Injectable porcine bone demineralized and digested extracellular matrix-PEGDA hydrogel blend for bone regeneration.

Extracellular matrix (ECM) has a major role in the structural support and cellular processes of organs and tissues. Proteins extracted from the ECM have been used to fabricate different scaffolds for tissue engineering applications. The aims of the present study were to extract, characterize and fabricate a new class of hydrogel with proteins isolated from pig bone ECM and combine them with a synthetic polymer so it could be used to promote bone regeneration. Porcine bone demineralized and digested extracellular matrix (pddECM) containing collagen type I was produced, optimized and sterilized with high pressurized CO2 method. The pddECM was further blended with 20% w/v polyethylene glycol diacrylate (PEGDA) to create an injectable semi interpenetrating polymer network (SIPN) scaffold with enhanced physicochemical properties. The blend tackled the shortfall of natural polymers, such as lack of structural stability and fast degradation, preserving its structure in more than 90% after 30 days of incubation; thus, increasing the material endurance in a simulated physiological environment. The manufactured injectable hydrogel showed high cytocompatibility with hOb and SaOs-2 cells, promoting osteogenic proliferation within 21 days of culture. The hydrogel had a high compression modulus of 520 kPa, low swelling (5.3 mg/mg) and millimetric volume expansion (19.5%), all of which are favorable characteristics for bone regeneration applications.

Characterization of inter-crystallite peptides in human enamel rods reveals contribution by the Y allele of amelogenin.

Proteins of the inter-rod sheath and peptides within the narrow inter-crystallite space of the rod structure are considered largely responsible for visco-elastic and visco-plastic properties of enamel. The present study was designed to investigate putative peptides of the inter-crystallite space. Entities of 1-6 kDa extracted from enamel rods of erupted permanent teeth were analysed by mass spectrometry (MS) and shown to comprise N-terminal amelogenin (AMEL) peptides either containing or not containing exon 4 product. Other dominant entities consisted of an N-terminal peptide from ameloblastin (AMBN) and a series of the most hydrophobic peptides from serum albumin (ALBN). Amelogenin peptides encoded by the Y-chromosome allele were strongly detected in Enamel from male teeth. Location of N-terminal AMEL peptides as well as AMBN and ALBN, between apatite crystallites, was disclosed by immunogold scanning electron microscopy (SEM). Density plots confirmed the relative abundance of these products including exon 4+ AMEL peptides that have greater capacity for binding to hydroxyapatite. Hydrophilic X and Y peptides encoded in exon 4 differ only in substitution of non-polar isoleucine in Y for polar threonine in X with reduced disruption of the hydrophobic N-terminal structure in the Y form. Despite similarity of X and Y alleles of AMEL the non-coding region upstream from exon 4 shows significant variation with implications for segregation of processing of transcripts from exon 4. Detection of fragments from multiple additional proteins including keratins (KER), fetuin A (FETUA), proteinases and proteinase inhibitors, likely reflect biochemical events during enamel formation.

The role of proteoglycans in the nanoindentation creep behavior of human dentin.

Attempts to understand the mechanical behavior of dentin and other mineralized tissues have been primarily focused on the role of their more abundant matrix components, such as collagen and hydroxyapatite. The structural mechanisms endowing these biological materials with outstanding load bearing properties, however, remain elusive to date. Furthermore, while their response to deformation has been extensively studied, mechanisms contributing to their recovery from induced deformation remain poorly described in the literature. Here, we offer novel insights into the participation of proteoglycans (PG) and glycosaminoglycans (GAG) in regulating the nanoindentation creep deformation and recovery of mineralized and demineralized dentin. Accordingly, after the enzymatic digestion of either PGs and associated GAGs or only GAGs, the nanoindentation creep deformation of dentin increased significantly, while the relative recovery of both the mineralized and demineralized dentin dropped by 40-70%. In summary, our results suggest that PGs and GAGs may participate in a nanoscale mechanism that contributes significantly to the outstanding durability of dentin and possibly other mineralized tissues of similar composition.

Effect of tooth bleaching agents on protein content and mechanical properties of dental enamel.

This study investigated the effect of two bleaching agents, 16% carbamide peroxide (CP) and 35% hydrogen peroxide (HP), on the mechanical properties and protein content of human enamel from freshly extracted teeth. The protein components of control and treated enamel were extracted and examined on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Marked reduction of the protein matrix and random fragmentation of the enamel proteins after bleaching treatments was found. The mechanical properties were analyzed with Vickers indentations to characterize fracture toughness, and nanoindentation to establish enamel hardness, elastic modulus and creep deformation. Results indicate that the hardness and elastic modulus of enamel were significantly reduced after treatment with CP and HP. After bleaching, the creep deformation at maximum load increased and the recovery upon unloading reduced. Crack lengths of CP and HP treated enamel were increased, while fracture toughness decreased. Additionally, the microstructures of fractured and indented samples were examined with field emission gun scanning electron microscopy (FEG-SEM) showing distinct differences in the fracture surface morphology between pre- and post-bleached enamel. In conclusion, tooth bleaching agents can produce detrimental effects on the mechanical properties of enamel, possibly as a consequence of damaging or denaturing of its protein components.

Regulation of reactionary dentin formation by odontoblasts in response to polymicrobial invasion of dentin matrix.

Odontoblast synthesis of dentin proceeds through discrete but overlapping phases characterized by formation of a patterned organic matrix followed by remodelling and active mineralization. Microbial invasion of dentin in caries triggers an adaptive response by odontoblasts, culminating in formation of a structurally altered reactionary dentin, marked by biochemical and architectonic modifications including diminished tubularity. Scanning electron microscopy of the collagen framework in reactionary dentin revealed a radically modified yet highly organized meshwork as indicated by fractal and lacunarity analyses. Immuno-gold labelling demonstrated increased density and regular spatial distribution of dentin sialoprotein (DSP) in reactionary dentin. DSP contributes putative hydroxyapatite nucleation sites on the collagen scaffold. To further dissect the formation of this altered dentin matrix, the associated enzymatic machinery was investigated. Analysis of extracted dentin matrix indicated increased activity of matrix metalloproteinase-2 (MMP-2) in the reactionary zone referenced to physiologic dentin. Likewise, gene expression analysis of micro-dissected odontoblast layer revealed up-regulation of MMP-2. Parallel up-regulation of tissue inhibitor of metalloproteinase-2 (TIMP-2) and membrane type 1- matrix metalloproteinase (MT1-MMP) was observed in response to caries. Next, modulation of odontoblastic dentinogenic enzyme repertoire was addressed. In the odontoblast layer expression of Toll-like receptors was markedly altered in response to bacterial invasion. In carious teeth TLR-2 and the gene encoding the corresponding adaptor protein MyD88 were down-regulated whereas genes encoding TLR-4 and adaptor proteins TRAM and Mal/TIRAP were up-regulated. TLR-4 signalling mediated by binding of bacterial products has been linked to up-regulation of MMP-2. Further, increased expression of genes encoding components of the TGF-ß signalling pathway, namely SMAD-2 and SMAD-4, may explain the increased synthesis of collagen by odontoblasts in caries. These findings indicate a radical adaptive response of odontoblasts to microbial invasion of dentin with resultant synthesis of modified mineralized matrix.

Extracellular ATP dissociates nonmuscle myosin from P2X(7) complex: this dissociation regulates P2X(7) pore formation.

The P2X(7) receptor is a ligand-gated cation channel that is highly expressed on monocyte-macrophages and that mediates the pro-inflammatory effects of extracellular ATP. Dilation of the P2X(7) channel and massive K(+) efflux follows initial channel opening, but the mechanism of secondary pore formation is unclear. The proteins associated with P2X(7) were isolated by using anti-P2X(7) monoclonal antibody-coated Dynabeads from both interferon-gamma plus LPS-stimulated monocytic THP-1 cells and P2X(7)-transfected HEK-293 cells. Two nonmuscle myosins, NMMHC-IIA and myosin Va, were found to associate with P2X(7) in THP-1 cells and HEK-293 cells, respectively. Activation of the P2X(7) receptor by ATP caused dissociation of P2X(7) from nonmuscle myosin in both cell types. The interaction of P2X(7) and NMMHC-IIA molecules was confirmed by fluorescent life time measurements and fluorescent resonance of energy transfer-based time-resolved flow cytometry assay. Reducing the expression of NMMHC-IIA or myosin Va by small interfering RNA or short hairpin RNA led to a significant increase of P2X(7) pore function without any increase in surface expression or ion channel function of P2X(7) receptors. S-l-blebbistatin, a specific inhibitor of NMMHC-IIA ATPase, inhibited both ATP-induced ethidium uptake and ATP-induced dissociation of P2X(7)-NMMHC-IIA complex. In both cell types nonmuscle myosin closely interacts with P2X(7) and is dissociated from the complex by extracellular ATP. Dissociation of this anchoring protein may be required for the transition of P2X(7) channel to a pore.

Two-dimensional fluorescence difference gel electrophoretic analysis of Streptococcus mutans biofilms.

Compared with traditional two-dimensional (2D) proteome analysis of Streptococcus mutans grown as a biofilm from a planktonic culture at steady state (Rathsam et al., Microbiol. 2005, 151, 1823-1837), the use of 2D fluorescence difference gel electrophoresis (DIGE) led to a 3-fold increase in the number of identified protein spots that were significantly altered in their level of expression (P < 0.050). Of the 73 identified proteins, only nine were up-regulated in biofilm grown cells. The results supported the previously surmised hypothesis that general metabolic functions were down-regulated in response to a reduction in growth rate in mature S. mutans biofilms. Up-regulation of competence proteins without any concomitant increase in stress-responsive proteins was confirmed, while the levels of glucosyltransferase C (GtfC), involved in glucan formation, O-acetylserine sulfhyrylase (cysteine synthetase A; CsyK), implicated in the formation of [Fe-S] clusters, and a hypothetical protein encoded by the open reading frame, SMu0188, were also up-regulated.

Up-regulation of competence- but not stress-responsive proteins accompanies an altered metabolic phenotype in Streptococcus mutans biofilms.

Mature biofilm and planktonic cells of Streptococcus mutans cultured in a neutral pH environment were subjected to comparative proteome analysis. Of the 242 protein spots identified, 48 were significantly altered in their level of expression (P<0.050) or were unique to planktonic or biofilm-grown cells. Among these were four hypothetical proteins as well as proteins known to be associated with the maintenance of competence or found to possess a cin-box-like element upstream of their coding gene. Most notable among the non-responsive genes were those encoding the molecular chaperones DnaK, GroEL and GroES, which are considered to be up-regulated by sessile growth. Analysis of the rest of the proteome indicated that a number of cellular functions associated with carbon uptake and cell division were down-regulated. The data obtained were consistent with the hypothesis that a reduction in the general growth rate of mature biofilms of S. mutans in a neutral pH environment is associated with the maintenance of transformation without the concomitant stress response observed during the transient state of competence in bacterial batch cultures.

Expression of a streptococcal glucosyltransferase as a fusion to a solute-binding protein in Lactobacillus fermentum BR11.

BspA is a non-covalently anchored cystine-binding protein from Lactobacillus fermentum BR11. It has previously been used to present antigens derived from infectious organisms on the L. fermentum BR11 cell surface. In this study, the capacity of BspA to present a very large polypeptide was tested. A temperature sensitive plasmid was constructed that encodes a 175-kDa chimeric protein consisting of a fusion between BspA and an N-terminally truncated derivative of the Streptococcus salivarius ATCC 25975 glucosyltransferase GtfJ. This plasmid was introduced into the L. fermentum genome. Integrants were able to incorporate 20-40 nmol sucrose derived glucose into glucan per ml culture per optical density unit. The glucosyltransferase activity was external to the cytoplasmic membrane and bound to the cell. Unlike native BspA, the BspA-GtfJ fusion could not be removed from the cell by 5 M LiCl wash.