WO2012156641A1

WO2012156641A1 - Use of kappa-elastin for the reconstruction and/or regeneration of periodontal tissues

Info

Publication number: WO2012156641A1
Application number: PCT/FR2012/051089
Authority: WO
Inventors: Sandrine LORIMIER; Frank ANTONICELLI; Stéphane BARTHELEMI; William Hornebeck
Original assignee: Universite De Reims Champagne Ardenne (U.R.C.A.)
Priority date: 2011-05-19
Filing date: 2012-05-15
Publication date: 2012-11-22
Also published as: FR2975292A1; FR2975292B1

Abstract

The invention relates to the use of at least one kappa-elastin peptide as an active ingredient in a pharmaceutical or cosmetic composition intended for the reconstruction and/or regeneration of a soft tissue of the periodontium, among the gingival tissue and the dentoalveolar ligaments, and/or of a hard tissue of the periodontium, among the alveolar bone and the root cement.

Description

USE OF KAPPA-ELASTIN FOR THE REPAIR AND / OR REGENERATION OF PERIODONTAL FABRICS

The invention relates to soluble peptides of elastin used for the preparation of a pharmaceutical or cosmetic composition capable of repairing and / or regenerating at least one of the periodontal tissues. The invention also relates to a composition comprising at least one particular peptide of kappa-elastin. Context of the invention

Periodontal diseases are defined as multifactorial tissue inflammation. They are characterized by an evolutionary inflammation associated with a progressive destruction of the tissues of support of the tooth, leading to the possible loss of the latter.

Periodontal diseases affect 40 to 50% of the adult population in industrialized countries. Most often, as in the case of gingivitis, they begin to reach the superficial periodontal tissues (gingiva). If no treatment is put in place, the deeper tissues (bone, alveolo-dental ligament) are then affected. We then speak of periodontitis. In the terminal phase of the disease, the tooth can be lost. Thus, periodontal diseases are the cause of the extraction of 30 to 40% of teeth extracted in the dental office and this trend increases with the age of patients.

In addition, it is recognized that periodontal diseases may be related, more or less directly, to certain general health problems or systemic diseases, such as diabetes, cardiovascular disease, premature labor, etc.

In periodontal diseases, the inflammation is partly due to the appearance and proliferation of so-called "periodontal-pathogenic" bacteria, such as Porphyromonas Gingivalis, involved in the destruction of periodontal tissues. Upon the accumulation of bacterial biofilm in the sulcus, an inflammatory infiltrate is formed, characterized by the recruitment of leucocytes.

Several types of periodontal tissue cells, such as polymorphonuclear neutrophils, gingival fibroblasts, and osteoblasts, respond to bacterial aggression by secreting a variety of inflammatory (IL-1, PGE ₂ ) cause of overexpression of proteolytic enzymes involved in periodontal matrix degradation (Cox and garlic, 2006), resulting in tissue lysis. These enzymes, with collagenolytic and elastolytic activities, are essential for the pathological degradation of periodontal tissues. These enzymes are endowed with collagenolytic activity (MMPs-1, -13, etc.) but also with elastolytics (MMPs -2, 9, leukocyte elastase, etc.). Thus, 70% of the collagen in situ is destroyed after 4 to 7 days of bacterial accumulation, destruction to which is added the lysis of the elastic fibers of the gingival connective tissue. Induced elastolysis, in addition to its influence on tissue functionality, generates peptide fragments that have the ability to modulate the phenotype of many cell types.

Thus, periodontal diseases can lead to at least partial destruction of the anchoring structures of the tooth, including the gingiva, the alveolar bone, the dentoalveolar ligament and the root cementum.

At present, for the prevention and / or treatment of periodontal diseases, it is known, for example, to use techniques of periodontal gingival or bone surgery, such as gingivo-dental grafting or bone grafting. Such grafts require two operating sites, respectively a donor site where tissues are taken and the site to be treated, and cause substantial pain.

There is therefore a real need for less expensive and less painful alternatives to the prophylactic and / or therapeutic solutions currently available for the prevention and / or treatment of these periodontal inflammatory diseases.

Summary of the invention

The present invention makes it possible to overcome the disadvantages of the therapeutic solutions of the state of the art, and in particular provides an alternative to the surgical procedures currently used.

In fact, the inventors have discovered that so-called peptides derived from the partial hydrolysis of elastin, called kappa-elastin peptides, can improve the gingival tissue structure by promoting the synthesis of collagen and elastin. The inventors have also discovered that these peptides can act on the bone structure of the periodontium, in particular on osteoblastic differentiation, by promoting the expression of bone matrix proteins and by promoting the mineralization of the bone matrix. The invention thus aims in particular at the use of at least one kappa-elastin peptide to act bifunctionally on the soft tissues and the hard (bone) tissues of the periodontium.

Until now, the kappa-elastin peptides, commonly obtained by partial hydrolysis of elastin from animal nuchal ligament, are used in topical applications, to fight against the loss of elasticity and the aging of the superficial cells of the skin. There was no indication that these peptides could act on the periodontal tissues, such as the gingiva, since the fibroblasts (cells where kappa-elastin is to function) of the skin and gingiva exhibit many phenotypic differences. Moreover, there was no reason to believe that these peptides could allow regeneration of tissues, going beyond simple tissue repair.

Such a discovery is particularly surprising and allows to consider many oral applications, both at the preventive level by fighting against the aging of the periodontal tissues and by promoting the maintenance of the biological structure of the gingiva, that at the therapeutic level, by allowing not only healing but also the regeneration of soft and / or hard tissues.

Also, the invention proposes to use at least one of the kappa-elastin peptides for the preparation of a composition intended to promote the creation of absent or insufficient periodontal tissues by improving the production of matrix proteins such as collagen and elastin, and / or promoting bone mineralization.

The invention also proposes using at least one kappa-elastin peptide for the preparation of a composition intended to promote the regeneration and repair of degraded tissues during periodontal diseases, such as the gingival connective tissue and / or the alveolar bone tissue.

The invention also proposes using a composition according to the invention, or more generally a peptide according to the invention, to promote the increase in the height and / or the thickness of the attached gingiva, the role of which is important. in the maintenance of soft tissues on the dental surfaces, and / or bone mass. Such a use allows for example to avoid gingivo-dental grafts.

The subject of the invention is therefore a therapeutic, pharmaceutical or cosmetic composition comprising at least one kappa-elastin peptide for use in the repair and / or regeneration of at least one periodontal tissue in the human or non-human mammal .

The subject of the invention is also the use of at least one peptide of kappa-elastin, as active principle, in a therapeutic, pharmaceutical or cosmetic composition intended for the repair and / or regeneration of at least one periodontal tissue in the human or non-human mammal.

The subject of the invention is also a kappa-elastin peptide for use as an active principle, in a therapeutic, pharmaceutical or cosmetic composition intended for the repair and / or regeneration of at least one periodontal tissue in the body. mammalian human or nonhuman.

The subject of the invention is also the use of a composition according to the invention, or more generally a peptide according to the invention, for the repair and / or the regeneration of at least one periodontal tissue in the mammal human or non-human.

The subject of the invention is also any method of therapeutic and / or prophylactic and / or cosmetic treatment using a kappa-elastin peptide for the repair and / or regeneration of at least one periodontal tissue in the human or non-human mammal human. Advantageously, the composition according to the invention, or more generally the peptide according to the invention, is capable of acting on at least one soft tissue of the periodontium among the gingival tissue and the dentoalveolar ligaments. Alternatively or cumulatively, the composition according to the invention, or more generally the peptide according to the invention, is capable of acting on at least one hard tissue of the periodontium among the alveolar bone and the radicular cementum.

The inventors have discovered that the kappa-elastin peptides comprising at least six consecutive amino acids according to the consensus sequence (XiGX ₂ X ₂ PG) n (SEQ ID No. 1), n being an integer between 1 and 10, exhibit a three-dimensional elbow conformation that potentiates the action mediated by kappa-elastin.

In an exemplary embodiment of the invention, at least one kappa-elastin peptide comprising at least the sequence (XiGX ₂ X ₂ PG) n, in which X 1 represents any amino acid and X ₂ represents a non-polar amino acid and n is an integer between 1 and 10, preferably between 1 and 7, and more preferably n equal 3. More particularly, the invention proposes to use at least one kappa-elastin peptide having at least minus the sequence (VGVAPG) n (SEQ ID No. 2) and / or (PGAIPG) n (SEQ ID No. 3), where n is an integer between 1 and 10, preferably between 1 and 7, and more preferably n equal 3.

According to the invention, such peptides may comprise, to the right and to the left of these sequences and / or between each of these preferred sequences at least one other amino acid.

Advantageously, at least one peptide of the invention can be obtained by chemical synthesis, so that it is possible to overcome the drawbacks associated with the use of products derived from the proteolytic digestion of elastin originating from nuchal ligament. mammal.

In a particular example, all the peptides of kappa elastin obtained by partial hydrolysis of elastin are used. Thus, according to one embodiment, the composition according to the invention comprises all of the kappa-elastin peptides obtained by partial hydrolysis of elastin for their use as an active ingredient for the repair and / or regeneration of at least one periodontal tissue in the human or non-human mammal.

In one embodiment of the invention, the peptide or peptides of kappa-elastin are used at a concentration of at least 25 μg / ml, preferably at least 75 μg / ml and more preferably at least equal to 100 μg / ml. . Thus, a composition according to the invention advantageously comprises at least 25 μg / ml, preferably at least 75 μg / ml and more preferably at least equal to 100 μg / ml of kappa elastin peptides. Detailed description of the invention

In the oral cavity, the elasticity and resilience of tissues subject to frequent deformities and tensions, such as chewing and occlusal forces, are provided by elastic fibers. The rheological properties of these elastic fibers are particularly ensured by the elastin which constitutes the amorphous structure of the elastic fibers. Elastin is a major component of mature elastic fibers, particularly abundant in the gum (6%). In general, elastolysis leads to the production of particular elastin fragments. These peptides are called "matrikines" or "elastokines" because of their "cytokine-like" activity mediated by their binding to the EBP protein ("Elastin Binding Protein") (Duca et al., 2004), also called S-Gal . The inventors have discovered that kappa-elastin peptides are able to mimic the effects of elastin by binding to EBP receptors. In addition, the inventors have discovered that gingival fibroblasts, such as osteoblastic-type bone cells, possess on their plasma membrane the EBP receptor, and that the action of kappa-elastin passes mainly through this receptor.

Also, the inventors propose using one or more peptides of kappa elastin to promote the repair and / or regeneration of the biological structure of the periodontium. The invention more particularly relates to at least one kappa-elastin peptide for use as an active ingredient in the preparation of a pharmaceutical or cosmetic composition intended for the repair and / or regeneration of at least one tissue of the periodontium in the mammal. The invention also relates to a pharmaceutical or cosmetic composition comprising at least one kappa-elastin peptide for use as an active ingredient for the repair and / or regeneration of at least one periodontal tissue in the mammal. The invention also relates to any therapeutic and / or prophylactic and / or cosmetic treatment for the repair and / or regeneration of at least one periodontal tissue in the mammal using at least one peptide of kappa elastin. According to the invention, kappa-elastin corresponds to a mixture of soluble elastin peptides whose molecular weights vary between 1000 and 100000 Da. Preferably, about 60% of the peptides have a size between 1000 and 60000 Da.

Kappa-elastin mainly comprises non-polar amino acids, which allow the elasticity functions. More specifically, kappa-elastin contains 75% of hydrophobic (apolar) amino acids, and predominantly Glycine (which represents one amino acid in three), Alanine (which represents one amino acid out of 4), Proline and Valine (each of which represents one amino acid out of 9). Kappa-elastin comprises peptides of sequence (XiGX ₂ X ₂ PG) n.

Preferably, the at least one kappa-elastin peptide used has a molecular weight between 1000 and 60000 Da, in order to promote its penetration and / or diffusion into the tissue to which it is applied.

Kappa-elastin can in particular be obtained by partial hydrolysis of elastin extracted from the nuchal ligament of a bovine or other mammal, with 0.1M potassium hydroxide taken up in ethanol at 80 ° C., according to the method proposed by Jacob. et al. (Jacob et al., 1984), kappa-elastin then corresponding to all of the soluble peptides resulting from this hydrolysis.

According to the invention, it is possible to use all or part of the peptides resulting from such partial hydrolysis of elastin.

In all of the present description, when reference is made to kappa-elastin, it is meant all the kappa-elastin peptides obtained by partial hydrolysis of elastin. Similarly, when referring to a kappa-elastin peptide is meant any of the kappa-elastin peptides. When it is desired to refer to one or more particular peptides of kappa-elastin, they are specified.

The peptide or peptides of the invention may in particular come from kappa elastin purchased commercially.

The peptide or peptides of kappa-elastin according to the invention can also be obtained by chemical synthesis. In this case, the peptide or peptides comprises (s) at least one sequence conforming to the sequence of at least one kappa-elastin peptide obtained by partial hydrolysis of elastin.

In the case of a chemical synthesis, the amino acids of the peptide or peptides according to the present invention may be natural or not. By non-natural amino acid is meant an analogue or derivative of a natural amino acid. For example, an unnatural amino acid may have an elongated, shorter side chain, or variant having appropriate functional groups. Of course, stereoisomers L and D are contemplated. In addition, the peptide bonds can be modified to make them resistant to proteolysis. For example, at least one peptide bond (-CO-NH-) may be replaced by a divalent bond selected from (-CH ₂ -NH-), (-NH-CO-), (-CH ₂ -O-), (-CH ₂ -S-), (-CH ₂ -CH ₂ -), (-CO-CH ₂ -), (-CHOH-CH ₂ -), (-N = N-), and (-CH = CH).

The residues of the sequences described above may vary conservatively. By conservative, it is meant that the variant residue will have comparable physicochemical characteristics. Among the physicochemical characteristics taken into account are steric hindrance, polarity hydrophobicity, or charge.

Thus, in the invention, the term "kappa-elastin peptide" is understood to mean any peptide comprising at least one amino acid sequence identical to the amino acid sequence of a peptide derived from the partial hydrolysis of the kappa-elastin peptide. elastin, whether the peptide is derived from such hydrolysis or is obtained by chemical synthesis.

Preferably, the invention relates to at least one kappa-elastin peptide comprising at least six consecutive amino acids, in order to promote binding to EBP receptors, and its use for the repair and / or regeneration of at least one tissue of the periodontal. For example, according to the invention, at least one kappa-elastin peptide comprising at least six consecutive amino acids is used as the active principle for the production of a composition intended to repair and / or regenerate at least one tissue of the periodontal.

In the invention, by periodontal means the organ composed of two hard tissues (alveolar bone and root cementum) and two soft tissues (gum and dento-alveolar ligament). All these hard and soft tissues form the support of the tooth and participate in maintaining its function within the oral cavity.

In the invention, by periodontal repair means the healing of tissues of the tooth attachment system, without total restoration of the architecture and / or the function of the damaged tissue. By periodontal regeneration is meant a restoration of integrally damaged tissues, corresponding to the formation of a new cementing and / or the formation of new functional collagen fibers and / or the formation of a new alveolar bone.

According to the invention, at least one kappa-elastin peptide capable of acting on at least one periodontal soft tissue, among the gingival tissue and the dento-alveolar ligaments, and / or on at least one hard tissue of the periodontium is used. among the alveolar bone and the radicular cementum.

The gingiva, or gingival tissue, is an oral fibril that covers the alveolar bone and surrounds the teeth in their cervical area. Gingival connective tissue, or lamina propria, has a complex architecture that anticipates its functions. It consists essentially of collagen fibers (about 60% of the tissue volume), proteoglycans, fibronectin, elastin (about 6%), fibroblasts (about 5% of the total tissue volume, 65% of the total cells). , vessels, nerves and substances (about 35%). Type I collagen is the major collagenic component of the gingiva (91% of total collagen). Collagenic fibers, preferentially of type I, are organized in bundles of thick fibers and participate in the formation of the supra-alveolar gingival fibrous apparatus, giving the gingiva a certain tonicity and biomechanical resistance (Bartold and Garlic, 2000; Narayanan, 2006).

The inventors have shown that kappa-elastin peptides increase the expression of type I collagen by human gingival fibroblasts, suggesting that these peptides may promote healing and repair of gingival tissues. This healing and repair of gingival tissues could prevent or even compensate for tissue loss during periodontal disease.

The dentoalveolar ligament, or periodontal ligament, is a dense, highly vascularized fibrocellular connective tissue that occupies a space of about 0.25 mm (0.15 to 0.38 mm) between the root of the tooth and the tooth. bone cell. It is continuous with the gingival connective tissue at the alveolar ridge, and the pulpal connective tissue at the apical foramen. This tissue contiguity explains, on the one hand, the aggravation of superficial parodontopathies towards the deep periodontium and, on the other hand, the extension of infectious pulpal pathologies towards the supporting tissues of the tooth. Like all connective tissue, the periodontal ligament consists of a cell compartment and an extracellular compartment, itself comprising type I collagen fibers preferentially (80%), packaged in a fundamental substance. Although the fibroblastic component is predominant, the periodontal membrane contains a heterogeneous cell population such as fibroblasts, osteoblasts, cementoblasts, undifferentiated mesenchymal cells capable of differentiating into fibroblasts, for example osteoblasts. In addition, the dentoalveolar ligament shares common properties with the embryonic mesenchymal tissues that make it assimilate to fetal connective tissue.

The alveolar bone is the specialized part of the maxillary and mandibular bones containing the dental cells. It is the place of insertion of the extrinsic fibers of the dento-alveolar ligament, allowing the maintenance of the tooth in its alveolus. The bone matrix consists essentially of an inorganic matrix (67% of the total volume) comprising hydroxyapatite crystals (Cai ₀ (PO ₄ ) 6 (OH) ₂ ) and an organic matrix, predominantly containing collagen (80%). at 90%). Type I collagen is the most represented collagenic protein (90%>). Other non-collagenous proteins, including osteocalcin and osteonectin, are also contained in the bone matrix. Although fundamentally comparable to other bone sites in the body, alveolar bone is subject to rapid and continuous remodeling under the influence of physiological mesial migration of teeth, orthodontic forces, and masticatory forces. Its existence is determined by the presence of teeth. In a physiological state, the bone is in perpetual shuffling, involving a coordinated coupling between the degradation of the extracellular matrix and its associated mineral part, and the neosynthesis of matrix components.

The root cementum is an anatomical element of the tooth, which by its function is a component of the periodontium. The root cementum is the layer of mineralized tissue that covers the root surface of human teeth, including the apical foramen and occasionally small coronal areas. It is a highly specialized tissue that forms the interface between root dentine and connective tissue, desmodontal and gingival. It serves as a pole of insertion of ligamentous fibers to the root surface of the tooth, thus uniting it to the bone wall and the gingiva. It is an essential part of the system of attaching the tooth to its bone socket. The structure and composition of the radicular cement confer some analogies with bone tissue. However, it is not vascularized or innervated. It consists of 65% wet mass of inorganic substances (mainly in the form of Cai ₀ (PO ₄ ) 6 (OH) ₂ ), 50% dry mass, 23% o organic substances and 12% water. .

It is known that bone remodeling essentially involves the coordination of the activities of two distinct cell types, osteoblasts and osteoclasts, the former allowing bone formation and activation and differentiation of osteoclasts, the latter bone resorption.

The osteoblast is a multi-activity cell, involved in the formation of bone tissue. The osteoblast evolves according to a linear sequence of maturation, from the undifferentiated mesenchymal cell of the bone marrow to the osteoprogenitor cell, then to the stage of pre-osteoblast, of post-mitotic osteoblast and finally, of mature osteocyte immured in its mineralized bone matrix. In the early stages of osteoblastic differentiation, cells produce type I collagen, transcription factors such as Runx-2 (Runt-related transcription factor 2), Dlx5 (Distal-less homeobox 5), Msx2 (Msh homeobox 2) , certain enzymes such as those of the family of metalloproteinases (MMP-13 and MMP-14) ensuring the formation and reorganization of the bone matrix.

The inventors have shown that all or part of the peptides of kappa-elastin make it possible to increase the gene expression of transcription factors (Runx-2, Dlx5, Msx2, TAZ, cathenin, etc.) favorable to the differentiation of osteoblasts. Likewise, the inventors have shown that all or part of the peptides of kappa-elastin make it possible to increase the expression of proteins constituting the bone matrix (osteocalcin, bone sialoprotein, type I collagen, osteonectin, etc.).

The inventors have also shown that all or part of the kappa-elastin peptides make it possible to increase the bone alkaline phosphatase activity by the osteoblastic type cells, compared with untreated cell cultures.

The inventors have also shown that the peptides of kappa-elastin make it possible to direct the osteoblastic phenotype towards bone formation, in favoring the increase of the gene and protein expression of osteoprotegerin and the decrease of that of the RANK-L protein, favorable to the differentiation and the activation of osteoclasts, in relation to bone resorption. Likewise, the inventors have shown that all or part of the peptides of kappa-elastin make it possible to promote the differentiation of undifferentiated mesenchymal cells from the bone marrow into human osteoblasts, as well as the differentiation of human osteoblasts. The peptides that are the subject of the invention, as well as the compositions according to the invention comprising such peptides, have the advantage of providing an exogenous complement of elastin precursors, which penetrates into the tissues to which they are applied, which contain elastin or endogenous derivatives. Elastin is known to be synthesized as a soluble precursor: tropoelastin (Vrhovski 1998, Wise and Weiss 2009). Several cell types, including smooth muscle cells, endothelial cells and human gingival fibroblasts, secrete tropoelastin with a molecular weight of 70 kDa (Uitto et al., Tsuruga et al., 2002, 2005). In humans, the domain encoded by exon 24 of the tropoelastin gene has 6 repeats of the hexapeptide (VGVAPG) (SEQ ID NO: 2) which is considered to be the binding domain of elastin to its receptor (Boom and garlic., 2006). The elastin receptor binding sequence (designated V14) is (VVGSPSAQDEASPL) (SEQ ID NO: 5). The inventors have also discovered that the peptides of kappa-elastin comprising at least the sequence (XiGX ₂ X ₂ PG) n (SEQ ID No. 1), preferentially the sequence (VGVAPG) n (SEQ ID No. 2), and / or (PGAIPG) n (SEQ ID No. 3), and more preferably the sequence (VGVAPG) 3 (SEQ ID No. 4), have an increased capacity for repairing and / or regenerating periodontal tissues.

In the above sequences, X represents any amino acid and X ₂ is a nonpolar amino acid.

Preferably, X1 represents any amino acid selected from the twenty naturally occurring amino acids. More preferably, Xi is a non-polar amino acid. By non-polar amino acids is meant the nine hydrophobic amino acids, namely alanine, phenylalanine, glycine, isoleucine, leucine, methionine, proline, valine and tryptophan. Preferably, the non-polar amino acids are chosen from valine, glycine, alanine and proline.

It is understood that the two residues X ₂ are not necessarily identical.

In the above sequences, n is an integer between 1 and 10, preferably between 1 and 7, and more preferably n is equal to 3.

According to an exemplary embodiment of the invention, the peptide or peptides of kappa-elastin are used at a concentration of at least 25 μg / ml, preferably at least 75 μg / ml and more preferably at least equal to 100 μg / ml. .

The subject of the invention is also a therapeutic, cosmetic or pharmaceutical composition for the repair and / or regeneration of at least one periodontal tissue comprising at least one sequence peptide (XiGX ₂ X ₂ PG) n (SEQ ID No. 1), in which Xi represents any amino acid and X ₂ represents a non-polar amino acid, and n is an integer between 1 and 10, preferably between 1 and 7, and more preferably n is equal to 3. In one exemplary embodiment, X ₂ is a non-polar amino acid, preferably selected from valine, glycine, alanine and proline.

Advantageously, the concentration of sequence peptide (XiGX ₂ X ₂ PG) n (SEQ ID No. 1) in the composition according to the invention is at least equal to 25 μg / ml by weight of the composition, preferably at least 75 μg / ml and more preferably at least 100 μg / ml by weight of the composition.

In a particular example, the composition according to the invention comprises at least one sequence peptide (VGVAPG) 3 (SEQ ID No. 4). Advantageously, the concentration of peptide sequence (VGVAPG) 3 in said composition is at least equal to 25 μg / ml by weight of the composition, preferably at least 75 μg / ml and more preferably at least 100 μg / ml by weight of the composition.

The composition according to the invention can be liquid, in aqueous suspension or not, in powder, etc. For example, the composition according to the invention may be a toothpaste, a solution for mouthwash, a cream, a gel or other formulation allowing local, oral, topical application etc. The composition according to the invention may also be in the form of a formulation capable of being injected into a deep soft tissue of the periodontium.

The peptide or peptides according to the invention can be, for example, included in toothpastes, mouthwashes, as well as creams, gels, etc., or any formulation that can be applied to or injected into a tissue. of the periodontium.

For example, the composition according to the invention may be a composition intended for oral application to periodontal tissues having lost their elasticity and / or their tonicity and / or their elastic function necessary for their maintenance on the bone and the tooth.

The invention also relates to a method of therapeutic and / or prophylactic treatment of at least one periodontal tissue having lost its elasticity and / or its tonicity and / or its elastic function using a composition according to the invention, or more generally using less a peptide of kappa-elastin according to the invention.

The composition according to the invention may be a preventive composition, for example intended to fight against the aging of the periodontium and / or to allow the maintenance of the biological structure of the periodontal surface, especially superficial (gingiva), in order to fight more easily against bacterial attacks. and / or mechanical (dental brushing or other). Such a composition favors the increase of the synthesis of the proteins constituting the gingival lamina propria, such as type I collagen.

The invention also relates to a method of therapeutic and / or prophylactic treatment of at least one periodontal tissue against bacterial and / or mechanical attacks using a composition according to the invention, or more generally using at least one peptide according to the invention .

The composition according to the invention may also be a therapeutic composition, intended for example to regenerate the superficial periodontal (gingiva) and / or deep (bone), by increasing the production of matrix proteins, such as gingival and bone type collagen I, but also other proteins of the bone matrix. This application may be particularly interesting in the case of a significant periodontal periodontal loss due to periodontal diseases (filling intraosseous periodontal defects), but also after root resection, apicectomy and cystectomy.

The invention also relates to a method of therapeutic and / or prophylactic treatment of a periodontal matrix loss, for example following a periodontal disease or following a periodontal surgery, said method using a composition according to the invention, or more generally using at least one peptide according to the invention.

The composition according to the invention may also promote the healing and / or the bone regeneration of a dental cavity after avulsion of the tooth, after removal of bulky cysts or during paro-implant defects (filling of bone loss, improvement / acceleration of bone healing, maintaining the alveolar ridge, etc.).

The invention also relates to a method of therapeutic treatment of a dental cell, for the healing and / or bone regeneration of said cell, using a composition according to the invention, or more generally using at least one peptide according to the invention.

The invention also provides for the grafting of kappa-elastin peptides to natural compounds of the gingival and bone matrix, such as type I collagen and fibrin which have favorable effects on the differentiation of mesenchymal cells of undifferentiated bone marrow. towards an osteoblastic phenotype. Such a solution allows the targeting and targeted delivery of kappa-elastin peptides within the tissues to be regenerated. Thus released, peptides of kappa-elastin, associated with type I collagen or fibrin, can act favorably on the differentiation of cells of the periodontium.

The invention therefore also relates to such kappa-elastin peptides grafted to natural compounds, such as type I collagen or fibrin. Similarly, the invention relates to the use of at least one such grafted peptide, for producing a cosmetic or pharmaceutical composition for repairing and / or regenerating at least one periodontal tissue. Similarly, the invention relates to a cosmetic or pharmaceutical composition for the repair and / or regeneration of at least one periodontal tissue comprising at least one such grafted peptide as active principle. The invention also relates to a method for therapeutic and / or prophylactic and / or cosmetic treatment of the periodontium using at least one peptide of kappa-elastin grafted to a natural compound, such as type I collagen or fibrin.

The invention will now be illustrated with the aid of the figures and examples below. These are presented as an indication and in no way limitative of the invention.

Legend of figures

Figure 1: Demonstration of the EBP receptor on gingival fibroblasts and human osteoblastic bone cells.

Figure 1A: Observation by confocal microscopy (Biorad MRC600 microscope, Zeiss Axiophot) of EBP on the membrane surface of gingival fibroblasts.

Figure 1B: Detection of EBP by western blot at human gingival fibroblasts (FGHs). Dermal fibroblasts (FDH) are used as a control.

Figure 1C: Detection of EBP by western blot in bone cells of the osteoblastic type (COH). Foreskin fibroblasts (FPH) are used as a control.

Figure 2: Demonstration of the involvement of the EBP receptor in the phenotype response of periodontal cells to kappa-elastin (kE).

Figure 2A: Western blot analysis of the involvement of EBP in the synthesis of MMP-3 by human gingival fibroblasts treated with 100 μg / ml of kappa-elastin.

Figure 2B: Western blot analysis of the involvement of EBP in the synthesis of MMP-2 by osteoblastic-type human bone cells treated with 100 μg / ml of kappa-elastin.

FIG. 2C: RT-PCR analysis of the involvement of EBP in Runx-2 gene expression by osteoblastic-type human bone cells treated with 100 μg / ml of kappa-elastin.

Figure 3: RT-PCR analysis of the influence of kappa-elastin (kE: 100 μg / mL) on the gene expression of matrix proteins by human gingival fibroblasts cultured for 1, 4 and 7 days. Figure 3A: RT-PCR of Troela, COL1 and FIB1 carried out at 1, 4 and 7 days.

FIGS. 3B, 3C, 3D: Graphical representations of the gene expression variations of Troela, COL1 and FIB1 by human gingival fibroblasts treated or not with 100 μg / ml at 1, 4 and 7 days of culture. Witness: 100%.

Figure 4: RT-PCR analysis of the effect of kappa-elastin at increasing concentration on the gene expression of Runx-2 and bone matrix proteins by bone cells of the osteoblastic type. Figure 5: RT-PCR analysis of the effect of kappa-elastin on gene expression of transcription factors characteristic of osteoblast differentiation over a 28-day period.

FIG. 5A: RT-PCR of Runx-2, Dlx5, Msx2, Taz and 18 S carried out at 1, 7, 14 and 28 days.

5B, 5C, 5D, 5E: Graphical representations of the gene expression variations of Runx-2 (B), Dlx5 (C), Msx2 (D) and Taz (E) by osteoblastic bone cells treated or not treated with 100 μg / mL of kappa-elastin or 150 μg / mL of (VGVAPG) ₃ (V3) at 14 and 28 days of culture. Witness: 100%. Figure 6: RT-PCR analysis of the effect of kappa-elastin on the gene expression of bone matrix proteins characteristic of osteoblastic differentiation over a period of 28 days.

Figure 6A: RT-PCR of COL1, ON, OP, BSP, OC, Gal3 and 18 S made at 1, 7, 14 and 28 days

FIGS. 6B, 6C, 6D, 6E, 6F: Graphical Representations of Gene Expression Variations of COL1 (B), ON (C), Gal3 (D), BSP (E) and OC (F) by Bone Type Cells osteoblastic treated or not with 100 μg / mL of κΕ or 150 μg / mL of (VGVAPG) ₃ (V3) at 14 and 28 days of culture. Witness: 100%. Figure 7: Variation of alkaline phosphatase activity (ALP) expressed by osteoblastic cells in the presence of kappa-elastin or (VGVAPG) ₃ (V3) for 28 days of culture.

Figure 8: Analysis of the effects of kappa-elastin on factors modulating the activation and differentiation of osteoclasts, osteoprotegerin and RANKL. FIGS. 8A and 8B: RT-PCR analyzes of the effect of kappa-elastin on the expression of osteoprotegerin and RANKL over time.

FIG. 8C: OPG / RANKL ratio obtained from the OPG and RANKL levels assayed by ELISA kit.

Figure 9: Scanning electron microscope observation of osteoblastic bone cells cultured on collagen layers in the presence or absence of kappa-elastin (10 (g / ml) at 1, 7 and 21 days of culture. : control kE: kappa elastin White arrow: nodule Col: collagen fiber network (^) CO: bone cells.

Figure 10: Detection and determination of the composition of nodules by MET / Microanalysis X within collagen layers seeded osteoblastic bone cells at 21 days of culture. A: control culture; B: X-ray microanalysis spectra of control culture; C: cell culture treated with kappa-elastin (100 μg / ml); D: X-ray microanalysis spectra of culture treated with kappa-elastin (intensity in number of strokes as a function of energy in keV).

EXAMPLES

Material and method

Obtaining kappa-elastin: Kappa-elastin is prepared from purified elastin purified from bovine or horse origin, extracted from the ligament from the neck of the animal and then digested with 0.1 M potassium hydroxide. taken up in ethanol at 80 °, according to the protocol described in Jacob et al., 1985 ("Isolation and characterization of insoluble and kappa-elastins"). This method makes it possible to obtain soluble elastin peptides whose molecular weights vary between 1000 and 100000 Da (verified by gel-filtration column chromatography G 3000 SW using an HPLC apparatus). About 60% of the peptides have a size between 1000 and 60000 Da.

Kappa-elastin contains 4 predominant amino acids: Glycine, Alanine, Proline, Valine and peptides of sequence X ₁ GX ₂ X ₂ PG, in which Xi represents any amino acid and X ₂ represents a non-polar amino acid and preferentially Glycine, Alanine, Proline, or Valine. The existence of elastin bypass is demonstrated by the presence of isodesmosine and desmosine (3.8 residues per 1000). The coacervation temperature is 50 ° C to pH2-5.

Kappa-elastin can be advantageously lyophylized to ensure better preservation. It is highly soluble in water.

No toxicity was observed on the cells used having been treated with kappa-elastin concentrations of up to 200 μg / mL, with a recommended concentration of use of between 25 and 100 μg / mL. Obtaining gingival fibroblasts: The human gingival fibroblasts used come from patients aged 40 to 60, free of general and periodontal diseases, non-smokers.

Human gingival fibroblasts are prepared from gingival biopsies obtained during pre-prosthetic surgeries and treated using the explantation technique.

The cells are cultured in Dulbecco's Modified Eagle Medium supplemented with 10% fetal calf serum (FCS) and 1% Penicillin / Streptomycin.

For some experiments, culture medium without serum was used. The cells were assayed between the 3 ^rd and 6 ^th passage. For each of the cell lines, the experiments were performed in triplicate.

Obtaining osteoblastic bone cells: The osteoblastic bone cells used are derived from human bone marrow of patients aged 74 to 77, free of general diseases.

The human osteoblastic bone cells are prepared from bone biopsies obtained during orthopedic surgery and treated according to the explantation technique. Initially, the biopsies undergo several successive rinses with PBS, trypsin then collagenase, before they are cultured in Dulbecco's Modified Eagle Medium medium supplemented with 10% fetal calf serum (FCS) and 1% Penicillin / Streptomycin.

For some experiments, culture medium without serum was used. The cells were assayed between the 2 ^nd and 4 ^th passage.

For each of the cell lines, the experiments were performed in triplicate. First experiment: Involvement of the EBP receptor in the effects of kappa-elastin on periodontal cells

1- Detection of the EBP receptor

In a first step, the inventors sought to show the presence of EBP receptors on the plasma membrane of gingival fibroblasts and human osteoblastic bone cells.

For this, they used the electrophoretic technique (Western Blot) and / or confocal microscopy. The results are shown in FIG. 1. The cell extracts were recovered from cells grown at confluence in Dulbecco's Modified Eagle Medium supplemented with 10% fetal calf serum and 1% Penicillin / Streptomycin.

It is observed that the EBP receptor is homogeneously distributed on the cell membrane of human gingival fibroblasts and its presence is also detected inside the cell (Figure 1A).

The expression of the receptor was also confirmed by Western blotting using a polyclonal primary antibody directed against VI 4, peptide of 14 amino acids (VVGSPSAQDEASPL) (SEQ ID No. 5) corresponding to the attachment sequence of elastin found in the EBP receiver. Only a protein band with a molecular weight of around 67 kDa is identified as the EBP receptor (Figure 1B). It is the same for bone cells of the osteoblastic type (FIG.

1 C).

2- Involvement of the EBP receptor in the effects of kappa-elastin To evaluate the involvement of the EBP receptor in the effects of kappa-elastin on periodontal cells, a comparative study of the effects of the sequence peptide (VGVAPG) (SEQ ID NO: 2), lactose and V14 (competitive receptor of EBP) was conducted on these cells cultured for 24 hours without fetal calf serum.

The effects of (VGVAPG) (200 μΒ / ml), lactose (50 μΜ), a blocking antibody V14 (10 μg / ml) and U0126 (10 nM, an ERK1 / 2 inhibitor) were compared with the effect of kappa-elastin. The control corresponds to cells not treated with kappa-elastin.

The data presented in FIGS. 2A, 2B and 2C are the average of experiments carried out in triplicate on three cell lines, namely gingival fibroblasts (FIG. 2A) and human bone cells of the osteoblastic type (FIGS. 2B and 2C).

In the case of human gingival fibroblasts (FIG. 2 A), the induction of proMMP-3 is obtained using peptides (VGVAPG), although at a level lower than that obtained in the presence of kappa-elastin. Lactose, incubated for 1 hour with cells before they are treated with kappa-elastin, substantially reduces kappa-elastin-induced MMP-3 production.

To evaluate whether the interaction of EBP with kappa-elastin is dependent on ER ½ activation (Extracellular Signal-Regulated Kinases 1 and 2), an ER ½ inhibitor, U0126, was used. It is found that this inhibitor decreases the expression of proMMP-3 by about 60% compared to the effect of kappa-elastin alone.

In the case of osteoblastic bone cells cultured 24 hours without serum (FIG. 2B and 2C), the induction of proMMP-2 caused by kappa elastin is reduced by about 30% in the presence of V14, used. as a competitive receptor for EBP at a concentration of 10 μg / mL (Fig.2B).

The effect of V14 on the gene expression of the Runx-2 transcription factor, characteristic of the osteoblast, is more marked with a return of expression of Runx-2 almost identical to that obtained with the witness. Kappa-elastin and (VGVAPG) increase their expression relative to this same control (FIG. 2C).

The regulation of the effects of kappa-elastin thus mainly passes through the EBP receptor present on human gingival fibroblasts and human osteoblastic bone cells.

Second Experiments: Effects of kappa-elastin on the repair and regeneration of periodontal tissues

1- Effects of kappa-elastin on the production of proteins of the gingival conjunctive matrix The biological effects of kappa-elastin (100 μg / ml) were tested on human gingival fibroblasts grown on plastic, at confluence, over a period of 7 days in the presence of 2% Fetal Calf Serum (Figure 3).

The expression of gingival matrix proteins, including tropoelastin, type I collagen and fibrin-1, was evaluated by RT-PCR at 1, 4 and 7 days of culture.

It is found that kappa-elastin, at 100 μg / mL, induces an increase in tropoelastine expression compared with control without kappa-elastin at 4 and 7 days (kE / control ratio = 5.5 at day 4 and 1.25 to J7) (Figure 3B).

Note also that the gene expression of tropoelastin decreases very rapidly over time in the control culture and is very weakly detected at 4 and 7 days. In the presence of kappa-elastin, its expression is visible at 4 days and also decreases very strongly at 7 days while remaining higher than that obtained with the control culture without kappa-elastin.

Kappa-elastin at 100 μg / mL induces an increase in type I collagen expression by human gingival fibroblasts compared to the control culture, regardless of the day of culture but with the highest kE / control ratio. at ¹ day of culture (ratio kE / Control = 6 to Jl to J4 against 1.5 and 1.25 to J7) (Figure 3C).

Kappa-elastin at 100 μg / mL induces an increase in the expression of fibrin-1 by human gingival fibroblasts compared to the control culture on days 1 and 7 of culture (kE / T e ratio = 1.5 to 1J and 4 to J7) (Figure 3D).

Over time, gene expression of fibrin-1 by human gingival fibroblasts decreased, with or without kappa-elastin. However, it is significantly less important in the presence of kappa-elastin than in its absence.

These results suggest that kappa-elastin is able to promote not only healing and repair, but also the regeneration of gingival tissues, by preventing or even compensating for tissue loss occurring during periodontal diseases.

2- Effects of kappa-elastin on the differentiation of osteoblastic bone cells

2.1. Effect of kappa-elastin at increasing concentration (0, 25, 50, 100 μ / mL)

The biological effects of kappa-elastin used at various concentrations (25, 50 and 100 μg / mL) were tested on bone marrow mesenchymal cells and grown on plastic, at confluence, for 24 hours, without calf serum fetal (Figure 4).

These cells were cultured on plastic for 24 hours in the presence of 0, 25, 50 or 100 μg / ml of kappa-elastin, without fetal calf serum. (18S: 18S rRNA).

These effects were evaluated on the expression of the Runx-2 transcription factor and three early-expressed bone proteins during the osteoblastic bone cell differentiation state, type I collagen (COL1), osteonectin (ON) and osteopontin (OP). RT-PCR analysis was performed from three different cell lines. For each of the cell lines, the experimentation was done in triplicate (Figure 4).

It is observed that the increasing use of kappa-elastin (25, 50, 100 μg / mL) increases the expression of Runx-2 in a non-significant manner, compared with the control grown without peptides, while the expression of collagen-type I, just as the expressions of osteonectin and osteopontin significantly increase with an elastin peptide concentration of 25 μg / ml.

However, regardless of the concentration of elastin peptides used (25, 50 and 100 μg / mL), the gene expression for each of the 3 bone proteins does not vary significantly between them.

During 24 hours of culture, the osteoblastic cells grown on plastic at confluence were treated or not at increasing concentrations of (VGVAPG) ₃ (0 - 50 - 100 - 150 and 200 μg / mL) in the absence of FBS. RT-PCR analysis indicates that peptide (VGVAPG) ₃ induced dose-dependent, increased gene expression (ON, OP, OPG and MMP-1) by osteoblast cells (not shown). However, at a concentration of 200 μg / mL, the peptide (VGVAPG) ₃ causes a significant decrease in the gene expression of ΓΟΝ and ΓΟΡ, or even an inhibition of that of OPG and MMP-1 compared to the control. without peptide. This decrease in gene expression of ΓΟΝ, ΓΟΡ, OPG and MMP-1 could be due to a toxicity of the peptide administered at 200 μg / ml, or to an acceleration of bone cell differentiation. In the case where a toxicity is confirmed, a maximum concentration of 150 μg / mL of the peptide (VGVAPG) ₃ would be recommended.

2.2. Effect of kappa-elastin on the differentiation of osteoblastic bone cells.

Bone marrow-derived osteoblastic bone cells were cultured on a Type I collagen layer for a period of 28 days in the presence of 2% fetal calf serum, with or without 100 μg / ml kappa-elastin or with 150 μg / mL of (VGVAPG) ₃ . (18S: 18S rRNA: internal control, ND: Not detected, T: control, κΕ: kappa-elastin, V3: (VGVAPG) ₃ ). The cell extracts were analyzed at 1, 7, 14, 21 and 28 days of culture. The culture media are renewed every 7 days in half. For this, to 1 ml of cell culture supernatant, 500 are removed and replaced with 500 of fresh medium with or without kappa-elastin.

The gene expression profile of osteoblast cells cultured on the type I collagen layer was analyzed by RT-PCR to evaluate their stage of differentiation according to the presence or absence of kappa-elastin ( Figure 5). To ¹ day in the presence of kappa-elastin, a small decrease in the gene expression of different transcription factors (Runx2, Dlx5, Msx2 and TAZ) by osteoblasts is observed compared to that obtained with the control without peptides. It should be noted that Msx2 is little expressed by the cells, whatever the culture conditions, with or without peptides (FIG. 5A).

At 14 days of culture, the expression of Runx-2, Msx2 and Dlx5 transcription factors increases by about 40% in the presence of kappa-elastin compared to the control without peptides (FIGS. 5B, 5C and 5D). The expression of TAZ increases by 25% (FIGS. 5E).

While the effect of the peptides seems to perpetuate the 28 ^th day of culture for gene expression of Runx2, it seems to be reversing for the TAZ. At this stage, Dlx5 and Msx2 are no longer detected, whatever the culture conditions. These results were expected and characterize a state of advanced osteoblastic differentiation.

It is also important to note that PPARy (Peroxisome proliferator-activated receptor), a transcription factor specific for the adipocyte, is never expressed, with or without treatment.

Sox9 (sex determination region Y - box9), a marker of the chondrocyte, is it, weakly expressed at the beginning of kinetics before not being detected during the late days. Type II collagen, a chondrocyte marker more specific than Sox9 and not expressed by osteoblasts, is not detected in culture, regardless of culture conditions.

Comparable results are obtained with the consensus sequence peptide (X ₁ GX ₂ X ₂ PG), more particularly (VGVAPG) 3. The latter has effects on osteogenic transcription factors greater than those obtained with the control without peptide. The peptide (VGVAPG) ₃ appears more effective than kappa elastin.

While these transcription factors are involved in osteogenesis by promoting osteoblastic differentiation, the inventors analyzed by RT-PCR the expression profile of osteogenic protein markers such as osteocalcin and bone sialoprotein.

For this purpose, osteoblastic cells were cultured on a type I collagen layer without or with 100 μg / ml of kappa-elastin, or 150 μg / ml of (VGVAPG) ₃ .

18S: 18S rRNA; N.D.: Not detected; T: witness; κΕ: kappa-elastin; V3:

VGVAPG ₃ . COL1: Collagen type I; ON: osteonectin; OP: osteopontin; BSP: bone sialoprotein; Gal3: Galectin-3; OC: osteocalcin (Figure 6).

At ¹ day of culture, treating the cells with the kappa-elastin has little effect on protein genes expressed in early stages of osteoblast differentiation such as Type I collagen, osteonectin, and osteopontin compared to the control. In the case of Galectin-3, a decrease is even noted. It appears that kappa-elastin induces an increase in the gene expression of "late" proteins of osteoblastic differentiation such as bone sialoprotein and osteocalcin compared to the control (FIG. 6A).

The gene expression of these proteins is, moreover, detected in control cultures without peptide. At 7 ^th and 14 ^th day of culture, the expression of these osteogenic markers was significantly increased with kappa-elastin compared to the control without peptide (FIGS. 6B, 6C, 6D, 6E, 6F). It is however interesting to note that if osteopontin exhibits a gene expression profile similar to the other osteoblast markers at 24 hr of culture, it is no longer detected on the following days (FIG. 6A).

Similarly, the expression of bone sialoprotein (Figure 6E) and osteocalcin (Figure 6E) decreases with time growing significantly between ¹ and 28 ^th day, whatever the culture conditions. However, the expression of osteocalcin remains greater in the presence of kappa-elastin compared to the control.

Comparable results are obtained with the consensus sequence peptide (VGVAPG) ₃ . The latter has greater osteogenic effects than those obtained with the control without peptide. In the case of gene expression of certain bone proteins such as type I collagen and osteonectin, the peptide appears more effective than kappa-elastin. The assay for bone alkaline phosphatase activity (PAL) was performed from cell extracts of osteoblastic cells cultured on collagen layer and treated or not treated with 100 μg / ml of kappa-elastin, or 150 μg / ml of (VGVAPG ) ₃ (V3) for 28 days (Figure 7).

The assay of the PAL activity is carried out from cell extracts obtained from osteoblastic cells cultured on a type I collagen layer with or without 100 μg / ml of kappa-elastin or 150 μg / ml of V3.

At 24 hours of culture, a nonsignificant decrease in PAL activity was observed in the presence of kappa-elastin compared to control without peptide (34.72 IU / L against 36.67 IU / L, respectively).

At 7 days, then until 28 ^th day, the PAL activity is higher in cell cultures treated with kappa-elastin, and the peptide (VGVAPG) _3, compared to the peptide without control culture (14 days T = 19.45 IU / L, kE = 22.63 IU / L, V3 = 24.66 IU / L). 2.3. Effects of kappa-elastin on the regulation of osteoblast differentiation

RANKL (receptor activator for nuclear factor kappa - B ligand) and osteoprotegerin (OPG) are the keys, respectively of the agonist - antagonist system that regulates the fate of osteoclasts: differentiation, fusion, activation, survival and apoptosis of cells.

The present study was carried out by analyzing the RT-PCR expression profile of the OPG and RANKL genes by osteoblastic cells cultured on a type I collagen layer with or without 100 μg / ml of kappa-elastin during 1, 7, 14 and 28 days.

OPG: osteoprotegerin; RANKL: RANK ligand; 18S: 18S rRNA; N.D .:

Not detected; T: witness; κΕ: kappa-elastin (Figure 8).

At ¹ day of culture, gene expression of RANKL and osteoprotegerin is significantly less in the osteoblast cultures treated with kappa-elastin, compared to the peptide without control (Figure 8A).

From the ^7th day, gene expression of RANKL is not detected while the OPG continues and appears to increase over time (Figure 8A).

On the ^7th day, the OPG expression intensity in the presence of kappa-elastin is higher than that of the control without peptide.

At 14 and 28 days, the gene expression of OPG with kappa-elastin was significantly greater than that obtained with control without peptide (14J: kE = ± 30%, 28J: kE = ± 60%) (Figure 8B ).

The ratio OPG / RANKL is positive regardless of the day of culture of osteoblastic-type bone cells, treated or not with kappa-elastin (FIG. 8C). However, this ratio is higher in the presence of kappa-elastin, compared to that obtained with the control without peptides ¹ to the 21 ^th days of study.

At 28 ^th day, the result is reversed.

Thus, kappa elastin at 100 μ§ / ι Ι, directs the osteoblastic phenotype towards a bone formation while it allows the increase of the gene expression and protein of osteoprotegerin and a decrease of that of RANK-L, favorable to differentiation and activation of osteoclasts, in relation to bone resorption.

Kappa elastin at 100 μg / mL increases the gene expression of transcription factors favorable to the differentiation of osteoblasts (Runx-2, Dlx5, Msx1, TAZ, etc.) as well as that of proteins constituting the bone matrix (osteocalcin, bone sialoprotein, type I collagen, osteonectin, etc.) by these cells, compared to untreated cell cultures, whether on short cultures on plastic (24 hours of culture) or on long cultures on layer collagen type I (28 days of culture). It has been shown that kappa elastin promotes the differentiation of undifferentiated mesenchymal cells from bone marrow into human osteoblasts. Kappa elastin also promotes the differentiation of human osteoblasts.

The consensus sequence peptides (VGVAPG) ₃ have osteogenic properties substantially identical to those of kappa elastin.

Third experiment: Effects of kappa-elastin on mineralization of the bone matrix

Osteoblastic bone marrow-type bone cells were cultured on a Type I collagen layer for a period of 28 days in the presence of 2% fetal calf serum and more or less in the absence of kappa elastin (100 μg / ml). mL). Studies on bone mineralization were performed at 1, 7, 21 and 28 days of culture. Culture media were renewed every 7 days by half. For this, to 1 ml of cell culture supernatant, 500 were removed and replaced with 500 of fresh medium with or without kappa-elastin.

1- Demonstration of bone mineralization from observation by scanning electron microscopy (Figure 9)

At ¹ day of culture, the morphology of cells cultured in the presence of kappa-elastin (κΕ) is substantially identical to that of control cells cultured without peptide. The cells are essentially cuboid or slightly elongate in shape.

They are deposited and homogeneously distributed on a network of dense collagen fibers, with which they have very numerous cell-matrix interactions.

From the ^7th day of culture, the collagen network seems less apparent and identifiable in the cells, whatever the culture conditions (control, kE) that this phenomenon is more pronounced in the presence of kappa-elastin compared to the control without peptide.

At 21 days of culture, the network of collagen fibers is almost no longer visible under the control cells, whereas in the presence of kappa-elastin, the extracellular matrix appears denser and contains small rounded structures that merge with each other. to others, suggesting the coalescence of mineralization nodules. Similar results are obtained in the presence of (VGVAPG) ₃ .

2- Demonstration of bone mineralization by X microanalysis (Figure

10)

Collagen layers seeded with osteoblastic bone cells are observed in transmission electron microscopy (TEM), combined with X microanalysis to determine if kappa-elastin promotes bone mineralization. The composition of calcification nodules is determined by microanalysis

X.

Three points are analyzed in the two types of culture, treated or not with kappa-elastin: two, inside nodules of different size and one, within the matrix.

At 21 days of culture, TEM analysis of the control cultures shows some concretions present in the matrix which turn out to be calcium nodules on microanalysis X (FIGS. 10A and 10B). The extracellular matrix, apart from these concretions, does not reveal the presence of calcium.

In the presence of kappa-elastin, cell cultures develop nodules in greater amounts compared to the control. These reveal a calcium peak at X microanalysis (Figure 10D). Micro-X analysis of the matrix also reveals the presence of calcium to a lesser extent compared to nodules (Figure 10D).

3. Infrared spectroscopy analysis of the effects of kappa-elastin on bone mineralization after 28 days of collagen layer osteoblast culture

The inorganic matrix of bone is composed mainly of hydroxyapatite. Infrared spectroscopy was used as a semi-quantitative approach to the detection of this compound in osteoblastic cell cultures seeded on a type I collagen layer.

An integration method was used to calculate the differences between the surface areas present under the spectra and obtained from cells treated or not treated with kappa-elastin.

The area defined under the spectrum corresponding to kappa-elastin-treated cell cultures is approximately 25% larger than that obtained with control cells, suggesting an increase in the level of hydroxyapatite in the presence of kappa-elastin compared to non-kappa cells. treated and thus an increase in the mineralization process with kappa-elastin. In the case of the peptide (VGVAPG) 3, the defined area obtained is 30% greater than that of the control. Kappa-elastin promotes bone mineralization by osteoblast cells cultured on a type I collagen layer for 28 days of culture compared to untreated cell cultures. This has been demonstrated by Scanning Electron Microscopy, Scanning Electron Microscopy Transmission and X-ray microanalysis and infrared spectroscopy which have revealed the presence of more abundant calcification nodules in the presence of kappa-elastin compared to cultures without have been treated.

Kappa-elastin therefore makes it possible to promote gingival and bone matrix formation and, more particularly, in the case of bone tissue, promotes bone repair and even regeneration.

Sequence peptides (VGVAPG) 3 promote bone matrix formation and mineralization.

BIBLIOGRAPHY

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DUCA L., FLOQUET N., ALIX A.J.P., HAYE B., DEBELLE L. Crit. Rev. Oncol. Hematol. 2004, 49: 235-244.

TSURUGA E, IRIE K, SAKAKURA Y, YAJIMA T. J Dent Res, 2002, 81: 198-202.

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Claims

1- A pharmaceutical or cosmetic composition comprising at least one kappa-elastin peptide for use in the repair and / or regeneration of at least one periodontal tissue in the human or non-human mammal.

2. The composition according to claim 1, wherein at least one kappa-elastin peptide is capable of acting on at least one soft tissue of the periodontium among the gingival tissue and the dento-alveolar ligaments.

3. The composition according to claim 1, wherein at least one kappa-elastin peptide is capable of acting on at least one hard tissue of the periodontium among the alveolar bone and the radicular cementum.

4. Composition according to any one of the preceding claims for the inhibition of bone resorption of at least one hard tissue of the periodontium among the alveolar bone and the radicular cementum.

5. Composition according to any one of the preceding claims, in which at least one kappa-elastin peptide used has the sequence (XiGX ₂ X ₂ PG) n, in which Xi represents any amino acid and X ₂ represents a non-polar amino acid and n is an integer between 1 and 10, and preferably between 1 and 7.

6. Composition according to any one of the preceding claims, in which at least one kappa-elastin peptide used has the sequence (VGVAPG) 3.

7. Composition according to any one of the preceding claims, in which at least one kappa-elastin peptide used is obtained by chemical synthesis. 8. Composition according to any one of the preceding claims, in which all the kappa-elastin peptides obtained by partial hydrolysis of elastin are used. 9. Composition according to any one of the preceding claims, in which the peptide (s) of kappa-elastin used are at a concentration of at least 25 μg / ml, preferably at least 75 μg / ml and more preferably at least equal to at 10 (g / ml.