Solution blow spinning of highly deacetylated chitosan nanofiber scaffolds for dermal wound healing.

Biocompatible fibrous scaffolds based on highly deacetylated chitosan were fabricated using high-throughput solution blow spinning. Scanning electron microscopy analysis revealed that the chitosan nanofiber scaffolds had ultrafine and continuous fibers (300-1200 nm) with highly interconnected porous structures (30-75% porosity), mimicking some aspects of the native extracellular matrix in skin tissue. Post-treatment of as-spun nanofibers with aqueous potassium carbonate solution resulted in a fibrous scaffold with a high chitosan content that retained its fibrous structural integrity for cell culture. Analysis of the mechanical properties of the chitosan nanofiber scaffolds in both dry and wet conditions showed that their strength and durability were sufficient for wound dressing applications. Significantly, the wet scaffold underwent remarkable elastic deformation during stretch such that the elongation at break dramatically increased to up to 44% of its original length, showing wavy fiber morphology near the break site. The culture of normal human dermal fibroblast cells onto scaffolds for 1-14 days demonstrated that the scaffolds were highly compatible and a suitable platform for cell adhesion, viability, and proliferation. Secretion profiles of wound healing-related proteins to the cell culture medium demonstrated that chitosan fibers were a promising scaffold for wound healing applications. Overall, the dense fibrous network with high porosity of the chitosan nanofiber scaffold and their mechanical properties indicate that they could be used to design and fabricate new materials that mimic the epidermis layer of natural skin.

Studies of Dynamic Binding of Amino Acids to TiO2 Nanoparticle Surfaces by Solution NMR and Molecular Dynamics Simulations.

Adsorption of biomolecules onto material surfaces involves a potentially complex mechanism where molecular species interact to varying degrees with a heterogeneous material surface. Surface adsorption studies by atomic force microscopy, sum frequency generation spectroscopy, and solid-state NMR detect the structures and interactions of biomolecular species that are bound to material surfaces, which, in the absence of a solid-liquid interface, do not exchange rapidly between surface-bound forms and free molecular species in bulk solution. Solution NMR has the potential to complement these techniques by detecting and studying transiently bound biomolecules at the liquid-solid interface. Herein, we show that dark-state exchange saturation transfer (DEST) NMR experiments on gel-stabilized TiO2 nanoparticle (NP) samples detect several forms of biomolecular adsorption onto titanium(IV) oxide surfaces. Specifically, we use the DEST approach to study the interaction of amino acids arginine (Arg), lysine (Lys), leucine (Leu), alanine (Ala), and aspartic acid (Asp) with TiO2 rutile NP surfaces. Whereas Leu, Ala, and Asp display only a single weakly interacting form in the presence of TiO2 NPs, Arg and Lys displayed at least two distinct bound forms: a species that is surface bound and retains a degree of reorientational motion and a second more tightly bound form characterized by broadened DEST profiles upon the addition of TiO2 NPs. Molecular dynamics simulations indicate different surface bound states for both Lys and Arg depending on the degree of TiO2 surface hydroxylation but only a single bound state for Asp regardless of the degree of surface hydroxylation, in agreement with results obtained from the analysis of DEST profiles.

Effect of proline-rich synthetic peptide-coated titanium implants on bone healing in a rabbit model.

PURPOSE: Previous studies have demonstrated the capacity of a designed proline-rich synthetic peptide to stimulate osteoblast differentiation and biomineralization in vitro. Therefore, the aim of the present study was to evaluate the osseointegration capacity of titanium (Ti) implants coated with these peptides in a rabbit model. MATERIALS AND METHODS: Four calibrated defects were prepared in the tibiae of three New Zealand rabbits, and the defects were randomized into a test group (peptide-modified machined Ti implant) and a control group (unmodified machined Ti implant). The performance in vivo was investigated after 4 weeks of implantation by real-time reverse transcriptase polymerase chain reaction of bone and inflammatory markers, microcomputed tomographic analysis of mineralized bone, and histologic examination. RESULTS: The peptides adsorbed in agglomerates on Ti and underwent a change in secondary structure upon adsorption, which induced an increase in surface wettability. Gene expression markers indicated that peptide-coated Ti implants had significantly decreased mRNA levels of tartrate-resistant acid phosphatase. A trend toward increased osteocalcin in the peri-implant bone tissue was also seen. Bone morphometric and histologic parameters did not show significant differences, although the peptide group showed a higher percentage of new bone histologically. CONCLUSIONS: Proline-rich peptides have potential as a biocompatible coating for promoting osseointegration of Ti implants by reducing bone resorption.

Hydrogen content in titanium and a titanium-zirconium alloy after acid etching.

Dental implant alloys made from titanium and zirconium are known for their high mechanical strength, fracture toughness and corrosion resistance in comparison with commercially pure titanium. The aim of the study was to investigate possible differences in the surface chemistry and/or surface topography of titanium and titanium-zirconium surfaces after sand blasting and acid etching. The two surfaces were compared by X-ray photoelectron spectroscopy, secondary ion mass spectroscopy, scanning electron microscopy and profilometry. The 1.9 times greater surface hydrogen concentration of titanium zirconium compared to titanium was found to be the major difference between the two materials. Zirconium appeared to enhance hydride formation on titanium alloys when etched in acid. Surface topography revealed significant differences on the micro and nanoscale. Surface roughness was increased significantly (p<0.01) on the titanium-zirconium alloy. High-resolution images showed nanostructures only present on titanium zirconium.

Coating of metal implant materials with strontium.

The aim of this study was to show that cathodic polarization can be used for coating commercial implant surfaces with an immobilized but functional and bioavailable surface layer of strontium (Sr). Moreover, this study assessed the effect of fluorine on Sr-attachment. X-ray photoelectron spectroscopy revealed that addition of fluorine (F) to the buffer during coating increased surface Sr-amounts but also changed the chemical surface composition by adding SrF2 alongside of SrO whereas pre-treatment of the surface by pickling in hydrofluoric acid appeared to hinder Sr-attachment. Assessment of the bio-availability hinted at a positive effect of Sr on cell differentiation given that the surface reactivity of the original surface remained unchanged. Additional SrF2 on the surface appeared to reduce undesired surface contamination while maintaining the surface micro-topography and micro-morphology. Anyhow, this surface modification revealed to create nano-nodules on the surface.

Adaptor protein complex 2-mediated, clathrin-dependent endocytosis, and related gene activities, are a prominent feature during maturation stage amelogenesis.

Molecular events defining enamel matrix removal during amelogenesis are poorly understood. Early reports have suggested that adaptor proteins (AP) participate in ameloblast-mediated endocytosis. Enamel formation involves the secretory and maturation stages, with an increase in resorptive function during the latter. Here, using real-time PCR, we show that the expression of clathrin and adaptor protein subunits are upregulated in maturation stage rodent enamel organ cells. AP complex 2 (AP-2) is the most upregulated of the four distinct adaptor protein complexes. Immunolocalization confirms the presence of AP-2 and clathrin in ameloblasts, with strongest reactivity at the apical pole. These data suggest that the resorptive functions of enamel cells involve AP-2 mediated, clathrin-dependent endocytosis, thus implying the likelihood of specific membrane-bound receptor(s) of enamel matrix protein debris. The mRNA expression of other endocytosis-related gene products is also upregulated during maturation including: lysosomal-associated membrane protein 1 (Lamp1); cluster of differentiation 63 and 68 (Cd63 and Cd68); ATPase, H(+) transporting, lysosomal V0 subunit D2 (Atp6v0d2); ATPase, H(+) transporting, lysosomal V1 subunit B2 (Atp6v1b2); chloride channel, voltage-sensitive 7 (Clcn7); and cathepsin K (Ctsk). Immunohistologic data confirms the expression of a number of these proteins in maturation stage ameloblasts. The enamel of Cd63-null mice was also examined. Despite increased mRNA and protein expression in the enamel organ during maturation, the enamel of Cd63-null mice appeared normal. This may suggest inherent functional redundancies between Cd63 and related gene products, such as Lamp1 and Cd68. Ameloblast-like LS8 cells treated with the enamel matrix protein complex Emdogain showed upregulation of AP-2 and clathrin subunits, further supporting the existence of a membrane-bound receptor-regulated pathway for the endocytosis of enamel matrix proteins. These data together define an endocytotic pathway likely used by ameloblasts to remove the enamel matrix during enamel maturation.

Dimensional Ridge Preservation with a Novel Highly Porous TiO(2) Scaffold: An Experimental Study in Minipigs.

Despite being considered noncritical size defects, extraction sockets often require the use of bone grafts or bone graft substitutes in order to facilitate a stable implant site with an aesthetically pleasing mucosal architecture and prosthetic reconstruction. In the present study, the effect of novel TiO(2) scaffolds on dimensional ridge preservation was evaluated following their placement into surgically modified extraction sockets in the premolar region of minipig mandibles. After six weeks of healing, the scaffolds were wellintegrated in the alveolar bone, and the convex shape of the alveolar crest was preserved. The scaffolds were found to partially preserve the dimensions of the native buccal and lingual bone walls adjacent to the defect site. A tendency towards more pronounced vertical ridge resorption, particularly in the buccal bone wall of the nongrafted alveoli, indicates that the TiO(2) scaffold may be used for suppressing the loss of bone that normally follows tooth extraction.

Enhanced osteoblast differentiation on scaffolds coated with TiO2 compared to SiO2 and CaP coatings.

The aim was to compare the protein release from normal human osteoblasts (NHO) cultured on scaffolds with similar morphology but different coatings. Different ceramic coatings; TiO(2), SiO(2) and calcium phosphate (CaP); Ca(9)HPO(4)(PO(4))(5)OH, were applied to porous TiO(2) scaffolds prepared by polymer sponge replication. NHO were cultured on scaffolds in triplicates. The concentration of cytokines and Ca(2+), and alkaline phosphatase (ALP) activity in the cell media was quantified. The secretion of osteopontin, osteoprotegerin, vascular endothelial growth factor and interleukin-6 was higher from NHO on TiO(2) compared to SiO(2) and CaP. The secretion from cells on the three scaffolds was, however, either similar or lower than the control cells cultured on plastic. The Ca(2+) concentration was higher in cell media on CaP the first week, and no difference in ALP activity was observed. TiO(2) coating induced a higher secretion of factors indicating enhanced osteoblast differentiation as compared to CaP and SiO(2).

Bone formation in TiO2 bone scaffolds in extraction sockets of minipigs.

The osteoconductive capacity of TiO(2) scaffolds was investigated by analysing the bone ingrowth into the scaffold structure following their placement into surgically modified extraction sockets in Gottingen minipigs. Non-critical size defects were used in order to ensure sufficient bone regeneration for the evaluation of bone ingrowth to the porous scaffold structure, and sham sites were used as positive control. Microcomputed tomographic analysis revealed 73.6±11.1% of the available scaffold pore space to be occupied by newly formed bone tissue, and the volumetric bone mineral density of the regenerated bone was comparable to that of the native cortical bone. Furthermore, histological evidence of vascularization and the presence of bone lamellae surrounding some of the blood vessels were also observed within the inner regions of the scaffold, indicating that the highly interconnected pore structure of the TiO(2) scaffolds supports unobstructed formation of viable bone tissue within the entire scaffold structure. In addition, bone tissue was found to be in direct contact with 50.0±21.5% of the TiO(2) struts, demonstrating the good biocompatibility and osteoconductivity of the scaffold material.

Amelogenins modulate cytokine expression in LPS-challenged cultured human macrophages.

Amelogenins are enamel matrix proteins with a proven ability to restore tissues in patients with advanced periodontitis and chronic skin wounds. To explore the mechanisms of action of amelogenins in wound inflammation, the in vitro effect on the expression of selected cell mediators involved in inflammation and tissue repair from human monocyte-derived macrophages was studied. Macrophages were treated with amelogenins in serum-enriched medium with simultaneous lipopolysaccharide (LPS) stimulation, for 6, 24 and 72 h, and the conditioned culture medium was analysed for 28 different cytokines. Amelogenin treatment directed the LPS-induced release of both pro- and anti-inflammatory cytokines towards an alternatively activated macrophage phenotype. This change in activation was also demonstrated by the amelogenin-induced secretion of alternative macrophage activation-associated CC chemokine-1 (AMAC-1, also known as CCL18; p<0.001), a well-documented marker of alternative activation. Amelogenins were also shown significantly to increase the macrophage expression of vascular endothelial growth factor and, to a lesser but significant extent, insulin-like growth factor-1 after 24h of culture. The results of the present in vitro study show that monocyte-derived macrophages stimulated by inflammatory agonist LPS respond to the treatment with amelogenins by reducing the pro-inflammatory activity and increasing the expression of tissue repair mediators.

Ameloblastin expression and putative autoregulation in mesenchymal cells suggest a role in early bone formation and repair.

Ameloblastin is mainly known as a dental enamel protein, synthesized and secreted into developing enamel matrix by the enamel-forming ameloblasts. The function of ameloblastin in tooth development remains unclear, but it has been suggested to be involved in processes varying from regulating crystal growth to activity as a growth factor or partaking in cell signaling. Recent studies suggest that some enamel matrix proteins also might have important functions outside enamel formation. In this context ameloblastin has recently been reported to induce dentin and bone repair, as well as being present in the early bone and cartilage extracellular matrices during embryogenesis. However, what cells express ameloblastin in these tissues still remains unclear. Thus, the expression of ameloblastin was examined in cultured primary mesenchymal cells and in vivo during healing of bone defects in a "proof of concept" animal study. Real time RT-PCR analysis revealed human ameloblastin (AMBN) mRNA expression in human mesenchymal stem cells and primary osteoblasts and chondrocytes. Expression of AMBN mRNA was also confirmed in human CD34 positive cells and osteoclasts. Western and dot blot analysis of cell lysates and medium confirmed the expression and secretion of ameloblastin from mesenchymal stem cells, primary human osteoblasts and chondrocytes. Expression of ameloblastin was also detected in newly formed bone in experimental bone defects in adult rats. Together these findings suggest a role for this protein in early bone formation and repair.

A novel ultra-porous titanium dioxide ceramic with excellent biocompatibility.

The current study compares biocompatibility, cell growth and morphology, pore diameter distribution, and interconnectivity of a novel titanium dioxide (TiO(2)) bone graft substitute granules with three different commercially available bone graft granules Natix, Straumann BoneCeramic, and Bio-Oss. Human primary mesenchymal stem cells were cultured on the bone graft substitutes and cell viability and proliferation were evaluated after 1 and 3 days. The microstructural properties of the bone graft substitutes were evaluated by scanning electron microscopy, micro-computed tomography analysis, and mechanical testing. The cell viability and proliferation, porosity, interconnectivity, open pore size, and surface area-to-volume ratio of TiO(2) granules were significantly higher than commercial bone granules (Bio-Oss and Straumann BoneCeramic).

Fluoride reduces the expression of enamel proteins and cytokines in an ameloblast-derived cell line.

OBJECTIVE: To investigate the effects of two different fluoride concentrations on the expression of enamel proteins, alkaline phosphatase (ALP), cytokines and interleukins by an ameloblast-derived cell line. METHODS: Murine ameloblast-derived cells (LS-8), mouse odontogenic epithelia, were exposed to 1 or 5ppm sodium fluoride (NaF) (0.46 and 2.25ppm F, respectively) for 1, 3 and 7 days. The effect of NaF on the mRNA expression of enamel proteins was quantified; the secretion of cytokines, and interleukins, and the alkaline phosphatase (ALP) activity, into the cell culture medium was measured and compared to untreated controls. The effect on cell growth after 1- and 3-days in culture was measured using BrdU incorporation. RESULTS: Fluoride at 2.25ppm reduced mRNA expression of the structural enamel matrix proteins amelogenin (amel), ameloblastin (ambn), enamelin (enam), and the enamel protease matrix metallopeptidase-20 (MMP-20). Similarly several vascularisation factors (vascular endothelial growth factor (VEGF), monocyte chemoattractant proteins (MCP-1) and interferon inducible protein 10 (IP-10), was also reduced by 2.25ppm fluoride. ALP activity and proliferation were stimulated by 0.46ppm fluoride but inhibited by 2.25ppm fluoride. CONCLUSIONS: These results indicate that fluoride may impact on the expression of structural enamel proteins and the protease responsible for processing these proteins during the secretory stage of amelogenesis and go some way to explaining the mineralization defect that characterises fluorotic enamel.

In vivo performance of titanium implants functionalized with eicosapentaenoic acid and UV irradiation.

The aim of this study was to design implant surfaces that attach less to bone but at the same time improve osseous healing for use as temporary bone fracture plates. The strategy was to combine the nonadhesive properties of smooth titanium (Ti) surfaces with the differentiative and anti-inflammatory properties of eicosapentaenoic acid (EPA). Machined Ti implant surfaces coated with a layer of EPA, with or without UV irradiation, were characterized by X-ray photoelectron spectroscopy, and their in vivo performance was evaluated in New Zealand White rabbits. The performance of the functionalised implants was analyzed after 10 weeks of healing by mechanical pull-out testing, molecular biology, and histological and microcomputed tomography analysis. The results indicate that surface functionalization with UV light can reduce bone attachment and volumetric bone mineral density in the peri-implant bone tissue. The presence of EPA on the surfaces enhanced this effect further. Gene expression of bone formation markers showed a trend toward higher mRNA levels in all EPA treated groups. The histological analyses demonstrated lower inflammation in the UV-irradiated group and immature bone formation in all the groups. In conclusion, surface functionalization of Ti implants with UV light and EPA could be a biocompatible coating for reduced bone bonding ability of Ti while promoting bone formation.

Rosuvastatin promotes osteoblast differentiation and regulates SLCO1A1 transporter gene expression in MC3T3-E1 cells.

Rosuvastatin (RSV) is a synthetic statin with favourable pharmacologic properties including minimal metabolism, hepatic selectivity and enhanced inhibition of HMG-CoA reductase. An induction of osteoblast differentiation has been reported in vitro with lipophilic statins but not with RSV, which, like pravastatin, is relatively hydrophilic compared with other statins. To mediate its action, an active transport mechanism via solute carrier (SLC) transporters from the SLC16, SLC21/SLCO and SLC22 gene family - specifically Slc16a1, Slco1a1, Slco2b1 and Slc22a8 - may be present to allow effective entry in osteoblastic cells. In this study, we demonstrate that RSV induced osteoblast differentiation, as measured by increased BMP-2 gene expression and secretion, and ALP activity in MC3T3-E1 osteoblast cells, without significantly affecting cell proliferation within the concentration range of 0.001-10 µM. Low concentrations of RSV (0.001-0.01 µM) were protective against cell death whereas higher concentrations (10-100 µM) showed cytotoxicity. Moreover, MC3T3-E1 osteoblasts expressed high levels of Slco1a1 and Slc16a1 mRNA and low levels of Slco2b1 and Slc22a8 mRNA, when compared with kidney and liver tissues from mice. Slco1a1 gene expression increased 12-fold during osteoblast differentiation and was further regulated after RSV treatment. In conclusion, as for other statins, RSV promotes osteoblast differentiation, and also, demonstrated for the first time, regulates the expression of Slco1a1, which may constitute the transport system for RSV across the cell membrane in mature osteoblasts.

Ultra-porous titanium oxide scaffold with high compressive strength.

Highly porous and well interconnected titanium dioxide (TiO(2)) scaffolds with compressive strength above 2.5 MPa were fabricated without compromising the desired pore architectural characteristics, such as high porosity, appropriate pore size, surface-to-volume ratio, and interconnectivity. Processing parameters and pore architectural characteristics were investigated in order to identify the key processing steps and morphological properties that contributed to the enhanced strength of the scaffolds. Cleaning of the TiO(2) raw powder removed phosphates but introduced sodium into the powder, which was suggested to decrease the slurry stability. Strong correlation was found between compressive strength and both replication times and solid content in the ceramic slurry. Increase in the solid content resulted in more favourable sponge loading, which was achieved due to the more suitable rheological properties of the ceramic slurry. Repeated replication process induced only negligible changes in the pore architectural parameters indicating a reduced flaw size in the scaffold struts. The fabricated TiO(2) scaffolds show great promise as load-bearing bone scaffolds for applications where moderate mechanical support is required.

Amelogenin is phagocytized and induces changes in integrin configuration, gene expression and proliferation of cultured normal human dermal fibroblasts.

Fibroblasts are central in wound healing by expressing important mediators and producing and remodelling extracellular matrix (ECM) components. This study aimed at elucidating possible mechanisms of action of the ECM protein amelogenin on normal human dermal fibroblasts (NHDF). Amelogenin at 100 and 1000 microg/ml increased binding of NHDF via several integrins, including alphavbeta3, alphavbeta5 and alpha5beta1. Further, both surface interaction and cellular uptake of amelogenin by NHDF was observed using scanning and transmission electron microscopy. Gene microarray studies showed >8-fold up or down-regulation of genes, of which most are involved in cellular growth, migration and differentiation. The effect of amelogenin was exemplified by increased proliferation over 7 days. In conclusion, the beneficial effects of amelogenin on wound healing are possibly conducted by stimulating fibroblast signalling, proliferation and migration via integrin interactions. It is hypothesized that amelogenin stimulates wound healing by providing connective tissue cells with a temporary extracellular matrix.

In vivo expression of osteogenic markers and bone mineral density at the surface of fluoride-modified titanium implants.

The aim of the present study was to investigate the biological mechanisms of the functional attachment of fluoride-modified titanium implants to cortical bone by studying the association of the pull-out test results with gene expression of osteoblast (runx2, osteocalcin, collagen-I and IGF-I), osteoclast (TRAP, H(+)-ATPase and calcitonin receptor) and inflammation (TNF-alpha, IL-6 and IL-10) markers from peri-implant bone tissue using real-time RT-PCR, following a 4- and 8-week healing period. After implant detachment, wound fluid from the implant site was collected for LDH and ALP activity analysis. A new method to study volumetric bone mineral density (vBMD) of sub-implant cortical bone was developed using micro-computed tomography. Our results show lower LDH activity and TRAP mRNA levels in fluoride implants after 4 weeks of healing, yet no differences were found either on the pull-out force or expression of bone formation marker genes. After 8 weeks of healing, both pull-out, vBMD and osteocalcin, runx2 and collage type I gene expression were higher in fluoride implants. In conclusion, fluoride-modified implants seem to modulate both inflammation and bone resorption/formation events at the bone-implant interface, suggesting that these biological effects are an intrinsic part of the clinical performance of this surface.

Bioinformatic analysis and molecular modelling of human ameloblastin suggest a two-domain intrinsically unstructured calcium-binding protein.

Ameloblastin (AMBN) was originally believed to be an enamel-specific extracellular matrix glycoprotein secreted by ameloblasts. Recently, AMBN expression was also detected in developing mesenchymal dental hard tissues, in trauma-induced reparative dentin, and during early craniofacial bone formation. The function and structure of AMBN still remain ambiguous, and there are no known proteins with similar primary sequences. We therefore performed a bio-informatic analysis of AMBN to model ab initio the three-dimensional structure of the molecule. The results suggest that AMBN is a two-domain, intrinsically unstructured protein (IUP). The analysis did not reveal any regions with structural similarity to known receptor-ligand systems, and did not identify any higher-order structures similar to functional regions in other known sequences. The AMBN model predicts 11 defined regions exposed on the surface, internalizing the rest of the molecule including a human-specific insert. Molecular dynamics analysis identified one specific and several non-specific calcium-binding regions, mostly at the C-terminal part of the molecule. The model is supported by previous observations that AMBN is a bipolar calcium-binding molecule and hints at a possible role in protein-protein interactions. The model provides information useful for further studies on the function of AMBN.

Determination of protein regions responsible for interactions of amelogenin with CD63 and LAMP1.

The enamel matrix protein amelogenin is secreted by ameloblasts into the extracellular space to guide the formation of highly ordered hydroxyapatite mineral crystallites, and, subsequently, is almost completely removed during mineral maturation. Amelogenin interacts with the transmembrane proteins CD63 and LAMP (lysosome-associated membrane protein) 1, which are involved in endocytosis. Exogenously added amelogenin has been observed to move rapidly into CD63/LAMP1-positive vesicles in cultured cells. In the present study, we demonstrate the protein region defined by amino acid residues 103-205 for CD63 interacts not only with amelogenin, but also with other enamel matrix proteins (ameloblastin and enamelin). A detailed characterization of binding regions in amelogenin, CD63 and LAMP1 reveals that the amelogenin region defined by residues PLSPILPELPLEAW is responsible for the interaction with CD63 through residues 165-205, with LAMP1 through residues 226-251, and with the related LAMP2 protein through residues 227-259. We predict that the amelogenin binding region is: (i) hydrophobic; (ii) largely disordered; and (iii) accessible to the external environment. In contrast, the binding region of CD63 is likely to be organized in a '7' shape within the mushroom-like structure of CD63 EC2 (extracellular domain 2). In vivo, the protein interactions between the secreted enamel matrix proteins with the membrane-bound proteins are likely to occur at the specialized secretory surfaces of ameloblast cells called Tomes' processes. Such protein-protein interactions may be required to establish short-term order of the forming matrix and/or to mediate feedback signals to the transcriptional machinery of ameloblasts and/or to remove matrix protein debris during enamel biomineralization.