WO1999011221A1

WO1999011221A1 - Use of antioxidant compounds and/or compounds bearing thiol groups for reducing the toxicity of biological materials

Info

Publication number: WO1999011221A1
Application number: PCT/FR1998/001871
Authority: WO
Inventors: Lena Stanislawski
Original assignee: Institut National De La Sante Et De La Recherche Medicale (Inserm); Universite Rene Descartes
Priority date: 1997-09-01
Filing date: 1998-08-31
Publication date: 1999-03-11
Also published as: FR2767696A1

Abstract

The invention concerns the use of at least one antioxidant compound and/or a compound bearing thiol groups as substances for reducing the toxicity of a biological material comprising a polymer matrix.

Description

ANTI-OXIDIZING COMPOUNDS AND / OR CARRIERS OF THIOL GROUPS FOR REDUCING TOXICITY OF BIOMATERIALS

The present invention relates to the use of antioxidant compounds and / or carriers of thiol groups as substances intended to reduce the toxicity of biomaterials comprising a polymer matrix. Biomaterials are used in particular to manufacture prostheses, implantable or non-implantable artificial organs, restorative, bonding and sealing materials. These can be metals, ceramics, polymers, carbon derivatives and medical devices that often need to be sealed. Biomaterials must be tolerated by the body and not have toxic effects for the same organism.

However, it appeared that biomaterials comprising a polymer matrix are capable of causing undesirable toxic effects. The authors of the present invention have shown in particular that glass-ionomer cements (CVI), in particular CVI modified by resins (CVI-resins), available on the dental product market sometimes exhibited such toxic effects, characterized by a apoptosis of the pulp cells which can lead to the destruction of the dental pulp in the medium term.

CVIs consist of an aluminosilicate filler and a polymer matrix carrying negatively charged groups (ionomer). The polymer matrix of conventional CVIs can in particular be polyacrylic acid. The polymerization is induced by a chemical initiator. In CVI-resins of the following generations, the polymer matrix consists of a copolymer formed by ionomer polymer units generally derived from polyacrylic acid and polymer units of a resin generally of polymethacrylate type obtained by in situ polymerization of monomers d methacrylic acid, the copolymer being further crosslinked. The hardening of these CVI-resins is obtained only by photopolymerization. The designation "CVI" below also refers conventional chemopolymerizing CVIs than photopolymerizing CVI-resins.

Generally, the filler is presented to the practitioner in powder form and the matrix in liquid form, to be mixed with the powder extemporaneously. The entire load and the matrix can also be presented in the form of pre-dosed capsules that can be activated using vibrators.

In the field of dentistry, CVI constitute materials of choice as: - cement for sealing prosthetic parts,

- protective backgrounds under composites and silver amalgams in deep cavities,

- restorative biomaterials, both in pediatric and geriatric dentistry. CVIs, and in particular CVI-resins, have many advantages.

They have a fairly good adhesion power to the dentinal and cementary walls, which limits the preparation by milling of the preparations by the practitioner. After a phase of contraction of setting and cracking, the CVI-resins take up water, from saliva and / or free water from dental tissues and exhibit an expansion. They then occupy all the useful volume, becoming homogeneous, without cracks or cracks appearing. They are therefore relatively waterproof. CVIs have the advantage of releasing fluorides continuously. As a result, they are capable of controlling bacterial colonization at the interface between the tooth and the biomaterial. Their potential to prevent recurrence of caries is at the heart of the interest of practitioners. CVIs can be recharged with fluorides, simply by using mouthwashes or fluoridated toothpastes. Their bacteriostatic and therefore cariostatic power can be maintained during time. This implies that it is not necessary to change them to maintain this preventive action.

CVIs are also easy to use in the clinic.

These various properties and qualities make CVIs, and in particular CVI-resins, particularly interesting for the reduction of carious pathologies in populations of developing countries. The implementation of such products after minimal milling can indeed be carried out by nurses or by health personnel who have not followed the course of dental surgery studies, but are simply trained in these prevention / filling tasks. This minimal atraumatic dentistry is bound to have a great future in developing countries.

The authors of the present invention have demonstrated that CVIs, during their initial intake and in the days that follow, can be the source of toxic effects on the pulp tissues, in particular by the release of toxic compounds such as as the acid components and the residual monomers used in the composition of the CVI-resins.

In vivo, dentin, on which the CVIs are applied, acts as a barrier against these toxic components and thus protects the pulp tissue. However, this protection is only ensured when the dentin layer is sufficiently thick, namely at least 1 mm. However, it is impossible, before a dental surgery operation, to assess the residual thickness of dentin. In addition, in the case of deep caries, the residual dentin layer is extremely thin, even non-existent.

In view of the numerous advantages that these biomaterials confer, the reduction of the cytotoxicity for which they are responsible represents a major challenge for their development. In order to counter the known toxicity of the monomers used, it has been proposed to prepolymerize the monomer during the manufacture of CVI-resins (Wilson et al, The International Journal of Prosthodentics, 1990, vol. 3, n.5, pp 425-429). However, this solution is not satisfactory because the CVI-resins thus obtained have unsaturated groups which are themselves harmful.

The authors of the present invention have surprisingly discovered that a considerable reduction in the toxic effects induced by biomaterials comprising a polymer matrix was obtained by using at least one compound chosen from the class of antioxidants and / or compounds carrying thiol groups .

The present invention therefore relates to the use of at least one antioxidant compound and / or carrier of thiol groups as a substance intended to reduce the toxicity of biomaterials comprising a polymer matrix.

By "polymer matrix" is meant a matrix consisting of an already formed polymer or the components, such as monomers, intended to form a polymer by polymerization, said polymerization generally being carried out in situ.

The present invention therefore also relates to the use of antioxidant compounds and / or carriers of thiol groups as substances intended to reduce the toxicity of biomaterials comprising a polymer matrix obtained by polymerization of monomers in situ.

By "antioxidant" is meant any compound capable of trapping free radicals. These compounds active on the reduction of the cytotoxicity induced by biomaterials comprising a polymer matrix can be used alone or as a mixture. Among these, antioxidants comprising one or more thiol groups are particularly preferred. Reduced glutathione, which occurs naturally in cells, and N-acetyl cysteine, which is an acetylated amino acid, have been shown to be particularly effective in reducing cellular toxicity.

Said biomaterials comprising a polymer matrix can be any biomaterial comprising a polymer matrix useful in orthopedic or dental surgery, and in particular products for restoring dental substances such as in particular glass-ionomer cements (CVI), in particular CVI-resins, amelodentine adhesives, adhesion promoters, adhesives and composite resins.

Said antioxidant and / or carrier compound of thiol groups used in accordance with the invention can be applied simultaneously with said biomaterial comprising a polymer matrix. It can also be applied sequentially, by adsorption on the surface requiring such treatment, prior to the application of said biomaterial, and / or optionally mixed with a surface preparation agent.

The present invention also relates to a biomaterial composition comprising at least one antioxidant compound and / or carrier of thiol groups and a biomaterial comprising a polymer matrix.

In the case of CVI-resins, said antioxidant and / or carrier compound of thiol groups can be advantageously added to the liquid phase containing the monomer to be polymerized to form the resin.

The present invention also relates to a composition intended to reduce the toxicity of a biomaterial comprising a polymer matrix, said composition comprising at least one antioxidant compound and / or carrier of thiol groups, and a surface preparation agent.

In dental surgery, this surface preparation agent, also called a "primer", can in particular be 10% polyacrylic acid. It is applied to the walls of the cavity to be closed and makes it possible to eliminate the layer of residual debris, and to improve the adhesion of the biomaterials to the dental surfaces. This surface preparation agent, which may have a very low pH, can also exert an etching action. It is also possible to adsorb said antioxidant compound and / or carrier of thiol groups on the surface in contact with the biomaterial. In dental surgery, said antioxidant and / or carrier compound of thiol groups can therefore be adsorbed on the walls of the cavity to be closed before application of the biomaterial, which can in particular be a CVI, in particular a CVI-resin.

The present invention also relates to a kit useful in orthopedic or dental surgery comprising

- A first container containing at least one antioxidant compound and / or carrier of thiol groups;

- a second container containing either a biomaterial comprising a polymer matrix or a surface preparation agent; and

- optionally a third container containing a surface preparation agent.

The present invention also relates to a method of surgical and / or therapeutic treatment comprising the steps consisting in:

- Apply to the surface of the organism requiring such treatment a biomaterial composition comprising at least one antioxidant compound and / or carrying thiol groups and a biomaterial comprising a polymer matrix;

- And / or adsorb on the surface of the organism requiring such treatment at least one antioxidant compound and / or carrier of thiol groups then apply a biomaterial comprising a polymer matrix;

-and / or applying to the surface of the organism requiring such treatment a composition comprising at least one antioxidant compound and / or carrier of thiol groups and a surface preparation agent, then a biomaterial comprising a polymer matrix.

The following examples and figures illustrate the invention without limiting it. Legend of figures:

FIG. 1 represents the percentage of in vitro viability of human dermal fibroblasts (FIG. 1A) and of human pulp cells (FIG. 1B) in the presence of CVI, as a function of the reduced glutathione concentration (GSH), relative to control cells without CVI or inhibitory antioxidant.

FIG. 2 represents the percentage of in vitro viability of human dermal fibroblasts (FIG. 2A) and human pulp cells (FIG. 2B) in the presence of CVI, as a function of the concentration of

N-acetyl cysteine (NAC) compared to control cells without CVI or inhibitory antioxidant.

EXAMPLE 1 Reduced glutathione inhibitory effect on CVI-induced cell toxicity.

at. Materials and methods :

Preparation of cell cultures:

The cytotoxicity of biomaterials was assessed by a test

"MITI" on human fibroblasts in vitro on the one hand and on human pulp cells in vitro on the other hand. This "MTT" test is a colorimetric test based on the quantification of mitochondrial dehydrogenases, enzymes which signify cell viability.

Pulp cells were obtained from cultures of pulp explants from healthy donors. This is "hospital waste", obtained after the extraction of germs from wisdom teeth or premolars, the extractions of these teeth coming within the usual framework of orthodontic treatments. Preparation of CVI pellets under standardized conditions:

CVIs are light-cured under standardized conditions according to the protocol defined by the manufacturers. The size of the samples obtained is also controlled, due to the use of a silicone mold allowing the preparation of a series of samples which do not vary in size. The volume and the taking of the samples of each cement being thus well defined, these photopolymerized samples are placed in the culture medium at different times and for determined times so as to establish a kinetics of diffusion of the products released by these biomaterials. The concentration of the culture medium containing the eluate is also controlled. Put in the presence of cell cultures, the eluates interact with them. The viability of the pulp cells is reflected by the colorimetric test with MTT.

Different CVI-resins have been evaluated for their toxicity:

- Vitremer® (3M), - Fuji II LC® (GC Int.)

- Compoglass® (Vivadent), and

- Photac Fil® (ESPE)

These different CVIs were chosen because of their great use by dental surgeons.

Effects of reduced glutathione:

The cells subjected to the action of eluates from the biomaterials evaluated are cultured in the presence or in the absence of reduced glutathione. b. Results:

The protective effect of reduced glutathione (GSH) on the toxicity of CVI tested on human dermal fibroblasts and on human pulp cells is evaluated by the increase in cell viability expressed by the percentage of cell viability in the presence of biomaterial and d antioxidant compared to cell viability in the presence of biomaterial but without antioxidant (Table I).

The experiments were carried out three times and the results were confirmed.

Table I: Protective effect of GSH (10 mM) on the cytotoxicity of CVI

na = not determined

In the presence of GSH, the percentage of living cells is greater than in the absence of GSH. The cell membranes are intact, and the cytoplasm of these cells is clear.

The results obtained allow us to conclude that the cytotoxic effects of light-curing CVIs - effects linked to the release of molecules toxic to human pulp and dermal cells in culture - can be reduced up to 48% of the initial value for Fuji Il LC® and 61% for Compoglass® when GSH is added to the eluate obtained from CVI studied.

FIGS. 1A and 1B show that the reducing effect of cytotoxicity is obtained for doses of GSH of between 1 and 10 mM.

EXAMPLE 2:

Inhibitory effect of N-acetyl cysteine on CVI-induced cell toxicity.

at. Materials and methods :

Experiments identical to those carried out with GSH are carried out with N-acetyl cysteine (NAC). The materials and methods are identical to those described in Example 1.

b. Results

The protective effect of N-acetyl cysteine (NAC) on the toxicity of CVI tested on human dermal fibroblasts and on human pulp cells is evaluated by the increase in cell viability expressed by the percentage of cell viability in the presence of biomaterial and antioxidant compared to cell viability in the presence of biomaterial but without antioxidant (Table II).

The experiments were carried out three times and the results were confirmed. Table II: Protective effect of NAC (30 mM) on the cytotoxicity of CVI

na = not determined

FIGS. 2A and 2B show that the reducing effect of cytotoxicity is obtained for doses of NAC of between 0.1 and 30 mM.

Examples 1 and 2 show the inhibitory effect of GSH and NAC on CVI-induced cytotoxicity.

These two compounds having perfect harmlessness for the organism, they are particularly useful for improving the biocompatibility of biomaterials comprising a polymer matrix used in dentistry or general orthopedics.

Claims

1. Use of at least one antioxidant compound and / or carrier of thiol groups as a substance intended to reduce the toxicity of a biomaterial comprising a polymer matrix.

2. Use according to claim 1, wherein said biomaterial comprises a polymer matrix obtained by polymerization of monomers in situ.

3. Use according to any one of the preceding claims, in which said antioxidant compound is reduced glutathione or N-acetyl cysteine.

4. Use according to any one of the preceding claims, in which said biomaterial is chosen from a glass-ionomer cement (CVI), an amelo-dentin adhesive, and a composite resin.

5. Use according to any one of the preceding claims, in which said biomaterial is a CVI-resin.

6. Biomaterial composition comprising at least one biomaterial comprising a polymer matrix, and reduced glutathione or N-acetyl cysteine, or a mixture of reduced glutathione and N-acetyl cysteine.

7. Composition according to claim 6 wherein said biomaterial comprises a polymer matrix obtained by polymerization of monomers in situ.

8. Composition according to any one of claims 6 or 7 in which said biomaterial is chosen from a glass-ionomer cement (CVI), an amelo-dentin adhesive, and a composite resin.

9. Composition according to any one of claims 6 to 8 wherein said biomaterial is a CVI-resin.

10. Composition intended to reduce the toxicity of a biomaterial comprising a polymer matrix, said composition comprising at least one antioxidant compound and / or carrier of thiol groups, and a surface preparation agent.

1 1. The composition of claim 10 wherein said antioxidant compound is reduced glutathione or N-acetyl cysteine or a mixture of reduced glutathione and N-acetyl cysteine.

12. Kit useful in orthopedic or dental surgery including

- A first container containing at least one antioxidant compound and / or carrying thiol groups; - a second container containing either a biomaterial comprising a polymer matrix or a surface preparation agent; and

- optionally a third container containing a surface preparation agent.

13. Use according to any one of claims 1 to 5, wherein said antioxidant compound and / or carrier of thiol groups is in the form of a composition according to any one of claims 6 to 1 1.