Demineralized Collagen Matrix Research Articles

Applications of collagen extrafibrillar demineralization in dentin bonding

The existing dentin bonding systems based on acid-etching technique lead to the loss of both extrafibrillar and intrafibrillar minerals from dentin collagen, causing excessive demineralization. Because resin monomers can not infiltrate the intrafibrillar spaces of demineralized collagen matrix, degradation of exposed collagen and resin hydrolysis subsequently occur within the hybrid layer, which seriously jeopardizing the longevity of resin-dentin bonding. Collagen extrafibrillar demineralization can effectively avoid the structural defects within the resin-dentin interface caused by acid-etching technique and improve the durability of resin-dentin bonding, by preserving intrafibrillar minerals and selectively demineralizing extrafibrillar dentin. The mechanism and research progress of collagen extrafibrillar demineralization in dentin bonding are reviewed in the paper.

Retention of Intrafibrillar Minerals Improves Resin–Dentin Bond Durability

The concept of extrafibrillar demineralization involves selective removal of apatite crystallites from the extrafibrillar spaces of mineralized dentin without disturbing the intrafibrillar minerals within collagen. This helps avoiding activation of endogenous proteases and enables air-drying of partially demineralized dentin without causing collapse of completely demineralized collagen matrix that adversely affects resin infiltration. The objective of the present study was to evaluate the potential of quaternized carboxymethyl chitosan (QCMC)–based extrafibrillar demineralization in improving resin–dentin bond durability. Isothermal titration calorimetry indicated that QCMC synthesized by quaternization of O-carboxymethyl chitosan had moderate affinity for Ca2+ (binding constant: 8.9 × 104 M−1). Wet and dry bonding with the QCMC-based demineralization produced tensile bond strengths equivalent to the phosphoric acid (H3PO4)–based etch-and-rinse technique. Those bond strengths were maintained after thermocycling. Amide I and PO43− mappings of QCMC-conditioned dentin were performed with atomic force microscope–infrared spectroscopy (AFM-IR). Whereas H3PO4-etched dentin exhibited an extensive reduction in PO43− signals corresponding to apatite depletion, QCMC-conditioned dentin showed scattered dark areas and bright PO43− streak signals. The latter were consistent with areas identified as collagen fibrils in the amide I mapping and were suggestive of the presence of intrafibrillar minerals in QCMC-conditioned dentin. Young’s modulus mapping of QCMC-demineralized dentin obtained by AFM-based amplitude modulation–frequency modulation recorded moduli that were the same order of magnitude as those in mineralized dentin and at least 1 order higher than H3PO4-etched dentin. In situ zymography of the gelatinolytic activity within hybrid layers created with QCMC conditioning revealed extremely low signals before and after thermocycling, compared with H3PO4-etched dentin for both wet and dry bonding. Confocal laser scanning microscopy identified the antibacterial potential of QCMC against Streptococcus mutans and Enterococcus faecalis biofilms. Taken together, the QCMC-based demineralization retains intrafibrillar minerals, preserves the elastic modulus of collagen fibrils, reduces endogenous proteolytic activity, and inhibits bacteria biofilms to extend dentin bond durability.

A novel dentin bonding scheme based on extrafibrillar demineralization combined with covalent adhesion using a dry-bonding technique

Dentin bonding is a dynamic process that involves the penetration of adhesive resin monomers into the extrafibrillar and intrafibrillar demineralized collagen matrix using a wet-bonding technique. However, adhesive resin monomers lack the capacity to infiltrate the intrafibrillar space, and the excess water that is introduced by the wet-bonding technique remains at the bonding interface. This imperfectly bonded interface is inclined to hydrolytic degradation, severely jeopardizing the longevity of bonded clinical restorations. The present study introduces a dentin bonding scheme based on a dry-bonding technique, combined with the use of extrafibrillar demineralization and a collagen-reactive monomer (CRM)-based adhesive (CBA). Selective extrafibrillar demineralization was achieved using 1-wt% high-molecular weight (MW) carboxymethyl chitosan (CMCS) within a clinically acceptable timeframe to create a less aggressive bonding substance for dentin bonding due to its selectively extrafibrillar demineralization capacity. CMCS demineralization decreased the activation of in situ collagenase, improved the shrinking resistance of demineralized collagen, and thus provided stronger and more durable bonding than traditional phosphoric acid etching. The new dentin bonding scheme that contained CMCS and CBA and used a dry-bonding technique achieved an encouraging dentin bonding strength and durability with low technical sensitivity. This bonding scheme can be used to improve the stability of the resin-dentin interface and foster the longevity of bonded clinical restorations.

Evaluation of caries – protective effect of three recent dentin bonding agents, xeno v plus, tetric n bond ,one coat seven universal on demineralized root surface "- An in vitro study

Background and objectives: An area on the surface of tooth, apical to CEJ, where root caries are bound to occur undergoes clinically apparent dissolution of the calcified tissues. When dentin bonding agent is applied, it forms finger like resin tags protruding into patent dentin tubules When this resin penetrates the superficially demineralized collagen matrix, it forms a hybrid layerand prevents root surface lesions. Aim: To evaluate the caries protective effect of three recent DBAs on demineralized root surface. Materials and methods: 88 single rooted extracted human teeth were selected, root surfaces apical to CEJ were polished .Acid resistant nail paint wascoated over root surfaces exposing a rectangular window on the buccal surface. Three experimental groups made: Group 1 (XENO V PLUS), Group 2 (TETRIC N BOND), Group 3 (ONE COAT 7 UNIVERSAL). These groups were divided into 2 Subgroups (A and B). Adhesive was applied and light cured with air thinning. Over this second layer of adhesive was applied, light cured with /without air thinning. Each tooth samples were sliced. A light microscope was used to examine the caries like lesions.The statistical analysis was carried out. Results:All control group specimen showed lesions with a mean depth of 177.74μm. Subgroup B results were better than A and C. Summary and Conclusion: Demineralization on root surface can be impeded by DBA tested TetricN Bond which showed maximum cariesprotective effect Key words:Cementum, demineralization, dentine bonding agent

Monitoring demineralization and remineralization of human dentin by characterization of its structure with resonance-enhanced AFM-IR chemical mapping, nanoindentation, and SEM

ObjectiveThis research aimed at monitoring demineralization and remineralization of dentin and its collagen matrix at the nanoscale by amorphous, microcrystalline, and in situ formed hydroxyapatite. MethodsThe concurrent use of the resonance-enhanced atomic force microscopy coupled with infrared probe (AFM-IR) chemical mapping, nano-indentation, and scanning electron microscopy (SEM) provides a detailed insight into the structure of human dentin, as well as to the processes of its partial demineralization and remineralization. ResultsThe resonance-enhanced AFM-IR chemical mapping of dentin has shown to be a useful method to follow distribution of its collagen and hydroxyapatite components at the micro- and nanoscale levels, especially in conjunction with SEM imaging and nanoindentation.Dentin with a higher extent of natural dentin tubule occlusion tends to be harder and less elastic.The relative affinity of the collagen and hydroxyapatite components of dentin toward hydroxyapatite depends on its type (amorphous, microcrystalline, or formed in-situ).The gel mineralization technique allows for an even and controlled growth of hydroxyapatite guided by the completely demineralized collagen matrix of dentin. SignificanceThe observed trends of the affinity of collagen toward different forms of hydroxyapatite helps develop new remineralizing formulations. The employed methods of characterization may provide an insight to the natural processes of bone mineralization guided by its both hydroxyapatite and protein constituents.

Isocyanate-terminated urethane-based dental adhesive bridges dentinal matrix collagen with adhesive resin

Commercially available dental adhesives fail to chemically unite the demineralized collagen matrix with resinous materials within the resin-dentin interface. Sub-micron separations between the collagen fibrils and polymerized resin provide the backdrop for bond deterioration. Here, novel isocyanate-terminated urethane methacrylate precursors (UMP) were synthesized with the capacity to bond chemically to dentin collagen via covalent and hydrogen bonds. Collagen grafted with UMP also copolymerized with other methacrylate resin monomers, thereby producing a monoblock of chemically-linked biocomposite. The viscosity, degree of conversion and biocompatibility of UMP are comparable with commercially available resin monomers. An experimental adhesive containing 40% UMP demonstrated co-polymerization capability, good infiltration capacity and achieved higher immediate bond strength to dentin than the control commercially available adhesive. Improvement of dentin bonding by incorporation of UMP into dentin adhesives justifies future evaluation of the potential of these UMP-based adhesives in extending the longevity of resin-dentin bonds. Statement of SignificanceComposite-adhesive restorations have become an indispensable treatment modality in contemporary restorative dentistry. While the inability of these adhesives to bond chemically with collagen undermines the bond quality. This study describes a novel isocyanate-terminated urethane multi methacrylate precursors (UMP) which can bridge dentinal matrix collagen with adhesive resin by covalent and hydrogen bonds. Furthermore, an experimental UMP-based adhesive shows better co-polymerization capability, good infiltration capacity and higher immediate bond strength than the putatively effective adhesive Single Bond 2. The new chemical bonding mechanism based on UMP would theoretically produce more stable bonding interface that are more resistant to degradation.

The effect of NDGA-modified etchant on the enzymatic degradation resistance and mechanical properties of collagen matrix

Enzymatic degradation of demineralized collagen matrix seriously impairs durable resin-dentin bonding. In this study, we evaluated the effect of nordihydroguaiaretic acid (NDGA)-modified etchant on the resistance to enzymatic degradation and mechanical properties of demineralized collagen matrix. Dentin beams were randomly demineralized by following solutions: 1) 10% phosphoric acid (PhA) solution, 2) 10% dimethyl sulfoxide (DMSO)-PhA solution, 3) NDGA-modified etchant, and 4) proanthocyanidins (PA)-modified etchant. The demineralized dentin collagen was then digested by type I collagenase solution. The collagenase degradation resistance was evaluated by measuring loss of dry mass, hydroxyproline release, and elastic modulus change. The degradation rate and hydroxyproline release of dentin collagen treated with NDGA-modified etchant were significantly lower than those in the other groups (P<0.001). The elastic modulus of dentin beams treated with NDGA-modified etchant did not increase significantly. However, after 48h of collagenase degradation, the loss of elastic modulus of dentin beams treated with NDGA-modified etchant was significantly lower than that of the control group (P<0.001). The NDGA-modified etchant could improve the resistance to enzymatic degradation of type I collagenase degradation, and the stability of the mechanical properties of dentin collagen. This proof-of-concept study validates the etch-and-crosslink technique that improves durability of dentin bonding through simultaneous dentin etching and collagen crosslinking.

Permeation of intrinsic water into ethanol- and water-saturated, monomer-infiltrated dentin bond interfaces

ObjectivesThe aim of this study was to evaluate the formation of dentin bonding interfaces using the water-wet and the ethanol-wet techniques under simulated pulpal pressure, and to assess the effect of adhesive solvent and thermomechanical loading. MethodsFlat dentin surfaces were restored under 20mm-simulated pulpal pressure following two bonding approaches (water-wet and ethanol-wet bonding) in combination with dental adhesives containing ethanol (Single Bond Plus and Scotchbond Multi-Purpose) or acetone (One-Step Plus and All-Bond 2) as solvent. Half of the restorations of each subgroup were subjected to thermocycling followed by cyclic loading (three teeth per group). Bond strength was measured using the microtensile bond strength test and fitted to a Weibull distribution (α=0.05). Ultrastructural analyses of the interface and leakage/nanoleakage evaluation were performed using confocal scanning microscopy (CLSM) and transmission electron microscopy (TEM). ResultsWater permeation through dentin tubules during adhesive application prevented adequate penetration of adhesive monomers into the demineralized collagen matrix in both bonding techniques, but more severely for water-wet bonding. Acetone-solvated adhesives showed worse bonding performance and hybridization than ethanol-based systems when applied in the ethanol-wet mode, both before and after thermomechanical challenge. SignificanceThe ethanol-wet bonding technique helps to compensate for water permeation from dentin tubules during the bonding procedures to form more stable dentin bonds, especially when used in conjunction to ethanol-solvated systems.

Carious affected dentine: its behaviour in adhesive bonding.

Carious affected dentine (CAD) represents a very common substrate in adhesive dentistry. Despite its ability to interact with adhesive systems, the intrinsic character of CAD leads to lower bonding compared with sound dentine, regardless of the adhesive systems used. This low bonding may be more susceptible to leakage and hydrolysis of the interface by matrix metalloproteinases (MMPs). This systematic review aimed to determine current knowledge of CAD bonding, together with bond strength and MMP inhibitors' ability to prevent hybrid layer instability. MEDLINE/Pubmed, Scopus and The Cochrane Library databases were electronically searched for articles published from 1 January 1960 to 31 August 2014. Two reviewers independently screened and included papers according to predefined selection criteria. The electronic searches identified 320 studies. After title, abstract and full-text examinations, 139 articles met the inclusion criteria. Data highlighted that a poor resin saturation of the already demineralized collagen matrix in CAD is strictly related to nanoleakage in interdiffusion and is the basis of the progressive decrease in strength with hydrolysis by MMPs. The use of mild self-etching systems seems to be the more accredited method to establish bonding in CAD. Inhibitors of MMPs may ensure better performance of CAD bonding, allowing undisturbed remineralization of the affected matrix. CAD bonding needs further understanding and improvement, particularly to enhance the strength and durability of the hybrid layer.

Polymerization- and solvent-induced phase separation in hydrophilic-rich dentin adhesive mimic

Current dental resin undergoes phase separation into hydrophobic-rich and hydrophilic-rich phases during infiltration of the over-wet demineralized collagen matrix. Such phase separation undermines the integrity and durability of the bond at the composite/tooth interface. This study marks the first time that the polymerization kinetics of model hydrophilic-rich phase of dental adhesive has been determined. Samples were prepared by adding varying water content to neat resins made from 95 and 99wt.% hydroxyethylmethacrylate and 5 and 1wt.% (2,2-bis[4-(2-hydroxy-3-methacryloxypropoxy)phenyl1]-propane prior to light curing. Viscosity of the formulations decreased with increased water content. The photopolymerization kinetics study was carried out with a time-resolved Fourier transform infrared spectrometer. All of the samples exhibited two-stage polymerization behavior which has not been reported previously for dental resin formulation. The lowest secondary rate maxima were observed for water contents of 10–30wt.%. Differential scanning calorimetry (DSC) showed two glass transition temperatures for the hydrophilic-rich phase of dental adhesive. The DSC results indicate that the heterogeneity within the final polymer structure decreased with increasing water content. The results suggest a reaction mechanism involving both polymerization-induced phase separation and solvent-induced phase separation for the model hydrophilic-rich phase of dental resin.

Functional biomimetic analogs help remineralize apatite-depleted demineralized resin-infiltrated dentin via a bottom–up approach

Natural biominerals are formed through metastable amorphous precursor phases via a bottom–up, nanoparticle-mediated mineralization mechanism. Using an acid-etched human dentin model to create a layer of completely demineralized collagen matrix, a bio-inspired mineralization scheme has been developed based on the use of dual biomimetic analogs. These analogs help to sequester fluidic amorphous calcium phosphate nanoprecursors and function as templates for guiding homogeneous apatite nucleation within the collagen fibrils. By adopting this scheme for remineralizing adhesive resin-bonded, completely demineralized dentin, we have been able to redeposit intrafibrillar and extrafibrillar apatites in completely demineralized collagen matrices that are imperfectly infiltrated by resins. This study utilizes a spectrum of completely and partially demineralized dentin collagen matrices to further validate the necessity for using a biomimetic analog-containing medium for remineralizing resin-infiltrated partially demineralized collagen matrices in which remnant seed crystallites are present. In control specimens in which biomimetic analogs are absent from the remineralization medium, remineralization could only be seen in partially demineralized collagen matrices, probably by epitaxial growth via a top–down crystallization approach. Conversely, in the presence of biomimetic analogs in the remineralization medium, intrafibrillar remineralization of completely demineralized collagen matrices via a bottom–up crystallization mechanism can additionally be identified. The latter is characterized by the transition of intrafibrillar minerals from an inchoate state of continuously braided microfibrillar electron-dense amorphous strands to discrete nanocrystals, and ultimately into larger crystalline platelets within the collagen fibrils. Biomimetic remineralization via dual biomimetic analogs has the potential to be translated into a functional delivery system for salvaging failing resin–dentin bonds.

Application of hydrophobic resin adhesives to acid‐etched dentin with an alternative wet bonding technique

Hydrophilic dentin adhesives are prone to water sorption that adversely affects the durability of resin-dentin bonds. This study examined the feasibility of bonding to dentin with hydrophobic resins via the adaptation of electron microscopy tissue processing techniques. Hydrophobic primers were prepared by diluting 2,2-bis[4(2-hydroxy-3-methacryloyloxy-propyloxy)-phenyl] pro- pane/triethyleneglycol dimethacrylate resins with known ethanol concentrations. They were applied to acid-etched moist dentin using an ethanol wet bonding technique that involved: (1) stepwise replacement of water with a series of increasing ethanol concentrations to prevent the demineralized collagen matrix from collapsing; (2) stepwise replacement of the ethanol with different concentrations of hydrophobic primers and subsequently with neat hydrophobic resin. Using the ethanol wet bonding technique, the experimental primer versions with 40, 50, and 75% resin exhibited tensile strengths which were not significantly different from commercially available hydrophilic three-step adhesives that were bonded with water wet bonding technique. The concept of ethanol wet bonding may be explained in terms of solubility parameter theory. This technique is sensitive to water contamination, as depicted by the lower tensile strength results from partial dehydration protocols. The technique has to be further improved by incorporating elements of dentin permeability reduction to avoid water from dentinal tubules contaminating water-free resin blends during bonding.

Microporous, Demineralized Collagen Matrices in Intact Radicular Dentin Created by Commonly Used Calcium-depleting Endodontic Irrigants

The use of calcium-depleting solutions in restorative dentistry results in demineralization of intact coronal dentin beneath smear layers. Likewise, application of calcium-depleting irrigants as final rinses might create demineralized collagen matrices in intact radicular dentin after removal of root canal wall smear layers created during canal shaping. These unsupported collagen matrices collapse on desiccation and might be difficult to detect in scanning electron microscopy dehydration techniques traditionally used in endodontic research. Demineralized collagen matrices were identified, by using a hexamethyldisilazane dehydration protocol, on the surface of smear layer-depleted radicular dentin when NaOCl was used as initial rinse, followed by the use of 17% ethylenediaminetetraacetic acid or BioPure MTAD as the final rinse. Compaction of conventional root filling materials over a demineralized collagen matrix in instrumented dentin is analogous to performing the same procedure against the predentin collagen network in the uninstrumented parts of root canals. The potential consequences of this process are, however, unknown.

Permeability of marginal hybrid layers in composite restorations

The goal of adhesive dentistry is to restore the peripheral seal of dentin lost from removal of enamel. Unfortunately, the hybrid layer (HL) that is used to create that seal is permeable to small ions or molecules, even in the absence of detectable, interfacial gap formation via nanoleakage. This nanoleakage results from several mechanisms including incomplete infiltration of adhesive monomers into demineralized collagen matrix, presence of hydrophilic monomers, and insufficient removal of solvent or water that remains trapped inside the HL. These mechanisms lead to a porous interface with nanometer-sized channels that increase the permeability of the HL. The null hypothesis tested in this study was that water and acidic solution storage are able to alter in vitro the resin-dentin interface, further increasing the marginal hybrid layer (MHL) permeability. Class II cavities were made in vitro. The specimens were stored in water for 1 week and in lactic acid solution for 3 days. Polyvinyl siloxane impressions of restoration margins were taken before and after storage in water and lactic acid solution. Polyether replicas were obtained using the silicon impressions as molds. Replicas and original samples were observed under scanning electron microscopy. Lines of water droplets were detected on MHLs and overlying adhesive only after storage. Replicas obtained after acidic solution storage showed great numbers of irregularities such as gaps, voids, and degradation of the dentin-restoration surface margin, but also a great number of droplets. Dentin-restoration resin interfaces absorb water and are damaged by storage in dilute lactic acid. The presence of water droplets probably indicates water that flows out of the interface during the setting time of the impression and thus represents an index of marginal HL water permeability.

A nanoleakage perspective on bonding to oxidized dentin.

The mechanism responsible for sodium-hypochlorite-induced reduction in dentin bond strength and its reversal with reducing agents is unknown. This study examined the relationship between nanoleakage and reversal of compromised bonding to oxidized dentin. Acid-etched dentin was completely depleted of demineralized collagen matrix when sodium hypochlorite was used. Specimens were bonded with two single-bottle dentin adhesives. They were immersed in ammoniacal silver nitrate for 24 hrs before being processed for transmission electron microscopy. For both adhesives, tensile bond strengths of acid-etched dentin were significantly reduced after sodium hypochlorite treatment, but were reversed when sodium ascorbate was used. After sodium hypochlorite application, reticular nanoleakge patterns in hybrid layers were replaced by vertical, shag-carpet-like patterns along the demineralization front. This type of nanoleakage was completely eliminated after sodium ascorbate treatment with the materials tested. Residual sodium hypochlorite within the porosities of mineralized dentin may result in incomplete resin polymerization, and hence compromised bond strength.

Reversal of compromised bonding to oxidized etched dentin.

The mechanism responsible for hydrogen-peroxide- or sodium-hypochlorite-induced reductions in dentin bond strength is unknown. This in vitro study tested the hypothesis that these oxidizing agents were responsible by attempting to reverse the effect with sodium ascorbate, a reducing agent. Human dentin was treated with these oxidants before or after being acid-etched and with or without post-treatment with sodium ascorbate. They were bonded with either Single Bond or Excite. Hydrogen peroxide reduced the bond strengths of both adhesives, while sodium hypochlorite produced reduction in adhesion of only Single Bond (p < 0.05). Following treatment with sodium ascorbate, reductions in bond strength were reversed. Transmission and scanning electron microscopy showed partial removal of the demineralized collagen matrix only by sodium hypochlorite. The observed compromised bond strengths cannot be attributed to incomplete deproteinization and may be related to changes in the redox potential of the bonding substrates.

Micromorphological spectrum of acid-conditioned dentin following the application of a water-based adhesive

Objectives. The goal of this study was to illustrate the micromorphological spectrum along the resin–dentin interface when a water-based, dentin adhesive (Scotchbond Multi-purpose) was applied to acid-conditioned dentin under different dry and wet bonding conditions. Methods. Twenty-eight 1 mm dentin discs were each conditioned with 10% maleic acid for 15 s. Twenty-four of these discs were randomly divided into four groups, based upon the status of the remaining surface moisture: Group I (30 s dry); Group II (3 s dry); Group III (visibly moist) and Group IV (overwet). They were bonded using Scotchbond Multi-purpose. The remaining four discs were bonded using an experimental water-based primer containing 35 vol.% HEMA (Group V). Laminated dentin disc pairs were prepared for transmission electron microscopic examination. Results. In all groups, diffusion of the polyalkenoic acid copolymer component of the primer into acid-conditioned dentin was localized to the surface region of the hybrid layer. The remaining part of the hybrid layer (the subsurface region) appeared variable. In Group I (30 s dry), collagen fibrils were collapsed. In Group II (3 s dry) and Group III (visibly moist), stained collagen fibrils were surrounded by wide electron–lucent interfibrillar spaces. In Group IV (overwet), a marked diffusion gradient was observed representing dilution of part of the primer components. In addition, an electron-dense primer phase, containing electron–lucent globular domains was invariably observed, irrespective of the hydration status of the demineralized collagen matrix. This electron dense phase was absent when HEMA alone was used as the primer (Group V). Significance. With the use of a water-based adhesive, one could briefly air-dry the acid-conditioned dentin and allow the water in the primer to rehydrate the collapsed collagen matrix, without the risk of incomplete hybridization or tubular seal along the resin–dentin interface. However, care should be exercised to minimize dilution of the water-soluble primer component.

Identification of Lysyl Oxidase and TRAMP as the Major Proteins in Dissociative Extracts of the Demineralized Collagen Matrix of Porcine Dentine

Carbonated apatite (dahllite) is formed within and between collagen fibrils in the mineralization of connective tissues. However, the mechanism of crystal nucleation at these sites has not been resolved. To identify non-collagenous proteins that may be involved in the nucleation process we have utilized a dissociative extraction procedure to isolate proteins associated non-covalently with the de-mineralized collagen matrix of dentine isolated from tooth roots of adult porcine incisors. Following extraction of dentine fragments with 4M GuHCl (G1-extract) and 0.5M EDTA (E-extract), de-mineralized collagen matrix-associated proteins were isolated with a second series of extractions with 4M GuHCl (G2-extract). Analysis of the G2-extracts on SDS-PAGE revealed two major 32 kDa and 24 kDa protein bands, comprising >80% of the extracted non-collagenous proteins. The 32 kDa protein was purified by FPLC on hydroxyapatite and Mono Q resins, followed by HPLC reverse-phase chromatography. Small amounts of 26 kDa and 6 kDa proteins, which appear to represent proteolytically processed, disulphide-linked fragments of the 32 kDa protein, co-eluted with the major protein. The 32 kDa protein was identified as lysyl oxidase from amino acid sequence analysis of a 13 kDa CNBr peptide obtained from protein purified by preparative electrophoresis on SDS-PAGE. Fractionation of the 24 kDa protein on FPLC Mono Q resin generated <5 closely eluting protein peaks. The proteins from these peaks were similar in size, staining properties, amino acid composition and CNBr digestion patterns. Each protein was immunoreactive with antibodies raised against a tyrosine-rich acidic matrix protein (TRAMP), reported previously to co-purify with lysyl oxidase. These studies, therefore, show that lysyl oxidase, which is important in collagen cross-link formation, and proteins with properties of TRAMP, a protein that can modulate collagen fibrillogenesis, are the major proteins in dissociative extracts of de-mineralized porcine dentine.