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Small Heterodimer Partner (SHP) interacts with diverse transcription factors such as Runx2 and regulates many cellular events including differentiation, proliferation, and energy metabolism. SHP is reported to be a positive regulator of BMP2-induced bone formation. This study aimed to clarify the role of SHP in odontoblast differentiation and matrix mineralization. Rat tooth germs were isolated, and gene expression was determined by RT-PCR and real-time PCR. Localization of SHP protein expression was identified by immunofluorescent analysis. Primary human dental pulp cells (HDPCs) were cultured with BMP2 and/or Ad-siSHP. Matrix mineralization was evaluated by Alizarin red staining. Transient transfection experiment was performed with the SHP or Dlx5 expressional plasmids and the DSPP gene. In tooth germs from post-natal days 3 to 9, BMP-2 and SHP expression increased with DSPP and DMP1 mRNA expression. In an immunostaining study, SHP was expressed in odontoblasts and surrounding osteoblasts. When HDPCs were cultured with BMP2 in mineralization-inducing medium, SHP expression also increased with an increase in DSPP expression. Down-regulation of SHP by Ad-siSHP inhibited matrix mineralization. In transient transfection experiments, overexpression of SHP was shown to enhance DSPP promoter activity through interactions between SHP and Dlx5. These results suggest that SHP may mediate BMP2 signaling to promote mineralization of the dentin matrix. [ABSTRACT FROM PUBLISHER]

Recent studies have reported collagen cross-linking after exposure to riboflavin followed by ultraviolet-A (UVA) exposure. This study is the first to investigate the effect of a riboflavin-containing primer on adhesive interface stability and dentinal matrix metalloproteinase activity. Human dentin was etched with 35% phosphoric acid, treated with 0.1% riboflavin, exposed to UVA for 2 min, and bonded with a two-step etch-and-rinse adhesive. Adhesive was applied to control specimens without riboflavin/UVA. Specimens were subjected to microtensile bond strength tests and pulled to failure after storage for 24 hrs, 6 mos, or 1 yr. Interfacial nanoleakage was evaluated by light and transmission electron microscopy. To investigate dentinal matrix metalloproteinase activity, we performed correlative zymographic assays on protein extracts obtained from phosphoric-acid-etched dentin powder with or without riboflavin/UVA treatment and XP Bond. Ultraviolet-activated riboflavin treatment increased the immediate bond strength to dentin at all aging intervals (p < 0.05 vs. control) and decreased interfacial nanoleakage in aged specimens (1 yr; p < 0.05). Zymograms revealed that riboflavin/UVA pre-treatment inhibited dentinal matrix metalloproteinase activity (especially MMP-9). In conclusion, dentinal collagen cross-linking induced by riboflavin/UVA increased immediate bond strength, stabilized the adhesive interface, and inhibited dentin matrix metalloproteinases, thereby increasing the durability of resin-dentin bonds. [ABSTRACT FROM PUBLISHER]

Incompletely infiltrated collagen fibrils in acid-etched dentin are susceptible to degradation. We hypothesize that degradation can occur in the absence of bacteria. Partially demineralized collagen matrices (DCMs) prepared from human dentin were stored in artificial saliva. Control specimens were stored in artificial saliva containing proteolytic enzyme inhibitors, or pure mineral oil. We retrieved them at 24 hrs, 90 and 250 days to examine the extent of degradation of DCM. In the 24-hour experimental and 90- and 250-day control specimens, we observed 5- to 6- μm-thick layers of DCM containing banded collagen fibrils. DCMs were almost completely destroyed in the 250-day experimental specimens, but not when incubated with enzyme inhibitors or mineral oil. Functional enzyme analysis of dentin powder revealed low levels of collagenolytic activity that was inhibited by protease inhibitors or 0.2% chlorhexidine. We hypothesize that collagen degradation occurred over time, via host-derived matrix metalloproteinases that are released slowly over time. [ABSTRACT FROM AUTHOR]

Studies of odontoblast differentiation and function have been limited due to difficulties in obtaining sufficient numbers of intact cells. We describe a novel approach of laser-capture microdissection to obtain homogenous populations of pre-odontoblasts and odontoblasts from tissue sections of mouse molar cusp tips. Fixation, processing, and staining conditions were assessed for the optimal retrieval of total RNA from microdissected odontoblasts. Fluorometric assays and RT-PCR analysis of α1 (I) collagen, dentin sialophosphoprotein (Dspp), and osteocalcin (OC) confirmed that the total RNA from three-day-old captured odontoblasts was sufficient in quantity and quality. Odontoblast-specific gene expression was studied by RT-PCR analysis performed in a single streptavidin-coated tube. At E15.5, Days 0 and 3, gene expression in laser-captured odontoblasts resembled that seen in vivo by in situ hybridization. The use of LCM is thus a valuable means of retrieving quality RNA from discrete populations of odontoblasts at different stages of dentinogenesis. [ABSTRACT FROM AUTHOR]

In dentinogenesis, certain growth factors, matrix proteoglycans, and proteins are directly or indirectly dependent on growth hormone. The hypothesis that growth hormone up-regulates the expression of enzymes, sialoproteins, and other extracellular matrix proteins implicated in the formation and mineralization of tooth and bone matrices was tested by the treatment of Lewis dwarf rats with growth hormone over 5 days. The molar teeth were processed for immunohistochemical demonstration of bone-alkaline phosphatase, bone morphogenetic proteins-2 and -4, osteocalcin, osteopontin, bone sialoprotein, and E11 protein. Odontoblasts responded to growth hormone by more cells expressing bone morphogenetic protein, alkaline phosphatase, osteocalcin, and osteopontin. No changes were found in bone sialoprotein or E11 protein expression. Thus, growth hormone may stimulate odontoblasts to express several growth factors and matrix proteins associated with dentin matrix biosynthesis in mature rat molars. [ABSTRACT FROM AUTHOR]

Recent atomic force microscopy studies have shown that acid-etching dentin causes the surface to recede. The purpose of this work was to test the hypothesis that acidic solutions can cause contraction of demineralized dentin matrix. Small beams of dentin were cut from extracted human third molars. The central region of each specimen was completely demineralized in EDTA. Specimens held in a tester were immersed in acidic solutions, and the amount of tension was recorded. Test variables included gauge length, cross-sectional area, pre-strain, and pH. The results showed that immersion of demineralized dentin in acidic solutions caused tension that was directly related both to H+ concentration at pH < 2 and to pre-strain. The contractile stress development (ca. 0.2-0.4 MPa) was sufficient to cause a collapse of demineralized dentin matrix. The mechanism for this effect probably involves H+-induced conformation changes in the collagen matrix. [ABSTRACT FROM AUTHOR]

During resin-bonding procedures, dentin surfaces are treated with acidic conditioners to remove the smear layer and decalcify the surface to expose the collagen fibrils of the underlying matrix. These decalcified surfaces are then either air-dried or treated with dehydrating solvents, procedures which may modify the physical properties of the dentin matrix. The purpose of this study was to evaluate the effects of dehydration on the stiffness of the decalcified dentin matrix. Small (8 x 1.7 x 0.9 mm) beams of dentin were prepared from mid-coronal dentin of extracted human molars. The ends were covered with varnish for protection, and the specimens were placed in 0.5 M EDTA for 5 days to decalcify. The stiffness was measured by both the cantilever technique and by conventional stress-strain testing. Specimens tested by the cantilever technique were sequentially exposed to water, acetone, alcohol, HEMA, and glutaraldehyde. Specimens tested by conventional stress- strain testing were exposed either to water, acetone, or HEMA, or were allowed to air-dry. The results indicate that the stiffness of decalcified human dentin matrix is very low (ca. 7 MPa), if the specimens are wet with water. As they are dehydrated, either chemically in water-miscible organic solvents or physically in air, the stiffness increases 20- to 38- fold at low strains or three- to six-fold at high strains. These increases in modulus were rapidly reversed by rehydration in water. Exposure to glutaraldehyde also produced an increase in stiffness that was not reversible when the specimens were placed back in water. [ABSTRACT FROM AUTHOR]