoblasts, cementoblasts, ameloblasts, and osteoblasts all form mineralized tissues in the craniofacial complex, and all these cell types exhibit active Wnt signaling during postnatal life. We set out to understand the functions of this Wnt signaling, by evaluating the phenotypes of mice in which the essential Wnt chaperone protein, Wntless was eliminated. The deletion of Wls was restricted to cells expressing Osteocalcin (OCN), which in addition to osteoblasts includes odontoblasts, cementoblasts, and ameloblasts. Dentin, cementum, enamel, and bone all formed in OCN‐Cre;Wls^fl/fl mice but their homeostasis was dramatically affected. The most notable feature was a significant increase in dentin volume and density. We attribute this gain in dentin volume to a Wnt‐mediated misregulation of Runx2. Normally, Wnt signaling stimulates Runx2, which in turn inhibits dentin sialoprotein (DSP); this inhibition must be relieved for odontoblasts to differentiate. In OCN‐Cre;Wls^fl/fl mice, Wnt pathway activation is reduced and Runx2 levels decline. The Runx2‐mediated repression of DSP is relieved and odontoblast differentiation is accordingly enhanced. This study demonstrates the importance of Wnt signaling in the homeostasis of mineralized tissues of the craniofacial complex. © 2014 American Society for Bone and Mineral Research.     

Expression of decorin, biglycan, and collagen type I in human renal fibrosing disease

BACKGROUND: The extracellular matrix proteoglycans decorin and biglycan may have a pathogenic role in renal fibrosing disease via regulation of the activity of growth factors, such as transforming growth factor-beta, and effects on collagen type I fibrillogenesis. The expression of decorin and biglycan in human glomerular diseases characterized by mesangial sclerosis is unknown. METHODS: Decorin, biglycan, and collagen type I were localized immunohistochemically in human renal biopsy cases of amyloidosis (N = 18), diabetic nephropathy (N = 11), fibrillary glomerulonephritis (N = 5), immunotactoid glomerulopathy (N = 5), light-chain deposition disease (N = 4), idiopathic mesangial sclerosis (N = 4), and nephrosclerosis (N = 6), and in morphologically normal tissues obtained from tumor nephrectomies (N = 8). Decorin and biglycan mRNA synthesis was evaluated by in situ hybridization. RESULTS: Decorin and biglycan protein were not identified in normal glomeruli. Decorin accumulated in amyloid deposits, but not in deposits of fibrillary glomerulonephritis or immunotactoid glomerulopathy. Biglycan weakly accumulated in amyloid deposits, and both decorin and biglycan weakly stained mesangial nodules in cases of morphologically advanced light-chain deposition disease and diabetic nephropathy. In all analyzed cases, irrespective of the underlying disease, decorin and biglycan accumulated in glomeruli in areas of fibrous organization of the urinary space and in areas of tubulointerstitial fibrosis. Biglycan, but not decorin, accumulated in the neointima of arteriosclerotic blood vessels. Decorin and biglycan mRNA synthesis was detected at sites of proteoglycan accumulation in glomeruli, interstitium, and neointima. Collagen type I colocalized with decorin and biglycan deposits. CONCLUSIONS: Differences in extracellular matrix proteoglycan composition may be diagnostically useful in distinguishing morphologically similar diseases. Distinct patterns of proteoglycan expression may be related to modulation of specific growth factor activity in different glomerular diseases.     

Biglycan modulates osteoblast differentiation and matrix mineralization.

MC3T3-E1 cell-derived clones expressing higher (S) or lower (AS) levels of biglycan were generated and characterized. The processes of cell differentiation and matrix mineralization were accelerated in S but delayed in AS, indicating that BGN modulates osteoblastic cell differentiation. INTRODUCTION: Biglycan (BGN), a member of the small leucine-rich proteoglycan family, is one of the major proteoglycans found in bone and has been implicated in bone formation. In this study, the effects of over- or underexpression of BGN on osteoblastic cell phenotypes and matrix mineralization were studied. MATERIALS AND METHODS: MC3T3-E1 cells were transfected with vectors containing the BGN cDNA in a sense or antisense orientation to generate clones expressing higher (S clones) or lower (AS clones) levels of BGN. MC3T3-E1 cells and those transfected with an empty vector (EV) were used as controls. The levels of BGN synthesized by these clones were evaluated by Western blot analysis. Cell growth was analyzed by cell counting and cell differentiation by the gene expression patterns of several osteoblastic markers using quantitative real-time PCR. The abilities of these clones to form mineralized matrices were evaluated by in vitro and in vivo mineralization assays. Furthermore, the clones were treated with BMP-4 and their responsiveness was assessed. RESULTS: The cell growth in these clones was unaffected; however, osteoblast differentiation was significantly accelerated in S clones and suppressed in AS clones. The in vitro matrix mineralization in S clones was significantly enhanced but severely impaired in AS clones. When transplanted into immunodeficient mice, S clone transplants exhibited larger areas of lamellar bonelike matrices, whereas only minute amounts of woven bone-type structure was found in AS transplants. The response to BMP-4 was higher in S clones but poorer in AS clones compared with that of controls. CONCLUSIONS: BGN modulates osteoblast differentiation, possibly by regulating BMP signaling, and consequently matrix mineralization.     

Glomerular expression of biglycan and decorin and urinary levels of decorin in primary glomerular disease

AIMS: Recent studies have suggested that small leucine-rich proteoglycans (SLRP) of the extracellular matrix play a major role in modulating the activity of growth factors and in regulating the deposition of collagens. In this study, the expression of the SLRPs biglycan and decorin in the glomeruli of patients with primary glomerular disease (minimal change disease, IgA nephropathy, and membranous nephropathy) and urine immunoreactive levels examined. METHODS: Renal biopsy specimens were obtained from patients with minimal change disease, IgA nephropathy and membranous nephropathy. Immunohistochemical staining was performed on fresh-frozen samples using anti-biglycan and anti-decorin antibodies. Examination of urine proteoglycan excretion from a total of 26 patients and 8 normal volunteers was performed by indirect ELISA. RESULTS: In normal kidney samples, biglycan and decorin expression was found predominantly in the intrarenal arteries and tubulointerstitium, with only minimal expression in the glomeruli. Glomerular expression of these proteoglycans in glomerular disease was unchanged in all of the 4 patients examined with minimal change disease. In the case of IgA nephropathy or membranous nephropathy, some of the patients showed minimally increased immunostaining of either biglycan or decorin, but there were no signs of simultaneous upregulation of both proteoglycans. To further examine the changes in proteoglycan expression, ELISA was performed on urine samples. Urine biglycan levels were below detection levels, but high values of urine decorin immunoreactivity were found in the patients with glomerular disease. A significant negative correlation was found between urine decorin and creatinine clearance. CONCLUSION: These results suggest that distinct changes in the expression of the SLRPs biglycan and decorin may be seen in patients with primary glomerular disease. Moreover, the negative relationship between urine decorin levels and renal function supports the hypothesis that decorin may be involved in the pathophysiology of renal dysfunction in humans.     

Immunohistochemical Characterization of Rapid Dentin Formation Induced by Enamel Matrix Derivative

The purpose of this study was to examine the pulpal expression of dentin-related proteins during enamel matrix derivative (EMD)–induced reparative dentin formation in a pulpotomy model in pig incisors. Pulpotomies were performed on 72 lower incisors in 24 adult miniature swine. The exposed pulp tissue was treated with EMD or covered with a calcium hydroxide paste (Dycal^®). At predefined time-points, ranging from 4 days to 12 weeks, experimental teeth were extracted and examined by use of light microscopy, and expression of dentin-related proteins in the pulps was investigated by immunohistochemistry, using antibodies against type I collagen, dentin sialoprotein (DSP), sheathlin, and EMD. In all EMD-treated teeth a substantial amount of reparative dentin formation was observed. The amount of reparative dentin in calcium hydroxide–treated teeth was significantly smaller than in EMD-treated teeth (P < 0.005) and was less effective in bridging the pulpal wounds. Immunohistochemistry demonstrated that enamel matrix proteins were present in detectable amounts at the application site for about 4 weeks. Moreover, the expression of proteins related to dentin formation in the wounded pulp tissue was about 2 weeks advanced in EMD-treated teeth. These findings demonstrate that enamel matrix molecules have the capacity to induce rapid pulpal wound healing in pulpotomized teeth, and suggest that the longevity and continued presence of enamel matrix macromolecules at the application site can be utilized to stimulate growth and repair of dentin over a period consistent with a favorable clinical outcome.     

Transforming growth factor-beta regulates production of proteoglycans by mesangial cells

Accumulation of glomerular extracellular matrix is a prominent feature of most forms of progressive glomerular disease. Since some growth factors may play a role in extracellular matrix production, we examined the effects of transforming growth factor-beta (TGF-beta), interleukin 1, platelet derived growth factor, and tumor necrosis factor on the production of extracellular matrix components by cultured rat mesangial cells. In control experiments we found that mesangial cells produced two distinct proteoglycans identified as the small chondroitin/dermatan sulfate proteoglycans biglycan (PG I) and decorin (PG II) by showing that their mobility on SDS-PAGE changed upon digestion by chondroitinase ABC, and that they reacted with antibodies raised against synthetic peptides from the core protein sequence of human biglycan and decorin. Exposure to TGF-beta for 48 hours stimulated an 8- to 10-fold increase in the biglycan and decorin bands, and induced a structural change detected as a shift in electrophoretic mobility. TGF-beta did not demonstrably affect the production of other matrix proteins by the mesangial cells. The other growth factors tested had no comparable effect on the production of proteoglycans or other extracellular matrix components by these cells. Our results show that TGF-beta is unique among growth factors in its regulatory effects on mesangial cell proteoglycan production. The release or activation of TGF-beta during glomerular injury could mediate the accumulation of proteoglycans in the extracellular matrix and predispose the kidney to development of glomerulosclerosis.     

EMMPRIN/CD147 deficiency disturbs ameloblast–odontoblast cross-talk and delays enamel mineralization

Tooth development is regulated by a series of reciprocal inductive signaling between the dental epithelium and mesenchyme, which culminates with the formation of dentin and enamel. EMMPRIN/CD147 is an Extracellular Matrix MetalloPRoteinase (MMP) INducer that mediates epithelial–mesenchymal interactions in cancer and other pathological processes and is expressed in developing teeth. Here we used EMMPRIN knockout (KO) mice to determine the functional role of EMMPRIN on dental tissue formation. We report a delay in enamel deposition and formation that is clearly distinguishable in the growing incisor and associated with a significant reduction of MMP-3 and MMP-20 expression in tooth germs of KO mice. Insufficient basement membrane degradation is evidenced by a persistent laminin immunostaining, resulting in a delay of both odontoblast and ameloblast differentiation. Consequently, enamel volume and thickness are decreased in adult mutant teeth but enamel maturation and tooth morphology are normal, as shown by micro-computed tomographic (micro-CT), nanoindentation, and scanning electron microscope analyses. In addition, the dentino-enamel junction appears as a rough calcified layer of approximately 10 ± 5 μm thick (mean ± SD) in both molars and growing incisors of KO adult mice. These results indicate that EMMPRIN is involved in the epithelial-mesenchymal cross-talk during tooth development by regulating the expression of MMPs. The mild tooth phenotype observed in EMMPRIN KO mice suggests that the direct effect of EMMPRIN may be limited to a short time window, comprised between basement membrane degradation allowing direct cell contact and calcified matrix deposition.     

Dentin Sialoprotein (DSP) Has Limited Effects on In Vitro Apatite Formation and Growth

Sialoproteins such as bone sialoprotein (BSP) and dentin sialoprotein (DSP) accumulate at the mineralization fronts in bone and dentin, respectively, suggesting they have some function in the mineralization process. BSP, a highly phosphorylated protein rich in polyglutamate repeats, is an effective nucleator of hydroxyapatite (HA) formation in vitro. The present study examines the effect of DSP, a low phosphorylated but related sialoprotein, on the formation and growth of HA. In vitro, in a gelatin gel diffusion system, DSP at low concentrations (<25 μg/ml) slightly increased the yield of HA formed at 3.5 and 5 days, while at higher concentrations (50–100 μg/ml) it slightly inhibited accumulation. Fewer mineral crystals were formed in the presence of high concentrations of DSP but they tended to aggregate (making them appear larger by electron microscopic analysis) than those formed in DSP-free gels. X-ray diffraction line broadening analysis failed to show significant changes in c-axis crystal dimensions with increasing DSP concentration. When HA-seed crystals were coated with DSP before inclusion in the gelatin gel there was a reduction in mineral accumulation relative to HA-seeds which had not been coated with DSP, but the extent of inhibition was significantly less than that seen in this system with other mineralized tissue matrix sialoproteins, such as osteopontin or BSP. The low affinity of DSP for well-characterized seed crystals and the limited effect of this protein on HA formation and growth suggest that the role of DSP in dentin is not primarily that of a mineralization regulator. Received: 25 April 2000 / Accepted: 21 July 2000 / Online publication: 2 November 2000     

Increased Turnover of Small Proteoglycans Synthesized by Human Osteoblasts During Cultivation with Ascorbate and β-Glycerophosphate

The small proteoglycan decorin had been localized previously at the d-band in the gap zone of collagen fibrils in nonmineralizing tissues. In bone matrix this zone is proteoglycan free and is at least in some species the place where mineralization along collagen fibrils starts. To study the metabolism of the small proteoglycans decorin and biglycan under mineralizing conditions, osteoblasts from human nasal bone were cultured for several weeks in the presence or absence of β-glycerophosphate and ascorbate. An immediate consequence of the treatment was a reduced expression of decorin, as judged by immune precipitation, whereas the biosynthesis of biglycan was not affected. Pulse-chase experiments were performed with osteoblasts embedded in floating type I collagen gels. In the presence of β-glycerophosphate and ascorbate, a more rapid turnover of both proteoglycans was noted; the one of biglycan reached statistical significance. Indirect evidence for an enhanced rate of proteoglycan endocytosis was obtained. This effect was not seen in cultured skin fibroblasts. Thus, osteoblasts respond rapidly to mineralizing conditions with alterations of small proteoglycan biosynthesis and turnover. Received: 30 October 1995 / Accepted: 3 January 1997     

Up-regulation of extracellular matrix proteoglycans and collagen type I in human crescentic glomerulonephritis

BACKGROUND: The pathogenesis of crescentic glomerulonephritis (CGN) involves cellular migration and proliferation in the urinary space, frequently followed by fibrous organization. Extracellular matrix proteoglycans (PGs) may regulate these events via effects on cellular migration, interactions with growth factors, including transforming growth factor-beta (TGF-beta), and control of collagen fibrillogenesis. The expression of PG in human CGN is unknown. METHODS: Renal tissues from 18 patients with CGN were examined immunohistochemically for versican, decorin, biglycan and collagen type I, and were compared with morphologically normal tissues from six tumor nephrectomies. Synthesis of decorin, biglycan, and procollagen type I mRNAs was evaluated by in situ hybridization. RESULTS: Versican was strongly expressed in cellular crescents and periglomerular areas, whereas decorin and biglycan accumulated in collagen type I-enriched regions, including fibrocellular and fibrous crescents, and interstitial fibrosis. PG and collagen type I accumulation colocalized with myofibroblasts in crescents, periglomerular areas, and interstitium. CONCLUSIONS: The temporal and spatial patterns of expression demonstrated in this study provide evidence to support pathogenic roles for PG in the evolution of CGN. Based on known biological properties of this molecule, versican may facilitate migration of cells in developing crescents. Decorin and biglycan may contribute to progression of CGN, perhaps via interactions with collagen type I in the remodeled extracellular matrix.     

Effects of Bone CS-Proteoglycans, DS-Decorin, and DS-Biglycan on Hydroxyapatite Formation in a Gelatin Gel

The small leucine-rich bone proteoglycans, biglycan and decorin, can be purified by chromatography on hydroxyapatite columns, demonstrating their potential affinities for bone apatite. To determine their effects on in vitro apatite formation and growth, a mixture of the chondroitin-sulfate (CS) bone proteoglycans, or purified fractions of the dermatan sulfate (DS) containing proteoglycans, DS-decorin and DS-biglycan obtained from skin and articular cartilage, respectively, were analyzed in a gelatin gel diffusion system in which apatite formation occurs in the absence of proteins in a 3.5 day period. Low concentrations of the bone CS-proteoglycan mixture and low DS-biglycan concentrations (5–25 μg/ml) increased apatite formation relative to proteoglycan-free controls at 3.5 days. The CS-proteoglycan mixture was less effective at 50 μg/ml than at 10 μg/ml. DS-biglycan was similarly most effective at 5–25 μg/ml. At 5 days, when apatite growth and proliferation were assessed, 10 and 50 μg/ml of both CS-bone proteoglycan and DS-biglycan increased mineral yields. DS-decorin, in contrast, had no significant effect on mineral accumulation at any of these concentrations. In seeded growth experiments, 1 and 10 μg/ml CS-proteoglycan and 10 and 50 μg/ml DS-biglycan were significant effective inhibitors of mineral accretion, whereas DS-decorin showed no tendency to inhibit seeded growth. Using molar extinction coefficients to determine concentrations, the binding of DS-biglycan and DS-decorin to apatite (specific surface 54 m²/g) was determined using a Langmuir adsorption isotherm model. DS-biglycan had a greater affinity for apatite than DS-decorin (0.285 ml/μmol versus 0.0098 ml/μmol). DS-biglycan binding was more specific with fewer binding sites (3.5 μmol/m² compared with 18.2 μmol/m² for DS-decorin). Data suggest that of the small proteoglycans, biglycan may play a more significant role than decorin in the regulation of mineralization. Received: 10 June 1996 / Accepted: 25 April 1997     

Binding of Dentin Noncollagenous Matrix Proteins to Biological Mineral Crystals: An Atomic Force Microscopy Study

Noncollagenous matrix proteins (NCPs) of dental hard tissues (dentin, cementum) are involved, both temporally and spatially, in the mineralization of their collagen matrices. Two of the NCPs thought to initiate mineral nucleation and control crystal growth in dentin, are dentin phosphoproteins (DPP) and dentin sialoprotein (DSP). Control of crystal growth would depend on the binding capacity of these two molecules, which may be related to the charge domains on the crystals and/or the phosphorylation of the protein. Phosphophoryn (a highly phosphorylated DPP) and DSP were isolated, purified, and characterized from the immature root apicies of human teeth. Dephosphorylation of phosphophoryn was carried out using bovine intestinal alkaline phosphatase. Enamel crystals were prepared from the maturation stage of developing rat incisor enamel. Protein-coated crystals were prepared for viewing in an atomic force microscope fluid cell using tapping mode. Desorption of the proteins was achieved using a phosphate buffer and surface roughness measurements were obtained from all specimens. Time-lapsed images of the crystals showed "nanospheres" of protein distributed along the crystals but only the phosphophoryn-coated crystals showed a distinctive banding pattern, which was still visible after the phosphate desorption experiments. The surface roughness measurements were statistically greater (P <0.01) when compared to the control for only the phosphophoryn-coated specimens. It is hypothesized that the phosphophoryn binding may be associated with charge arrays on the crystal surface and its phosphorylation. Also, based on its affinity for the crystalsurfaces, phosphophoryn seems the most likely candidate for controlling dentin crystal growth and morphology.