Ivarne L.S. Tersariol

Optimizing dentin bond durability: control of collagen degradation by matrix metalloproteinases and cysteine cathepsins

OBJECTIVES Contemporary adhesives lose their bond strength to dentin regardless of the bonding system used. This loss relates to the hydrolysis of collagen matrix of the hybrid layers. The preservation of the collagen matrix integrity is a key issue in the attempts to improve the dentin bonding durability. METHODS Dentin contains collagenolytic enzymes, matrix metalloproteinases (MMPs) and cysteine cathepsins, which are responsible for the hydrolytic degradation of collagen matrix in the bonded interface. RESULTS The identities, roles and function of collagenolytic enzymes in mineralized dentin has been gathered only within last 15 years, but they have already been demonstrated to have an important role in dental hard tissue pathologies, including the degradation of the hybrid layer. Identifying responsible enzymes facilitates the development of new, more efficient methods to improve the stability of dentin-adhesive bond and durability of bond strength. SIGNIFICANCE Understanding the nature and role of proteolytic degradation of dentin-adhesive interfaces has improved immensely and has practically grown to a scientific field of its own within only 10 years, holding excellent promise that stable resin-dentin bonds will be routinely available in a daily clinical setting already in a near future.

Strategies to prevent hydrolytic degradation of the hybrid layer-A review.

OBJECTIVE Endogenous dentin collagenolytic enzymes, matrix metalloproteinases (MMPs) and cysteine cathepsins, are responsible for the time-dependent hydrolysis of collagen matrix of hybrid layers. As collagen matrix integrity is essential for the preservation of long-term dentin bond strength, inhibition of endogenous dentin proteases is necessary for durable resin-bonded restorations. METHODS Several tentative approaches to prevent enzyme function have been proposed. Some of them have already demonstrated clinical efficacy, while others need to be researched further before clinical protocols can be proposed. This review will examine both the principles and outcomes of techniques to prevent collagen hydrolysis in dentin-resin interfaces. RESULTS Chlorhexidine, a general inhibitor of MMPs and cysteine cathepsins, is the most tested method. In general, these experiments have shown that enzyme inhibition is a promising approach to improve hybrid layer preservation and bond strength durability. Other enzyme inhibitors, e.g. enzyme-inhibiting monomers, may be considered promising alternatives that would allow more simple clinical application than chlorhexidine. Cross-linking collagen and/or dentin matrix-bound enzymes could render hybrid layer organic matrices resistant to degradation. Alternatively, complete removal of water from the hybrid layer with ethanol wet bonding or biomimetic remineralization should eliminate hydrolysis of both collagen and resin components. SIGNIFICANCE Understanding the function of the enzymes responsible for the hydrolysis of hybrid layer collagen has prompted several innovative approaches to retain hybrid layer integrity and strong dentin bonding. The ultimate goal, prevention of collagen matrix degradation with clinically applicable techniques and commercially available materials may be achievable in several ways.

Cysteine Cathepsins in Human Dentin-Pulp Complex

INTRODUCTION Collagen-degrading matrix metalloproteinases (MMPs) are expressed by odontoblasts and present in dentin. We hypothesized that odontoblasts express other collagen-degrading enzymes such as cysteine cathepsins, and their activity would be present in dentin, because odontoblasts are known to express at least cathepsin D. Effect of transforming growth factor beta (TGF-beta) on cathepsin expression was also analyzed. METHODS Human odontoblasts and pulp tissue were cultured with and without TGF-beta, and cathepsin gene expression was analyzed with DNA microarrays. Dentin cathepsin and MMP activities were analyzed by degradation of respective specific fluorogenic substrates. RESULTS Both odontoblasts and pulp tissue demonstrated a wide range of cysteine cathepsin expression that gave minor responses to TGF-beta. Cathepsin and MMP activities were observed in all dentin samples, with significant negative correlations in their activities with tooth age. CONCLUSIONS These results demonstrate for the first time the presence of cysteine cathepsins in dentin and suggest their role, along with MMPs, in dentin modification with aging.

Cysteine Cathepsins in Human Carious Dentin

Matrix metalloproteinases (MMPs) are important in dentinal caries, and analysis of recent data demonstrates the presence of other collagen-degrading enzymes, cysteine cathepsins, in human dentin. This study aimed to examine the presence, source, and activity of cysteine cathepsins in human caries. Cathepsin B was detected with immunostaining. Saliva and dentin cysteine cathepsin and MMP activities on caries lesions were analyzed spectrofluorometrically. Immunostaining demonstrated stronger cathepsins B in carious than in healthy dentin. In carious dentin, cysteine cathepsin activity increased with increasing depth and age in chronic lesions, but decreased with age in active lesions. MMP activity decreased with age in both active and chronic lesions. Salivary MMP activities were higher in patients with active than chronic lesions and with increasing lesion depth, while cysteine cathepsin activities showed no differences. The results indicate that, along with MMPs, cysteine cathepsins are important, especially in active and deep caries.

MMP Activity in the Hybrid Layer Detected with in situ Zymography

Dentinal proteases are believed to play an important role in the degradation of hybrid layers (HL). This study investigated the HL gelatinolytic activity by in situ zymography and functional enzyme activity assay. The hypotheses were that HLs created by an etch-and-rinse adhesive exhibit active gelatinolytic activity, and MMP-2 and -9 activities in dentin increase during adhesive procedures. Etched-dentin specimens were bonded with Adper Scotchbond 1XT and restored with composite. Adhesive/dentin interface slices were placed on microscope slides, covered with fluorescein-conjugated gelatin, and observed with a multi-photon confocal microscope after 24 hrs. Human dentin powder aliquots were prepared and assigned to the following treatments: A, untreated; B, etched with 10% phosphoric acid; or C, etched with 10% phosphoric acid and mixed with Scotchbond 1XT. The MMP-2 and -9 activities of extracts of dentin powder were measured with functional enzyme assays. Intense and continuous enzyme activity was detected at the bottom of the HL, while that activity was more irregular in the upper HL. Both acid-etching and subsequent adhesive application significantly increased MMP-2 and -9 activities (p < 0.05). The results demonstrate, for the first time, intrinsic MMP activity in the HL, and intense activation of matrix-bound MMP activity with both etching and adhesive application.

Heparan sulfates and heparins: similar compounds performing the same functions in vertebrates and invertebrates?

The distribution and structure of heparan sulfate and heparin are briefly reviewed. Heparan sulfate is a ubiquitous compound of animal cells whose structure has been maintained throughout evolution, showing an enormous variability regarding the relative amounts of its disaccharide units. Heparin, on the other hand, is present only in a few tissues and species of the animal kingdom and in the form of granules inside organelles in the cytoplasm of special cells. Thus, the distribution as well as the main structural features of the molecule, including its main disaccharide unit, have been maintained through evolution. These and other studies led to the proposal that heparan sulfate may be involved in the cell-cell recognition phenomena and control of cell growth, whereas heparin may be involved in defense mechanisms against bacteria and other foreign materials. All indications obtained thus far suggest that these molecules perform the same functions in vertebrates and invertebrates.

Crotamine Mediates Gene Delivery into Cells through the Binding to Heparan Sulfate Proteoglycans

Recently we have shown that crotamine, a toxin from the South American rattlesnake Crotalus durissus terrificus venom, belongs to the family of cell-penetrating peptides. Moreover, crotamine was demonstrated to be a marker of centrioles, of cell cycle, and of actively proliferating cells. Herein we show that this toxin at non-toxic concentrations is also capable of binding electrostatically to plasmid DNA forming DNA-peptide complexes whose stabilities overcome the need for chemical conjugation for carrying nucleic acids into cells. Interestingly, crotamine demonstrates cell specificity and targeted delivery of plasmid DNA into actively proliferating cells both in vitro and in vivo, which distinguishes crotamine from other known natural cell-penetrating peptides. The mechanism of crotamine penetration and cargo delivery into cells was also investigated, showing the involvement of heparan sulfate proteoglycans in the uptake phase, which is followed by endocytosis and peptide accumulation within the acidic endosomal vesicles. Finally, the permeabilization of endosomal membranes induced by crotamine results in the leakage of the vesicles contents to the cell cytosol.

Development of new heparin-like compounds and other antithrombotic drugs and their interaction with vascular endothelial cells

The anticlotting and antithrombotic activities of heparin, heparan sulfate, low molecular weight heparins, heparin and heparin-like compounds from various sources used in clinical practice or under development are briefly reviewed. Heparin isolated from shrimp mimics the pharmacological activities of low molecular weight heparins. A heparan sulfate from Artemia franciscana and a dermatan sulfate from tuna fish show a potent heparin cofactor II activity. A heparan sulfate derived from bovine pancreas has a potent antithrombotic activity in an arterial and venous thrombosis model with a negligible activity upon the serine proteases of the coagulation cascade. It is suggested that the antithrombotic activity of heparin and other antithrombotic agents is due at least in part to their action on endothelial cells stimulating the synthesis of an antithrombotic heparan sulfate.

Abundance of MMPs and Cysteine Cathepsins in Caries-affected Dentin

Degradation of dentin matrix components within caries dentin has been correlated with the activity of host-derived proteases, such as matrix metalloproteases (MMPs) and cysteine cathepsins (CTs). Since this relationship has not been fully established, we hypothesized that the abundance of MMPs and CTs in caries-affected dentin must be higher than in intact dentin. To test this premise, we obtained 5 slices (200 µm) from 5 intact teeth and from 5 caries-affected teeth (1 slice/tooth) and individually incubated them with primary antibodies for CT-B, CT-K, MMP-2, or MMP-9. Negative controls were incubated with pre-immune serum. Specimens were washed and re-incubated with the respective fluorescent secondary antibody. Collagen identification, attained by the autofluorescence capture technique, and protease localization were evaluated by multi-photon confocal microscopy. The images were analyzed with ZEN software, which also quantitatively measured the percentages of collagen and protease distribution in dentin compartments. The abundance of the test enzymes was markedly higher in caries-affected than in intact dentin. CT-B exhibited the highest percentage of co-localization with collagen, followed by MMP-9, MMP-2, and CT-K. The high expression of CTs and MMPs in caries-affected teeth indicates that those host-derived enzymes are intensely involved with caries progression.

Cysteine proteinase activity regulation. A possible role of heparin and heparin-like glycosaminoglycans.

Papain is considered to be the archetype of cysteine proteinases. The interaction of heparin and other glycosaminoglycans with papain may be representative of many mammalian cysteine proteinase-glycosaminoglycan interactions that can regulate the function of this class of proteinases in vivo. The conformational changes in papain structure due to glycosaminoglycan interaction were studied by circular dichroism spectroscopy, and the changes in enzyme behavior were studied by kinetic analysis, monitored with fluorogenic substrate. The presence of heparin significantly increases the α-helix content of papain. Heparin binding to papain was demonstrated by affinity chromatography and shown to be mediated by electrostatic interactions. The incubation of papain with heparin promoted a powerful increase in the affinity of the enzyme for the substrate. In order to probe the glycosaminoglycan structure requirements for the papain interaction, the effects of two other glycosaminoglycans were tested. Like heparin, heparan sulfate, to a lesser degree, was able to decrease the papain substrate affinity, and it simultaneously induced α-helix structure in papain. On the other hand, dermatan sulfate was not able to decrease the substrate affinity and did not induce α-helix structure in papain. Heparin stabilizes the papain structure and thereby its activity at alkaline pH.