EP1959970A1 - Use of a polysaccharide which is excreted by the vibrio dlabolicus species for the regeneration and protection of the periodontium - Google Patents

Use of a polysaccharide which is excreted by the vibrio dlabolicus species for the regeneration and protection of the periodontium

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Publication number
EP1959970A1
EP1959970A1 EP06841874A EP06841874A EP1959970A1 EP 1959970 A1 EP1959970 A1 EP 1959970A1 EP 06841874 A EP06841874 A EP 06841874A EP 06841874 A EP06841874 A EP 06841874A EP 1959970 A1 EP1959970 A1 EP 1959970A1
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EP
European Patent Office
Prior art keywords
use according
polysaccharide
collagen
matrix
cells
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP06841874A
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German (de)
French (fr)
Inventor
Karim Senni
Corinne Sinquin
Sylvia Colliec-Jouault
Gaston-Jacques Godeau
Jean Guezennec
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Institut Francais de Recherche pour lExploitation de la Mer (IFREMER)
Original Assignee
Institut Francais de Recherche pour lExploitation de la Mer (IFREMER)
Universite Paris 5 Rene Descartes
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Application filed by Institut Francais de Recherche pour lExploitation de la Mer (IFREMER), Universite Paris 5 Rene Descartes filed Critical Institut Francais de Recherche pour lExploitation de la Mer (IFREMER)
Publication of EP1959970A1 publication Critical patent/EP1959970A1/en
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/20—Polysaccharides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/04—Drugs for skeletal disorders for non-specific disorders of the connective tissue
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]

Definitions

  • the present invention relates to the regeneration of non-mineralized connective tissue of the periodontium.
  • EPS Exopolysaccharide producing bacteria
  • HE800 is an EPS produced by the Vibrio diabolicus strain. Its weight-average molar mass is approximately 800,000 g / mol in the native state. It is characterized by an original linear repeating osidic sequence consisting of 4 osidic residues:
  • HE800 has been described in the international application on behalf of IFREMER published under the number WO9838327 as well as in the following articles: Raguénippo et al, Int J Syst Bact, 1997, 47, 989-995 and Rougeaux et al, Carbohyd. Res, 1999, 322, 40-45. Many applications for this exopolysaccharide have been described. By way of example of application, mention may be made of international application WO0202051, which describes the beneficial properties of HE800 in bone healing. To date, no application of HE800 is known with regard to the regeneration of the non-mineralized connective tissue of the periodontium.
  • the periodontium is a set of tissues whose purpose is to support and maintain the tooth in its socket. It consists of two soft tissues, the gum and the periodontal ligament (desmodont), and two calcified tissues, the cementum and the alveolar bone. This organ has the particularity of being located in an environment, where it is subjected to
  • the oral cavity is indeed, a humid environment sheltering a commensal bacterial flora.
  • the integrity of the periodontium depends essentially on the balance between the oral tissues and this bacterial flora. Any destabilization of this relationship promotes the proliferation of pathogenic flora which can lead to the destruction of periodontal tissues. To meet these constraints, the periodontium is constantly being remodeled.
  • the gum is the covering fabric of the periodontium, it thus constitutes a protection, against the bacterial attacks, for the elements of the periodontium which it covers (cement, desmodont, and alveolar bone). Histologically, this tissue consists of a connective tissue covered with an epithelium of ectodermal origin.
  • the gingival connective tissue consists of an extracellular gingival matrix very similar in terms of macromolecular content to that of the dermis.
  • the gum unlike the dermis, is in direct relation with different mineralized tissues and presents several types of collagen fibers binding the gum to the alveolar bone, to the cementum or to other fibers linked to the neighboring tooth.
  • Fibrillar collagens represent 50 to 60% of the proteins found in the gingival connective tissue. Phenotypic analyzes show that these collagens are 91% type I, 8% type III and less than 1% type V.
  • the extracellular matrix constitutes the reinforcement of the connective tissue. It gives the fabric its shape, its mechanical resistance, its flexibility and ensures important physiological functions. It is also necessary for maintaining the differentiated state of the cells which synthesize and reshape it.
  • IH membrane receptors such as integrins
  • resident cells such as fibroblasts which allows it, depending on its condition, to control migration, proliferation or metabolic activities.
  • these cells can reshape the matrix that surrounds them. They can then express proteases to degrade it or resynthesize new matrix components.
  • the extracellular matrix is therefore in constant equilibrium (degradation-resynthesis).
  • This balance can be irreparably upset during certain pathologies. Indeed, in inflammatory syndromes, the destructive power of resident cells is exacerbated under the influence of inflammation cells, the latter, after activation, also degrade the matrix. Other pathologies can contribute to the deregulation of the matrix dynamics such as fibrosis during which the expression of one or more matrix components is exacerbated.
  • the proper restructuring of the extracellular matrix is the guarantor of tissue regeneration.
  • the organization of the collagen framework is an essential element of this tissue restructuring. Indeed, these collagens constitute the majority protein class of the extracellular matrices and in particular those of the periodontium.
  • the gum unlike the dermis, is subject to a large and constant remodeling. This remodeling is the consequence of the coexistence between the bacterial plaque depositing on the tooth and the gingival tissue, and the mechanical stress to which the gum is subjected during chewing. These factors more or less directly affect the gingival fibroblasts which represent the majority of the cells present in the gingival connective tissue.
  • This permanent activation results in a very large phenotypic heterogeneity of the gingival fibroblasts.
  • myofibroblasts proliferate in the event of an inflammatory process.
  • the fibroblast is the key cell of tissue homeostasis.
  • fibroblastic heterogeneity has an advantage in physiological conditions, it can prove to be extremely deleterious in the context of periodontal pathologies settling in chronicity. Indeed, any lasting alteration in cell balance can lead to the unwanted activation of certain cell phenotypes such as, for example, the proliferation of myofibroblasts at the expense of fibroblasts.
  • High molecular weight hyaluronic acid is commonly used to treat many oral conditions.
  • EP0444492 describes the use of hyaluronic acid to treat inflammatory diseases of the oral cavity, such as gingivitis.
  • WO2005000321 describes the use of hyaluronic acid to treat canker sores in the oral cavity.
  • hyaluronic acid is used in different formulations, there may be mentioned, for example, gengigel®, which is in the form of a spray or mouthwash.
  • Hyaluronic acid which is a product of animal origin, is produced from animal extract or by genetic engineering.
  • the object of the present invention is to identify a compound capable of preserving periodontal tissue homeostasis and / or of promoting the restructuring, that is to say the restoration, of an altered collagen framework of non-mineralized connective tissues of the periodontium, and to favor the proliferation of the gingival fibroblasts in order to restore 1 gingival homeostasis.
  • Such a compound will thus have a protective and regenerative activity on the non-mineralized connective tissues of the periodontium and will facilitate the restoration of tissue homeostasis.
  • a polysaccharide of average molar mass by weight of between 500,000 and 2,000,000 g / mol characterized by a linear repetitive osidic sequence comprising the following 4 osidic residues:
  • [(-3) -D GlcNac ⁇ (1-4) D GIcA ⁇ (1-4) D GIcA ⁇ (1-4) D GalNac ⁇ (1-)] has the following properties: it induces a selection of fibroblast strains , it stimulates the mobilization and proliferation of fibroblasts in the extracellular matrix, it accelerates collagen fibrillation and thus promotes the reconstitution of the connective matrix. Therefore, this polysaccharide accelerates regeneration by accelerating the restructuring of connective tissues. It leads to complete regeneration so that the appearance of pathological situations of fibrotic or inflammatory type is avoided.
  • This polysaccharide allows the restructuring of the collagen framework of the non-mineralized connective tissues of the
  • the polysaccharide is particularly suitable for the following applications: the regeneration of the connective tissues of the periodontium as well as the treatment of oral pathologies, in particular those linked to an inflammatory or traumatic state.
  • the polysaccharide allows the production of collagen matrix with improved properties.
  • the collagen framework of the collagen matrices comprising the polysaccharide has better resistance to physical factors such as temperature and mechanical stresses.
  • it promotes the culture of gingival fibroblasts and allows the production of gum substitute.
  • the subject of the present invention is the use of a polysaccharide or a salt of this polysaccharide with a weight average molar mass of between 500,000 and 2,000,000 g / mol, preferably between 700,000 and 900,000 g / mol , characterized by a linear repetitive osidic sequence comprising the following 4 oidic residues:
  • the polysaccharide can be in the form of a salt.
  • the polysaccharide is a polysaccharide excreted by the Vibrio diabolicus species or a derivative obtained from it. Methods of preparation have been described in the following documents: WO 98/38327, Raguénippo et al, Int J Syst Bact, 1997, 47, 989-995 and Rougeaux et al, Carbohyd. Res, 1999, 322, 40-45.
  • derivatives of average molar mass by weight of between 500,000 and 2,000,000 g / mol can be obtained by partial depolymerization, by bridging and / or by chemical modifications, in particular, by sulfation and / or by acetylation.
  • WO0046252 describes a method for bridging hyaluronic acid, typically this method could be adapted to generate bridged derivatives of the polysaccharide excreted by the species Vibrio diabolicus.
  • One embodiment of the invention relates to the manufacture of a composition, of a medicament, of a medical device for treating an oral pathology of the non-mineralized connective tissue of the periodontium.
  • oral pathology is linked to an inflammatory state or to a traumatic state.
  • the oral pathology is chosen from the group consisting of periodontitis, gingivitis, gingival fibrosis, gingival recession, canker sore, recurrent oral aphtosis, foot-and-mouth disease, and bullous pathologies.
  • composition or the drug produced is intended for topical administration at the periodontium level.
  • topical composition is meant a composition which acts at a determined point and which is directly applicable on the oral mucosa.
  • composition and the medicament may be in the form of a topical composition for oral use, in particular in the form of a gel, a solution of an emulsion or a spray.
  • a topical composition according to the invention is produced in a manner known per se.
  • a topical composition according to the invention contains the polysaccharide at a concentration between 0.005 and 10% by weight on the total weight of the composition, more preferably at a concentration between 0.01 and 5% by weight.
  • a gel according to the invention may comprise sorbitol, maltitol, xylitol and / or sodium carboxymethylcellulose.
  • composition or same drug can be used to impregnate an oral dressing.
  • a toothpaste, a mouthwash, a spray, a denture glue and an oral dressing comprising the polysaccharide.
  • a toothpaste, a mouthwash or a spray according to the invention contains the polysaccharide at a concentration of between 0.005 and 1% by weight on the total weight of the composition, more preferably at a concentration of between 0 , 01 and 0.1% by weight.
  • a toothpaste according to the invention may comprise, for example, one or more of the following compounds: sorbitol, maltitol, xylitol and / or sodium carboxymethylcellulose.
  • a mouthwash according to the invention may contain one or more of the following excipients: polysorbate 60, saccharin
  • a mouthwash according to the invention may also contain an additional active agent such as, for example, hexetidine.
  • the polysaccharide can be used as the sole active agent or accompanied by other active agents such as, for example, an antibacterial, an antibiotic , vitamins or trace elements.
  • the invention relates to a collagen matrix comprising the polysaccharide according to the invention.
  • the collagen of the matrix is a collagen chosen from the group consisting of type I, III, V collagens or a mixture of these.
  • the collagen is a type I collagen.
  • the techniques commonly used for the manufacture of collagen matrices from acid-soluble fibrillar collagens In the presence of the polysaccharide according to the invention, the acid-soluble fibrillar collagens fibrillate naturally after neutralization of the pH.
  • the collagen matrix according to the invention can be obtained by bridging the polysaccharide according to the invention with the collagen. To carry out the bridging, a person skilled in the art will use the techniques commonly used for the bridging of polysaccharides with collagen.
  • EP1374857 is an illustration of a usable bypass technique.
  • the matrix may also comprise a growth factor which promotes colonization of the matrix by the gingival fibroblasts and the reconstitution of the connective tissue.
  • the growth factor may be chosen from the group consisting of TGF-beta, PDGF, FGFs, BMPs (bone morphogenetic proteins), VEGF, CTGF (connective tissue growth factor).
  • the matrix can serve as an absorbable medical device or an implant.
  • Such a matrix will allow mechanical and functional replacement of damaged structures with a minimum of undesirable reactions.
  • This matrix once implanted in the tissue to be regenerated will serve as a guiding structure and will mark out the regenerative potential of the tissue.
  • the matrix will promote the penetration of fibroblasts in an orderly fashion after the graft of the matrix, while encouraging these same fibroblasts to produce their own extracellular matrix.
  • the matrix may also comprise gingival fibroblasts so as to constitute a gum substitute.
  • This substitute can be implanted in vivo.
  • it is the gingival fibroblasts of the patient on which the graft is to be performed which serve to colonize the matrix.
  • the invention relates to a cell culture support, characterized in that the surface of the support on which the cells are cultivated comprises the polysaccharide according to the invention.
  • the polysaccharide is in the form of a film, a membrane or a three-dimensional honeycomb structure.
  • the invention relates to a method for culturing gingival fibroblasts, characterized in that said fibroblasts are cultured on a matrix according to the invention or on a support as described above.
  • the HE800 strain is cultivated on 2216E medium [OPPENHEIMER,
  • the bacteria are separated from the must by centrifugation at 20,000 g for 2 hours, then the polysaccharide is precipitated from the supernatant using pure ethanol, then several ethanol / water washes are carried out in increasing proportions of ethanol, according to the method described by TALMONT et al. [Food Hydrocolloids 5, 171-172 (1991)] or VINCENT et al. [Appl. About. Microbiol. , 60, 4134-4141 (1994)].
  • the polysaccharide obtained is dried at 30 ° C. and stored at room temperature. 2.5 g of purified polysaccharide per liter of culture were thus obtained.
  • the cultures are carried out in a medium, known as complete, composed of Dubelco MEM Glutamax I containing 100 U / ml of penicillin 100 ⁇ g / ml of streptomycin and 2 ⁇ g / ml of fungizone (Gibco BRL Cergy Pontoise France) supplemented or not (medium deficiency) in fetal calf serum (SVF).
  • a medium known as complete, composed of Dubelco MEM Glutamax I containing 100 U / ml of penicillin 100 ⁇ g / ml of streptomycin and 2 ⁇ g / ml of fungizone (Gibco BRL Cergy Pontoise France) supplemented or not (medium deficiency) in fetal calf serum (SVF).
  • a medium known as complete, composed of Dubelco MEM Glutamax I containing 100 U / ml of penicillin 100 ⁇ g / ml of strepto
  • the dermal biopsies used are cultured within 3 hours of their collection by the practitioner.
  • the samples used are obtained after circumcision, from the foreskin of clinically healthy children.
  • Gingival biopsies are taken from young patients (under 30) free of pathologies. These biopsies are located at the level of the attached gum of premolars extracted for orthodontic reasons. These gums are also declared clinically healthy by the practitioner.
  • These biopsies are tissue fragments detached during the extraction and which did not require any modification of the operating procedure.
  • the dermal and gingival samples are rinsed twice in a DMEM medium containing a higher than normal concentration of antibiotic (penicillin 6x, streptomycin 4x, fungizone2x) then they are cut into very small explants ( ⁇ 2mm 2 ). These explants are placed with a sterile Pasteur pipette or with the tip of the scalpel, in a 25 cm 2 culture flask, parenchymal side on the plastic. The box is raised and left for 15 minutes in this position so that the explants, dry, adhere to the plastic.
  • antibiotic penicillin 6x, streptomycin 4x, fungizone2x
  • the explants that have adhered are covered with a few drops of DMEM supplemented with 20% in fetal calf serum (SVF).
  • the culture dish is then placed overnight in an incubator at 37 ° C., under an atmosphere composed of 5% CO 2 and 95% air.
  • the next day the supernatant is replaced with fresh medium comprising 20% of FCS, it is subsequently renewed every week. After three weeks the fibroblasts have colonized the entire bottom of the dish (the
  • kLkirLJA u i "l ⁇ ij keratinocytes present in the explant do not adhere under these culture conditions
  • transplanting is carried out.
  • the explants are removed using forceps, the cells are rinsed twice with PBS, then trypsinized (Trypsin EDTA Gibco). Trypsinization is then stopped by the addition of DMEM comprising 10% of FCS.
  • the cells are counted on the counter (Coulter) and then reseeded in several culture dishes. They are, at this time, considered in the first pass and are maintained in a complete environment containing 10% of SVF. When the cells are again confluent, another passage is carried out according to the same procedure, until the start of the experiments.
  • Surfacting is carried out by depositing 200 ⁇ l of a 2 mg / ml solution of HE800 at the bottom of the culture wells (24-well dish, 2 cm 2 ).
  • the culture dish is placed in a culture hood on a heating plate adjusted to 37 ° C for at least 5 hours. After evaporation, a film of HE800 is formed at the bottom of the box.
  • the gingival fibroblasts are seeded at the rate of 10,000 cells per well and cultured for 7 days. A cell count is carried out every day, certain wells are fixed for the morphological study and the immuno-detection of the ⁇ -actin of smooth muscles.
  • the collagen used is an acid-soluble type I collagen (2 mg / ml) obtained from rat tail (Institut Jacques
  • the culture dish (24-well dish, or labtek, 2 cm 2 per well) is placed in a culture hood on a heating plate adjusted to 37 ° C., for at least 5 hours. After evaporation, a collagen film with or without HE800 is formed at the bottom of the box. Fibroblasts are seeded on these films in order to ensure the biocompatibility of the new culture surface.
  • the collagen films and the composite films are fixed with absolute ethanol -20 ° C. and then rehydrated to be colored with sirius red ((Junquera coloring, specific for collagens).
  • sirius red ((Junquera coloring, specific for collagens).
  • the laths are made with the same collagen I as that used to form the collagen films.
  • the gel containing the cells and which is being polymerized is poured into a petri dish 5 cm in diameter.
  • HE800 is added to the collagen before the addition of the cells at a rate of 150 ⁇ g, 300 ⁇ g or 600 ⁇ g per lattice (respectively 5%, 10%, and 20% of the total amount of collagen) 1.4.1) Preparation of the stock solution
  • the lattices are recovered fixed in paraformaldehyde and then prepared for inclusion in paraffin. 7 ⁇ m thick sections are then made in a microtome. The specific colorings of these sections allow us to observe
  • Staining with hemalun-eosin makes it possible to distinguish the cells of the matrix which surrounds them.
  • hemalun colors the nuclei of cells in black blue, while eosin colors more or less intense red cytoplasms and extracellular structures (eosinophils).
  • the contrast thus created makes it possible to distinguish each cell under a microscope equipped with a CDD camera connected to a semi-automatic image analyzer.
  • the cells found in the fields defined by the microscopic magnification are then counted in the lattices at 11 and 40 days. A dozen fields per cut were analyzed.
  • each lattice is considered to be cylindrical, the periphery of the lattice being defined as a crown of 10 ⁇ m in thickness (equivalent to the diameter of two cellular foundations) representing 2% of the total volume of the lattice.
  • FEPfLLE PE EKLE 26 1.5. Indirect immuno-detection of oc-actin in smooth muscles.
  • the fixed cells are re-permeabilized in 70% ethanol (20 min), then rehydrated in PBS (10 min). Endogenous peroxidases are blocked with a methanol solution (30%), H 2 O 2 (0.3%). This operation is followed by rinsing with PBS (2 min) and then blocking of the non-specific antigenic sites with a PBS / 1% skimmed milk solution (1 h). The cultures are then incubated with a primary antibody (mouse IgG) directed against human ⁇ -actin (1/30; 50 min) and then rinsed with PBS (3 x 10 min).
  • a primary antibody mouse IgG
  • the cells are then incubated in the dark for 60 min with a biotinylated anti-mouse IgG antibody (1/200), rinsed with PBS (3 x 10 min) and then incubated with streptavidin coupled to peroxidase (1/200 ).
  • the products used come from the firm DAKO.
  • the control experiments concerning the immunodetection of the ⁇ -actin of smooth muscles were carried out by omitting the primary antibody and / or by using a secondary antibody of another animal species than that which made it possible to obtain the antibody. primary.
  • the culture surfaces were treated with HE800, in order to form a polysaccharide film at the bottom of the dishes. It is observed, during the first days of culture (Days 2 and 4) that the number of cells seeded in the boxes coated with HE800 is much lower than the number of those in the control boxes (Table I). On the last day of the experiment, on the other hand, we notice an inversion of these results (Tables I and II). The curves presented show that the cells cultured on HE ⁇ OO film observe a lag time before entering the exponential growth phase, longer than that expressed by the cells cultured on plastic. On the other hand, while the number of cells in the control cultures reached a plateau on the later days of the experiment (cf. Table III), the cultures on HE800 film continue to proliferate. The observations made during the culture or after fixing of the cells make it possible to advance hypotheses as to the cellular behaviors expressed in the different culture conditions:
  • the cultures on HE ⁇ OO film are characterized during the first days of cultures by the presence of numerous cells which do not adhere to the support. This non-adhesion may explain the delay in proliferation observed during cell counts in these cultures.
  • control cells are distributed uniformly in the box, without any particular orientation, while the cells sown on HE800 film are organized in cords in the center of the box.
  • the immuno-cytochemical labeling concerning smooth muscle ⁇ -actin shows:
  • the various films deposited are composed of:
  • the films (3) show for the films (3) the appearance of a dense network composed of long filaments.
  • the films (2) have some much shorter fibers, while the films (1) have practically none.
  • the 3 films are colored Sirius red, but only the film (3) shows a fibrillar network deflecting polarized light.
  • HE800 promotes the formation of collagen fibers, but also allows better resistance of the collagen frame in the face of physical factors such as temperature and mechanical stresses.
  • the cells are cultured in a collagen matrix (three-dimensional culture model) in order to best mimic the cell / matrix interactions observed in the connective tissue.
  • the first parameter studied is the lattice retraction speed:
  • the retraction curves for control laths and lattices comprising 1 ⁇ E800 are similar. Despite these similarities, we notice that the HE800 laths have a speed
  • the number of cells present in each lattice varies, at the two culture times, between 180,000 and 250,000 cells.
  • the number of cells at the periphery represents 2 to 12% of the total number of cells.
  • Tables IV, V and VI show the number of cells per volumetric unit (mm 3 ) present in the entire lattice and in its different regions.
  • the total volumetric cell densities of the laths after 11 and 40 days are between 3200 and 5900 cells / mm 3 (see Table IV). These values are comparable to those found in normal human connective tissue as previously described (Miller et al., Exp Dermatol. 2003 Aug; 12 (4): 403-11).
  • the physiological cellularity of the control lattices and of the lattices comprising HE800 therefore attests to the validity of the culture model used and to the compatibility of HE800 with this physiological model.
  • the total cell density (see Table IV) of the control lattices does not vary regardless of the culture time. On the 11th day of culture, the total cell density of the HE800 lattices is 25 to 40% lower than those of the control lattices. On the 40 th day of culture, the cell densities of the control lattices and of the HE800 lattices are equivalent. The variations in cell densities observed inside the laths (cf. Table V) reproduce exactly those of the entire lath. The topological organization of the cells of the
  • the cell densities of the peripheral crown are 4 times greater than those of the interior of the lath and show little variation between the equivalent connective connective tissues and the equivalent connective tissues comprising HE800 (Table VI).
  • the overall cellularity of the HE800 lattices is lower than that of the control lattices in the early days of culture and then becomes equivalent to the late days of culture.
  • the number of cells decreases during the culture time, this reduction is particularly accentuated in the lattices comprising HE800 (decrease by 2 to 3.5 times in the number of cells). This decrease can be explained by a loss of adhesion of the peripheral cells which detaches from the extracellular matrix and / or a massive migration of these cells inside. This explains the overall gains in cellularity, over time, in lattices containing HE800.
  • Table V Cell density inside the lattice cell number per mm 3 .
  • HE800 promotes the proliferation of dermal fibroblasts in the extracellular matrix and / or promotes their mobilization, that is to say the selection, migration and massive penetration of peripheral cells. 2.4.3) State of the collagen matrix:
  • the Sirius red coloring makes it possible to specifically color the collagens, in the skin for example its collagens appear in the form of a loose filamentary structure, colored in red.
  • the Sirius red staining of the histological sections after observation in transmitted light and polarized light shows that the addition of HE800 during the formation of the lattice allows the formation of a much denser matrix and at much shorter times than in the lattis witnesses.
  • the density of the control collagen matrix after 40 days of culture is equivalent to that observed in the collagen matrices formed in the presence of HE800 at 11 days of culture. This effect on density is much greater at the lowest doses (10%, 5%).
  • Electron microscopy was performed on equivalent connective tissue cultured for 11 days. A good ultrastructural state of the cells was observed, either in the controls or in the laths formed in the presence of the different concentrations of HE ⁇ OO.
  • the laths comprising 20% of HE800 make it possible to observe some fibrillar elements taken in a gel consisting of the exopolysaccharide.
  • the laths comprising 10% and 5% of exopolysaccharides are very different, in effect many collagen fibers are present, they are distributed throughout the lattice and present, for some,
  • I / HE800 accelerates collagen fibrillation and promotes the constitution of an extracellular matrix.

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Abstract

The invention relates to the use of a polysaccharide which is excreted by the Vibrio diabolicus species for the regeneration and protection of the non-mineralised connective tissue of the periodontium.

Description

Utilisation d/ un polysaccharide excrété par l'espèce V±bx±o d±àbol±cus à des fins de régénération et de protection du parodonte  Use of a polysaccharide excreted by the species V ± bx ± o d ± àbol ± cus for the purposes of regeneration and protection of the periodontium
La présente invention se rapporte à la régénération du tissu conjonctif non minéralisé du parodonte. The present invention relates to the regeneration of non-mineralized connective tissue of the periodontium.
Des bactéries productrices d' exopolysaccharide (EPS) ont été isolées parmi des microorganismes provenant des écosystèmes hydrothermaux profonds. HE800 est un EPS produit par la souche Vibrio diabolicus . Sa masse molaire moyenne en poids est d'environ 800 000 g/mol à l'état natif. Il est caractérisé par une séquence osidique répétitive linéaire originale constituée par 4 résidus osidiques : Exopolysaccharide producing bacteria (EPS) have been isolated from microorganisms from deep hydrothermal ecosystems. HE800 is an EPS produced by the Vibrio diabolicus strain. Its weight-average molar mass is approximately 800,000 g / mol in the native state. It is characterized by an original linear repeating osidic sequence consisting of 4 osidic residues:
[(-3)-D GlcNac β (1-4) D GIcA β (1-4) D GIcA β (1-4) D GalNac α (l-)]n  [(-3) -D GlcNac β (1-4) D GIcA β (1-4) D GIcA β (1-4) D GalNac α (l -)] n
HE800 a été décrit dans la demande internationale au nom de IFREMER publiée sous le numéro WO9838327 ainsi que dans les articles suivants : Raguénès et al, Int J Syst Bact, 1997 , 47, 989-995 et Rougeaux et al, Carbohyd. Res, 1999, 322, 40- 45. De nombreuses applications pour cet exopolysaccharide ont été décrites. A titre d'exemple d'application, on peut citer la demande internationale WO0202051, qui décrit les propriétés bénéfiques de HE800 en cicatrisation osseuse. Aucune application de HE800 n'est à ce jour connue en ce qui concerne la régénération du tissu conjonctif non minéralisé du parodonte. HE800 has been described in the international application on behalf of IFREMER published under the number WO9838327 as well as in the following articles: Raguénès et al, Int J Syst Bact, 1997, 47, 989-995 and Rougeaux et al, Carbohyd. Res, 1999, 322, 40-45. Many applications for this exopolysaccharide have been described. By way of example of application, mention may be made of international application WO0202051, which describes the beneficial properties of HE800 in bone healing. To date, no application of HE800 is known with regard to the regeneration of the non-mineralized connective tissue of the periodontium.
Le parodonte est un ensemble de tissus dont le but est le soutien et le maintien de la dent dans son alvéole. Il est constitué de deux tissus mous que sont la gencive et le ligament parodontal (desmodonte) et de deux tissus calcifiés, le cément et l'os alvéolaire. Cet organe à la particularité de se situer dans un environnement, où il est soumis à de The periodontium is a set of tissues whose purpose is to support and maintain the tooth in its socket. It consists of two soft tissues, the gum and the periodontal ligament (desmodont), and two calcified tissues, the cementum and the alveolar bone. This organ has the particularity of being located in an environment, where it is subjected to
fei.ï. continuelles agressions (bactériennes, mécaniques, chimiques) . La cavité buccale est en effet, un milieu humide abritant une flore bactérienne commensale. L'intégrité du parodonte dépend essentiellement de l'équilibre entre les tissus buccaux et cette flore bactérienne. Toute déstabilisation de cette relation favorise la prolifération d'une flore pathogène pouvant amener à la destruction des tissus parodontaux. Pour répondre à ces contraintes le parodonte est en constant remodelage. fei.ï. continual attacks (bacterial, mechanical, chemical). The oral cavity is indeed, a humid environment sheltering a commensal bacterial flora. The integrity of the periodontium depends essentially on the balance between the oral tissues and this bacterial flora. Any destabilization of this relationship promotes the proliferation of pathogenic flora which can lead to the destruction of periodontal tissues. To meet these constraints, the periodontium is constantly being remodeled.
La gencive est le tissu de recouvrement du parodonte, elle constitue ainsi une protection, contre les attaques bactériennes, pour les éléments du parodonte qu'elle recouvre (cément, desmodonte, et os alvéolaire) . Histologiquement, ce tissu est constitué d'un tissu conjonctif recouvert d'un épithélium d'origine ectodermique . Le tissu conjonctif gingival est constitué d'une matrice extracellulaire gingivale très semblable quant au contenu macromoléculaire à celle du derme. La gencive contrairement au derme est en relation directe avec différents tissus minéralisés et présente plusieurs types de fibres collagéniques liant la gencive à l'os alvéolaire, au cément ou à d'autres fibres liées à la dent voisine. The gum is the covering fabric of the periodontium, it thus constitutes a protection, against the bacterial attacks, for the elements of the periodontium which it covers (cement, desmodont, and alveolar bone). Histologically, this tissue consists of a connective tissue covered with an epithelium of ectodermal origin. The gingival connective tissue consists of an extracellular gingival matrix very similar in terms of macromolecular content to that of the dermis. The gum, unlike the dermis, is in direct relation with different mineralized tissues and presents several types of collagen fibers binding the gum to the alveolar bone, to the cementum or to other fibers linked to the neighboring tooth.
Les collagènes fibrillaires représentent 50 à 60% des protéines retrouvées dans le tissu conjonctif gingival. Les analyses phénotypiques montrent que ces collagènes sont à 91% de type I, 8% type III et moins de 1% de type V.  Fibrillar collagens represent 50 to 60% of the proteins found in the gingival connective tissue. Phenotypic analyzes show that these collagens are 91% type I, 8% type III and less than 1% type V.
La matrice extracellulaire constitue l'armature du tissu conjonctif. Elle confère au tissu leur forme, leur résistance mécanique, leur souplesse et assure des fonctions physiologiques importantes. Elle est aussi nécessaire au maintien de l'état différencié des cellules qui la synthétisent et la remodèlent. Par l'intermédiaire de The extracellular matrix constitutes the reinforcement of the connective tissue. It gives the fabric its shape, its mechanical resistance, its flexibility and ensures important physiological functions. It is also necessary for maintaining the differentiated state of the cells which synthesize and reshape it. Through
ç IH récepteurs membranaires tels que les intégrines, elle est en étroite association avec les cellules résidentes comme les fibroblastes ce qui lui permet, selon son état, d'en contrôler la migration, la prolifération ou les activités métaboliques. En retour, ces cellules peuvent remodeler la matrice qui les environne. Elles peuvent alors exprimer des protéases pour la dégrader ou resynthétiser de nouveaux composants matriciels. La matrice extracellulaire est donc en constant équilibre (dégradation-resynthèse) . Cet équilibre peut être irrémédiablement bouleversé lors de certaines pathologies . En effet, dans les syndromes inflammatoires, le pouvoir destructeur des cellules résidentes est exacerbé sous l'influence des cellules de l'inflammation, ces dernières, après activation dégradent aussi la matrice. D'autres pathologies peuvent contribuer au dérèglement de la dynamique matricielle telles que les fibroses au cours desquelles l'expression d'un ou de plusieurs composants matriciels est exacerbée . ç IH membrane receptors such as integrins, it is in close association with resident cells such as fibroblasts which allows it, depending on its condition, to control migration, proliferation or metabolic activities. In return, these cells can reshape the matrix that surrounds them. They can then express proteases to degrade it or resynthesize new matrix components. The extracellular matrix is therefore in constant equilibrium (degradation-resynthesis). This balance can be irreparably upset during certain pathologies. Indeed, in inflammatory syndromes, the destructive power of resident cells is exacerbated under the influence of inflammation cells, the latter, after activation, also degrade the matrix. Other pathologies can contribute to the deregulation of the matrix dynamics such as fibrosis during which the expression of one or more matrix components is exacerbated.
Dans ce contexte, la bonne restructuration de la matrice extracellulaire est le garant de la régénération tissulaire. L'organisation de la trame collagénique est un élément essentiel de cette restructuration tissulaire. En effet, ces collagènes constituent la classe protéique majoritaire des matrices extracellulaires et en particulier celles du parodonte .  In this context, the proper restructuring of the extracellular matrix is the guarantor of tissue regeneration. The organization of the collagen framework is an essential element of this tissue restructuring. Indeed, these collagens constitute the majority protein class of the extracellular matrices and in particular those of the periodontium.
La gencive, contrairement au derme, est soumise à un important et constant remodelage. Ce remodelage est la conséquence de la coexistence entre la plaque bactérienne se déposant sur la dent et le tissu gingival, et du stress mécanique auquel est soumise la gencive lors de la mastication. Ces facteurs affectent plus ou moins directement les fibroblastes gingivaux qui représentent la majorité des cellules présentes dans le tissu conjonctif gingival. Ces  The gum, unlike the dermis, is subject to a large and constant remodeling. This remodeling is the consequence of the coexistence between the bacterial plaque depositing on the tooth and the gingival tissue, and the mechanical stress to which the gum is subjected during chewing. These factors more or less directly affect the gingival fibroblasts which represent the majority of the cells present in the gingival connective tissue. These
E U ;;_t.π (RÈGLE 2β fibroblastes sont en grande partie responsables du remodelage observé dans la gencive saine. Pour maintenir l'attache de la gencive au cément et à l'os alvéolaire les fibroblastes gingivaux doivent être capables de répondre constamment aux contraintes qui s'exercent sur le tissu qui les abrite. EU ;; _ t.π (RULE 2β Fibroblasts are largely responsible for the remodeling seen in healthy gums. To maintain the attachment of the gum to the cementum and the alveolar bone, the gingival fibroblasts must be able to constantly respond to the stresses exerted on the tissue that shelters them.
Cette activation permanente se traduit par une très grande hétérogénéité phénotypique des fibroblastes gingivaux. On distingue notamment les myofibroblastes des fibroblastes, les myofibroblastes prolifèrent en cas de processus inflammatoire. Le fibroblaste est la cellule clef de 1' homéostasie tissulaire. This permanent activation results in a very large phenotypic heterogeneity of the gingival fibroblasts. A distinction is made in particular between myofibroblasts and fibroblasts, myofibroblasts proliferate in the event of an inflammatory process. The fibroblast is the key cell of tissue homeostasis.
Si cette hétérogénéité fibroblastique présente un avantage dans des conditions physiologiques, elle peut se révéler extrêmement délétère dans le cadre de pathologies parodontales s' installant dans la chronicité. En effet, toute altération durable de l'équilibre cellulaire peut entraîner 1' activation non désirée de certains phénotypes cellulaires comme par exemple la prolifération des myofibroblastes au dépend des fibroblastes.  If this fibroblastic heterogeneity has an advantage in physiological conditions, it can prove to be extremely deleterious in the context of periodontal pathologies settling in chronicity. Indeed, any lasting alteration in cell balance can lead to the unwanted activation of certain cell phenotypes such as, for example, the proliferation of myofibroblasts at the expense of fibroblasts.
L'acide hyaluronique de haut poids moléculaire est couramment utilisé pour traiter de nombreuses pathologies buccales.  High molecular weight hyaluronic acid is commonly used to treat many oral conditions.
EP0444492 décrit l'utilisation de l'acide hyaluronique pour traiter les maladies inflammatoires de la cavité buccale, comme la gingivite. WO2005000321 décrit l'utilisation de l'acide hyaluronique pour traiter les aphtes de la cavité buccale. Pour ces traitements, l'acide hyaluronique est utilisé sous différentes formulations, on peut citer à titre d'exemple le gengigel® qui se présente sous forme de spray ou de bain de bouche. EP0444492 describes the use of hyaluronic acid to treat inflammatory diseases of the oral cavity, such as gingivitis. WO2005000321 describes the use of hyaluronic acid to treat canker sores in the oral cavity. For these treatments, hyaluronic acid is used in different formulations, there may be mentioned, for example, gengigel®, which is in the form of a spray or mouthwash.
L'acide hyaluronique qui est un produit d'origine animale est produit à partir d'extrait d'animaux ou bien par génie génétique . L'objet de la présente invention est d'identifier un composé capable de préserver l' homéostasie tissulaire parodontale et/ou de favoriser la restructuration, c'est-à-dire la restauration, d'une trame collagénique altérée des tissus conjonctifs non minéralisés du parodonte, et de favoriser la prolifération des fibroblastes gingivaux afin de rétablir 1' homéostasie gingivale. Hyaluronic acid, which is a product of animal origin, is produced from animal extract or by genetic engineering. The object of the present invention is to identify a compound capable of preserving periodontal tissue homeostasis and / or of promoting the restructuring, that is to say the restoration, of an altered collagen framework of non-mineralized connective tissues of the periodontium, and to favor the proliferation of the gingival fibroblasts in order to restore 1 gingival homeostasis.
Un tel composé présentera ainsi une activité protectrice et régénératrice sur les tissus conjonctifs non minéralisés du parodonte et facilitera le rétablissement de l' homéostasie tissulaire .  Such a compound will thus have a protective and regenerative activity on the non-mineralized connective tissues of the periodontium and will facilitate the restoration of tissue homeostasis.
Les inventeurs ont mis en évidence, de façon surprenante et inattendue, qu'un polysaccharide de masse molaire moyenne en poids comprise entre 500 000 et 2 000 000 g/mol, caractérisé par une séquence osidique répétitive linéaire comprenant les 4 résidus osidiques suivants : The inventors have shown, surprisingly and unexpectedly, that a polysaccharide of average molar mass by weight of between 500,000 and 2,000,000 g / mol, characterized by a linear repetitive osidic sequence comprising the following 4 osidic residues:
[(-3)-D GlcNac β (1-4) D GIcA β (1-4) D GIcA β (1-4) D GalNac α (1-)] présente les propriétés suivantes : il induit une sélection des souches fibroblastiques, il stimule la mobilisation et la prolifération des fibroblastes dans la matrice extracellulaire, il accélère la fibrillation collagénique et favorise ainsi la reconstitution de la matrice conjonctive. De ce fait, ce polysaccharide accélère la régénération en accélérant la restructuration des tissus conjonctifs. Il permet d'aboutir à une régénération complète de sorte que l'apparition de situations pathologiques de type fibrotique ou inflammatoire est évitée.  [(-3) -D GlcNac β (1-4) D GIcA β (1-4) D GIcA β (1-4) D GalNac α (1-)] has the following properties: it induces a selection of fibroblast strains , it stimulates the mobilization and proliferation of fibroblasts in the extracellular matrix, it accelerates collagen fibrillation and thus promotes the reconstitution of the connective matrix. Therefore, this polysaccharide accelerates regeneration by accelerating the restructuring of connective tissues. It leads to complete regeneration so that the appearance of pathological situations of fibrotic or inflammatory type is avoided.
Ce polysaccharide permet la restructuration de la trame collagénique des tissus conjonctifs non minéralisés du  This polysaccharide allows the restructuring of the collagen framework of the non-mineralized connective tissues of the
KOILLE K LΛ,s, (E≥€LE 26J parodonte, et il constitue un support permettant l'adhésion et la prolifération cellulaire des fibroblastes gingivaux. KOILLE K LΛ, s, (E≥ € LE 26J periodontium, and it constitutes a support allowing the adhesion and the cellular proliferation of the gingival fibroblasts.
Ainsi, de par ses propriétés le polysaccharide est particulièrement adapté aux applications suivantes : la régénération des tissus conjonctifs du parodonte ainsi que le traitement de pathologies buccales, en particulier celles liées à un état inflammatoire ou à un état traumatique. Thus, due to its properties, the polysaccharide is particularly suitable for the following applications: the regeneration of the connective tissues of the periodontium as well as the treatment of oral pathologies, in particular those linked to an inflammatory or traumatic state.
De plus, de par ces mêmes propriétés, le polysaccharide permet la fabrication de matrice de collagène aux propriétés améliorées. En effet, la trame collagénique des matrices de collagènes comprenant le polysaccharide présente une meilleure résistance devant des facteurs physiques tels que la température et les contraintes mécaniques. Enfin, il favorise la culture des fibroblastes gingivaux et permet la réalisation de substitut de gencive. In addition, by these same properties, the polysaccharide allows the production of collagen matrix with improved properties. Indeed, the collagen framework of the collagen matrices comprising the polysaccharide has better resistance to physical factors such as temperature and mechanical stresses. Finally, it promotes the culture of gingival fibroblasts and allows the production of gum substitute.
La présente invention a pour objet l'utilisation d'un polysaccharide ou d'un sel de ce polysaccharide de masse molaire moyenne en poids comprise entre 500 000 et 2 000 000 g/mol, préférentiellement comprise entre 700 000 et 900 000 g/mol, caractérisé par une séquence osidique répétitive linéaire comprenant les 4 résidus osidiques suivants : The subject of the present invention is the use of a polysaccharide or a salt of this polysaccharide with a weight average molar mass of between 500,000 and 2,000,000 g / mol, preferably between 700,000 and 900,000 g / mol , characterized by a linear repetitive osidic sequence comprising the following 4 oidic residues:
[(-3)-D GlcNac β (1-4) D GIcA β (1-4) D GIcA β (1-4) D GalNac α (1-)] pour la fabrication d'une composition, d'un médicament ou d'un dispositif médical ayant une activité protectrice et/ou régénératrice sur les tissus conjonctifs non minéralisés du parodonte .  [(-3) -D GlcNac β (1-4) D GIcA β (1-4) D GIcA β (1-4) D GalNac α (1-)] for the manufacture of a composition, of a medicament or of a medical device having a protective and / or regenerative activity on the non-mineralized connective tissues of the periodontium.
Typiquement le polysaccharide peut se présenter sous la forme d'un sel. Typiquement le polysaccharide est un polysaccharide excrété par l'espèce Vibrio diabolicus ou un dérivé obtenu à partir de celui-ci. Des modes de préparation ont été décrits dans les documents suivants : WO 98/38327, Raguénès et al, Int J Syst Bact, 1997, 47, 989-995 et Rougeaux et al, Carbohyd. Res, 1999, 322, 40-45. A titre d'exemple, des dérivés de masse molaire moyenne en poids comprise entre 500 000 et 2 000 000 g/mol peuvent être obtenus par dépolymérisation partielle, par pontage et/ou par des modifications chimiques, en particulier, par sulfatation et/ou par acétylation. A titre d'exemple, WO0046252 décrit une méthode de pontage de l'acide hyaluronique, typiquement cette méthode pourra être adaptée pour générer des dérivés pontés du polysaccharide excrété par l'espèce Vibrio diabolicus. Typically the polysaccharide can be in the form of a salt. Typically the polysaccharide is a polysaccharide excreted by the Vibrio diabolicus species or a derivative obtained from it. Methods of preparation have been described in the following documents: WO 98/38327, Raguénès et al, Int J Syst Bact, 1997, 47, 989-995 and Rougeaux et al, Carbohyd. Res, 1999, 322, 40-45. By way of example, derivatives of average molar mass by weight of between 500,000 and 2,000,000 g / mol can be obtained by partial depolymerization, by bridging and / or by chemical modifications, in particular, by sulfation and / or by acetylation. By way of example, WO0046252 describes a method for bridging hyaluronic acid, typically this method could be adapted to generate bridged derivatives of the polysaccharide excreted by the species Vibrio diabolicus.
Un mode de réalisation de l'invention concerne la fabrication d'une composition, d'un médicament, d'un dispositif médical pour traiter une pathologie buccale du tissu conjonctif non minéralisé du parodonte. One embodiment of the invention relates to the manufacture of a composition, of a medicament, of a medical device for treating an oral pathology of the non-mineralized connective tissue of the periodontium.
En particulier la pathologie buccale est liée à un état inflammatoire ou à un état traumatique.  In particular, oral pathology is linked to an inflammatory state or to a traumatic state.
Préférentiellement la pathologie buccale est choisie dans le groupe consistant en la parodontite, la gingivite, la fibrose gingivale, la récession gingivale, l'aphte, l'aphtose buccale récidivante, les maladies aphteuses, et les pathologies bulleuses .  Preferably, the oral pathology is chosen from the group consisting of periodontitis, gingivitis, gingival fibrosis, gingival recession, canker sore, recurrent oral aphtosis, foot-and-mouth disease, and bullous pathologies.
Typiquement la composition ou le médicament fabriqué est destiné une administration topique au niveau du parodonte. Par composition topique, on entend une composition qui agit en un point déterminé et qui est applicable directement sur la muqueuse buccale. Typically the composition or the drug produced is intended for topical administration at the periodontium level. By topical composition is meant a composition which acts at a determined point and which is directly applicable on the oral mucosa.
26} La composition et le médicament peuvent être sous la forme d'une composition topique à usage buccal, en particulier sous la forme d'un gel, d'une solution d'une émulsion ou d'un spray. Typiquement une composition topique selon l'invention est fabriquée de manière connue en soi . A titre d' exemple une composition topique selon l'invention contient le polysaccharide à une concentration comprise entre 0,005 et 10% en poids sur le poids total de la composition, plus préférentiellement à une concentration comprise entre 0,01 et 5% en poids. 26} The composition and the medicament may be in the form of a topical composition for oral use, in particular in the form of a gel, a solution of an emulsion or a spray. Typically a topical composition according to the invention is produced in a manner known per se. By way of example, a topical composition according to the invention contains the polysaccharide at a concentration between 0.005 and 10% by weight on the total weight of the composition, more preferably at a concentration between 0.01 and 5% by weight.
Un gel selon l'invention pourra comprendre du sorbitol, du maltitol, du xylitol et/ou de la carboxymethylcellulose sodique .  A gel according to the invention may comprise sorbitol, maltitol, xylitol and / or sodium carboxymethylcellulose.
Cette même composition ou ce même médicament peut servir à imprégner un pansement buccal. This same composition or same drug can be used to impregnate an oral dressing.
Un mode de réalisation de l'invention concerne une pâte dentifrice, un bain de bouche, un spray, une colle pour denture et un pansement buccal comprenant le polysaccharide. Typiquement l'homme du métier pourra utiliser les techniques usuelles pour la mise au point de ces produits. A titre d'exemple une pâte dentifrice, un bain de bouche ou un spray selon l'invention contient le polysaccharide à une concentration comprise entre 0,005 et 1% en poids sur le poids total de la composition, plus préférentiellement à une concentration comprise entre 0,01 et 0,1% en poids. Une pâte dentifrice selon l'invention pourra comprendre à titre d'exemple un ou plusieurs des composés suivants : du sorbitol, du maltitol, du xylitol et/ou de la carboxymethylcellulose sodique. Typiquement, un bain de bouche selon l'invention pourra contenir un ou plusieurs des excipients suivants : le polysorbate 60, la saccharine One embodiment of the invention relates to a toothpaste, a mouthwash, a spray, a denture glue and an oral dressing comprising the polysaccharide. Typically a person skilled in the art will be able to use the usual techniques for the development of these products. By way of example, a toothpaste, a mouthwash or a spray according to the invention contains the polysaccharide at a concentration of between 0.005 and 1% by weight on the total weight of the composition, more preferably at a concentration of between 0 , 01 and 0.1% by weight. A toothpaste according to the invention may comprise, for example, one or more of the following compounds: sorbitol, maltitol, xylitol and / or sodium carboxymethylcellulose. Typically, a mouthwash according to the invention may contain one or more of the following excipients: polysorbate 60, saccharin
p™ F.,' 1} t ? f m /in (ζ r sodique, le salicylate de méthyle, l'essence de girofle ; l'essence d'anis, l'essence d'eucalyptus, l'acide citrique, le menthol. Un bain de bouche selon l'invention pourra également contenir un agent actif supplémentaire comme par exemple l' hexétidine. p ™ F., '1} t? fm / in (ζ r sodium, methyl salicylate, clove essence; anise essence, eucalyptus essence, citric acid, menthol. A mouthwash according to the invention may also contain an additional active agent such as, for example, hexetidine.
Pour toutes les compositions, médicaments, pâtes dentifrices, bains de bouche, sprays, colles pour denture selon l'invention, le polysaccharide peut être utilisé comme l'unique agent actif ou accompagné d'autres agents actifs comme par exemple un antibactérien, un antibiotique, des vitamines ou des oligoélements . For all the compositions, medicaments, toothpastes, mouthwashes, sprays, adhesives for toothing according to the invention, the polysaccharide can be used as the sole active agent or accompanied by other active agents such as, for example, an antibacterial, an antibiotic , vitamins or trace elements.
Selon un autre de réalisation, l'invention concerne une matrice de collagène comprenant le polysaccharide selon 1' invention. According to another embodiment, the invention relates to a collagen matrix comprising the polysaccharide according to the invention.
Typiquement le collagène de la matrice est un collagène choisi dans le groupe constitué des collagènes de type I, III, V ou d'un mélange de ceux-ci. Préférentiellement le collagène est un collagène de type I.  Typically the collagen of the matrix is a collagen chosen from the group consisting of type I, III, V collagens or a mixture of these. Preferably, the collagen is a type I collagen.
Typiquement pour fabriquer une telle matrice de collagène, l'homme du métier utilisera les techniques couramment utilisées pour la fabrication des matrices de collagènes à partir de collagènes fibrillaires acido-solubles . En présence du polysaccharide selon l'invention, les collagènes fibrillaires acido-solubles se fibrillent naturellement après neutralisation du pH. Alternativement la matrice de collagène selon l' invention pourra être obtenue par pontage du polysaccharide selon l'invention avec le collagène. Pour réaliser le pontage l'homme du métier utilisera les techniques couramment utilisées pour le pontage de polysaccharides avec le collagène. EP1374857 est une illustration d'une technique de pontage utilisable. Typically to manufacture such a collagen matrix, those skilled in the art will use the techniques commonly used for the manufacture of collagen matrices from acid-soluble fibrillar collagens. In the presence of the polysaccharide according to the invention, the acid-soluble fibrillar collagens fibrillate naturally after neutralization of the pH. Alternatively, the collagen matrix according to the invention can be obtained by bridging the polysaccharide according to the invention with the collagen. To carry out the bridging, a person skilled in the art will use the techniques commonly used for the bridging of polysaccharides with collagen. EP1374857 is an illustration of a usable bypass technique.
De manière avantageuse, la matrice pourra comprendre en outre un facteur de croissance qui favorise la colonisation de la matrice par les fibroblastes gingivaux et la reconstitution du tissu conjonctif. Advantageously, the matrix may also comprise a growth factor which promotes colonization of the matrix by the gingival fibroblasts and the reconstitution of the connective tissue.
Préférentiellement le facteur de croissance pourra être choisi dans le groupe consistant en TGF-beta, PDGF, FGFs, BMPs (bone morphogenetic proteins), VEGF, CTGF (connective tissue growth factor) . Preferably, the growth factor may be chosen from the group consisting of TGF-beta, PDGF, FGFs, BMPs (bone morphogenetic proteins), VEGF, CTGF (connective tissue growth factor).
Typiquement la matrice pourra servir de dispositif médical résorbable ou d'implant. Une telle matrice permettra le remplacement mécanique et fonctionnel de structures endommagées avec un minimum de réactions indésirables. Cette matrice une fois implantée dans le tissu à régénérer servira de structure de guidage et balisera le potentiel régénératif du tissu. La matrice favorisera la pénétration des fibroblastes de façon ordonnée après la greffe de la matrice, tout en incitant ces mêmes fibroblastes à produire leur propre matrice extracellulaire.  Typically, the matrix can serve as an absorbable medical device or an implant. Such a matrix will allow mechanical and functional replacement of damaged structures with a minimum of undesirable reactions. This matrix once implanted in the tissue to be regenerated will serve as a guiding structure and will mark out the regenerative potential of the tissue. The matrix will promote the penetration of fibroblasts in an orderly fashion after the graft of the matrix, while encouraging these same fibroblasts to produce their own extracellular matrix.
Selon un mode de réalisation préférée de l'invention la matrice pourra comprendre en outre des fibroblastes gingivaux de façon à constituer un substitut de gencive. Ce substitut pourra être implanté in vivo. De manière avantageuse, ce sont les fibroblastes gingivaux du patient sur lequel la greffe doit être effectuée qui servent à coloniser la matrice. According to a preferred embodiment of the invention, the matrix may also comprise gingival fibroblasts so as to constitute a gum substitute. This substitute can be implanted in vivo. Advantageously, it is the gingival fibroblasts of the patient on which the graft is to be performed which serve to colonize the matrix.
Selon un autre mode de réalisation, l'invention concerne un support de culture cellulaire caractérisé en ce que la surface du support sur laquelle les cellules sont cultivées comprend le polysaccharide selon l'invention. Typiquement le polysaccharide est sous forme d'un film, d'une membrane ou d'une structure alvéolée tridimensionnelle. According to another embodiment, the invention relates to a cell culture support, characterized in that the surface of the support on which the cells are cultivated comprises the polysaccharide according to the invention. Typically the polysaccharide is in the form of a film, a membrane or a three-dimensional honeycomb structure.
Selon un autre mode de réalisation, l'invention concerne un procédé de culture de fibroblastes gingivaux, caractérisé en ce que les dits fibroblastes sont cultivés sur une matrice selon l'invention ou sur un support tel que décrit précédemment . According to another embodiment, the invention relates to a method for culturing gingival fibroblasts, characterized in that said fibroblasts are cultured on a matrix according to the invention or on a support as described above.
Le contenu de tous les documents cités doit être considéré comme faisant partie de la présente description. The content of all cited documents should be considered as part of this description.
La présente invention sera mieux illustrée ci-après à l'aide des exemples qui suivent. Ces exemples sont donnés uniquement à titre d'illustration de l'objet de l'invention, dont ils ne constituent en aucune manière une limitation. The present invention will be better illustrated below with the aid of the examples which follow. These examples are given solely by way of illustration of the subject of the invention, of which they in no way constitute a limitation.
EXEMPLES EXAMPLES
1. Matériels et Méthodes 1. Materials and Methods
1.1. Préparation de l' exopolysaccharide HE800 à partir de cultures de Vîbrio Diabolxcus (la souche HΞ800) . 1.1. Preparation of the exopolysaccharide HE800 from cultures of Vîbrio Diabolxcus (the strain HΞ800).
Des modes de préparation d'HE800 ont été décrits dans les documents suivants : WO 98/38327, Raguénès et al, Int J Syst Methods of preparing HE800 have been described in the following documents: WO 98/38327, Raguénès et al, Int J Syst
Bact, 1997, 47, 989-995 et Rougeaux et al, Carbohyd. Res,Bact, 1997, 47, 989-995 and Rougeaux et al, Carbohyd. Res,
1999, 322, 40-45. 1999, 322, 40-45.
a) Cultures de Vibrio diabolicus a) Vibrio diabolicus cultures
La souche HE800 est cultivée sur milieu 2216E [OPPENHEIMER, The HE800 strain is cultivated on 2216E medium [OPPENHEIMER,
J. Mar. Res. 11, 10-18 (1952)] enrichi avec du glucose (30 g/1) . La production est effectuée à 300C et à pH 7,4 en fermenteur de 2 litres contenant 1 litre du milieu 2216E- J. Mar. Res. 11, 10-18 (1952)] enriched with glucose (30 g / 1). The production is carried out at 30 ° C. and at pH 7.4 in a 2 liter fermenter containing 1 liter of medium 2216E-
FEtJfUE PI tziimxzzim (riae 2β glucose. Après 48 heures de culture, le moût présente une faible viscosité (de l'ordre de 40 centipoises à 60 rpm) . b) Purification de l ' exopolysaccharide FEtJfUE PI tziimxzzim (riae 2β glucose. After 48 hours of culture, the must has a low viscosity (of the order of 40 centipoises at 60 rpm). b) Purification of the exopolysaccharide
Les bactéries sont séparées du moût par une centrifugation à 20 000 g pendant 2 heures, puis le polysaccharide est précipité à partir du surnageant à l'aide d'éthanol pur, puis on effectue plusieurs lavages éthanol/eau en proportions croissantes d'éthanol, selon le procédé décrit par TALMONT et al. [Food Hydrocolloids 5, 171-172 (1991)] ou VINCENT et al. [Appl. Environ. Microbiol. , 60, 4134-4141 (1994)]. Le polysaccharide obtenu est séché à 300C et conservé à température ambiante. 2,5 g de polysaccharide purifié par litre de culture ont ainsi été obtenus. The bacteria are separated from the must by centrifugation at 20,000 g for 2 hours, then the polysaccharide is precipitated from the supernatant using pure ethanol, then several ethanol / water washes are carried out in increasing proportions of ethanol, according to the method described by TALMONT et al. [Food Hydrocolloids 5, 171-172 (1991)] or VINCENT et al. [Appl. About. Microbiol. , 60, 4134-4141 (1994)]. The polysaccharide obtained is dried at 30 ° C. and stored at room temperature. 2.5 g of purified polysaccharide per liter of culture were thus obtained.
1.2. Obtention des fibroblastes 1.2. Obtaining fibroblasts
Les expériences ont été réalisées sur des fibroblastes d'origine dermique et des fibroblastes d'origine gingivale. Ces deux types de fibroblastes adoptent vis-à-vis du HE800 un comportement très similaire, par conséquent les résultats obtenus avec les fibroblastes dermiques sont extrapolables aux fibroblastes gingivaux. The experiments were carried out on fibroblasts of dermal origin and fibroblasts of gingival origin. These two types of fibroblasts adopt a very similar behavior towards HE800, therefore the results obtained with dermal fibroblasts can be extrapolated to gingival fibroblasts.
1.2.1) Milieux de culture : 1.2.1) Culture media:
Les cultures sont effectuées dans un milieu, dit complet, composé, de Dubelco MEM Glutamax I contenant de 100 U/ml de pénicilline 100 μg/ml de streptomycine et 2μg/ml de fungizone (Gibco BRL Cergy Pontoise France) supplémenté ou non (milieu carence) en sérum de veau foetal (SVF) . 1.2.2) Origine des prélèvements tissulaires : The cultures are carried out in a medium, known as complete, composed of Dubelco MEM Glutamax I containing 100 U / ml of penicillin 100 μg / ml of streptomycin and 2 μg / ml of fungizone (Gibco BRL Cergy Pontoise France) supplemented or not (medium deficiency) in fetal calf serum (SVF). 1.2.2) Origin of tissue samples:
Les biopsies dermiques utilisées sont mises en culture dans les 3 heures qui suivent leur prélèvement par le praticien. Les prélèvements utilisés sont obtenus après circoncision, à partir de prépuces d'enfants cliniquement sains. Les biopsies gingivales sont prélevées sur des patients jeunes (moins de 30 ans) exempt de pathologies . Ces biopsies sont situées au niveau de la gencive attachée de prémolaires extraites pour raisons orthodontiques . Ces gencives sont de plus, déclarées cliniquement saines par le praticien. Ces biopsies sont des bribes tissulaires détachées au cours de l'extraction et qui n'ont demandé aucune modification de l'acte opératoire.  The dermal biopsies used are cultured within 3 hours of their collection by the practitioner. The samples used are obtained after circumcision, from the foreskin of clinically healthy children. Gingival biopsies are taken from young patients (under 30) free of pathologies. These biopsies are located at the level of the attached gum of premolars extracted for orthodontic reasons. These gums are also declared clinically healthy by the practitioner. These biopsies are tissue fragments detached during the extraction and which did not require any modification of the operating procedure.
1.2.3) Mise en culture : 1.2.3) Cultivation:
Les prélèvements dermiques et gingivaux sont rincés deux fois dans un milieu DMEM contenant une concentration d'antibiotique supérieure à la normale (pénicilline 6x, streptomycine 4x, fungizone2x) puis ils sont découpés en très petits explants (≈2mm2 ). Ces explants sont déposés à la pipette Pasteur stérile ou avec la pointe du scalpel, dans un flacon de culture de 25 cm2, coté parenchyme sur le plastique. La boîte est relevée et laissée 15 minutes dans cette position pour que les explants, à sec, adhèrent au plastique. The dermal and gingival samples are rinsed twice in a DMEM medium containing a higher than normal concentration of antibiotic (penicillin 6x, streptomycin 4x, fungizone2x) then they are cut into very small explants (≈2mm 2 ). These explants are placed with a sterile Pasteur pipette or with the tip of the scalpel, in a 25 cm 2 culture flask, parenchymal side on the plastic. The box is raised and left for 15 minutes in this position so that the explants, dry, adhere to the plastic.
Les explants ayant adhéré sont recouverts de quelques gouttes de DMEM supplémenté à 20 % en sérum de veau fœtal (SVF) . La boîte de culture est alors placée pendant une nuit dans un incubateur à 37 0C, sous une atmosphère composée de 5 % de CO2 et de 95 % d'air. Le lendemain le surnageant est remplacé par du milieu frais comprenant 20% de SVF, il est renouvelé par la suite toutes les semaines. Après trois semaines les fibroblastes ont colonisé tout le fond de la boîte (les The explants that have adhered are covered with a few drops of DMEM supplemented with 20% in fetal calf serum (SVF). The culture dish is then placed overnight in an incubator at 37 ° C., under an atmosphere composed of 5% CO 2 and 95% air. The next day the supernatant is replaced with fresh medium comprising 20% of FCS, it is subsequently renewed every week. After three weeks the fibroblasts have colonized the entire bottom of the dish (the
kLkirLJA u(i«l{i j kératinocytes présents dans l'expiant n'adhèrent pas dans ces conditions de culture), on procède alors au repiquage. Les explants sont retirés à l'aide d'une pince, les cellules sont rincées deux fois au PBS, puis trypsinisées (Trypsine EDTA Gibco) . La trypsinisation est alors stoppée par l'ajout de DMEM comprenant 10% de SVF. Les cellules sont comptées au compteur (Coulter) puis réensemencées dans plusieurs boîtes de culture. Elles sont, à ce moment, considérées en premier passage et sont entretenues dans un milieu complet contenant 10 % de SVF. Lorsque les cellules sont à nouveau confluentes un autre passage est effectué suivant la même procédure, et ce jusqu'au début des expérimentations. kLkirLJA u (i "l {ij keratinocytes present in the explant do not adhere under these culture conditions), then transplanting is carried out. The explants are removed using forceps, the cells are rinsed twice with PBS, then trypsinized (Trypsin EDTA Gibco). Trypsinization is then stopped by the addition of DMEM comprising 10% of FCS. The cells are counted on the counter (Coulter) and then reseeded in several culture dishes. They are, at this time, considered in the first pass and are maintained in a complete environment containing 10% of SVF. When the cells are again confluent, another passage is carried out according to the same procedure, until the start of the experiments.
1.3. Préparation des films et culture des fibroblastes 1.3. Film preparation and fibroblast culture
1.3.1) Préparation des films d'HE800 et culture des fibroblastes 1.3.1) Preparation of HE800 films and fibroblast culture
Un surfactage est effectué par le dépôt de 200μl d'une solution à 2mg/ml d'HE800 au fond des puits de culture (boîte 24 puits, 2cm2) . La boîte de culture est déposée sous hotte de culture sur une platine chauffante réglée à 37 °C, pendant au moins 5 heures. Après évaporation, un film d'HE800 se constitue au fond de la boîte. Les fibroblastes gingivaux sont ensemencés à raison de 10000 cellules par puits et cultivés pendant 7 jours. Une numération des cellules est effectuée chaque jour, certains puits sont fixés pour l'étude morphologique et l' immuno-détection de l' α-actine des muscles lisses . Surfacting is carried out by depositing 200 μl of a 2 mg / ml solution of HE800 at the bottom of the culture wells (24-well dish, 2 cm 2 ). The culture dish is placed in a culture hood on a heating plate adjusted to 37 ° C for at least 5 hours. After evaporation, a film of HE800 is formed at the bottom of the box. The gingival fibroblasts are seeded at the rate of 10,000 cells per well and cultured for 7 days. A cell count is carried out every day, certain wells are fixed for the morphological study and the immuno-detection of the α-actin of smooth muscles.
1.3.2) Préparation de films de collagène et de films composite collagène-HE800 et culture des fibroblastes : 1.3.2) Preparation of collagen films and collagen-HE800 composite films and culture of fibroblasts:
Le collagène utilisé est un collagène de type I acido-soluble (2mg/ml) obtenu à partir de queue de rat (Institut Jacques  The collagen used is an acid-soluble type I collagen (2 mg / ml) obtained from rat tail (Institut Jacques
1. W l L(UE UIZ Lf iiHilr !«>.L -. »L.i. - J 1 I[ui> i5-.G AXi/ Boy, Reims). Le surfactage des boîtes de culture s'effectue par le dépôt de 200μl d'un mélange de collagène (40μg total) et d'HE800 (5, 50, ou 200μg total). 1. W l L (UE UIZ Lf iiHilr! ">. L -." Li - J 1 I [ui> i5-.G AXi / Boy, Reims). The culture dishes are surfacted by depositing 200 μl of a mixture of collagen (40 μg total) and HE800 (5, 50, or 200 μg total).
La boîte de culture (boîte 24 puits, ou labtek, 2cm2 par puits) est déposée sous hotte de culture sur une platine chauffante réglée à 370C, pendant au moins 5 heures. Après évaporation, un film de collagène avec ou sans HE800 se constitue au fond de la boîte. Des fibroblastes sont ensemencés sur ces films afin de s'assurer de la biocompatibilité de la nouvelle surface de culture. The culture dish (24-well dish, or labtek, 2 cm 2 per well) is placed in a culture hood on a heating plate adjusted to 37 ° C., for at least 5 hours. After evaporation, a collagen film with or without HE800 is formed at the bottom of the box. Fibroblasts are seeded on these films in order to ensure the biocompatibility of the new culture surface.
1.3.3) Caractérisation de la structure des films : 1.3.3) Characterization of the structure of the films:
Les films de collagène et les films composites sont fixés à l'éthanol absolu -200C puis réhydratés pour être colorés au rouge sirius ( (coloration de Junquera, spécifique des collagènes). Ainsi, en rouge Sirius, sous lumière transmise tous les collagènes sont colorés, mais seuls les collagènes correctement fibrilles sont capables de dévier la lumière polarisée . The collagen films and the composite films are fixed with absolute ethanol -20 ° C. and then rehydrated to be colored with sirius red ((Junquera coloring, specific for collagens). Thus, in Sirius red, under light transmitted all the collagens are colored, but only properly fibrillated collagens are able to deflect polarized light.
1.4. Culture en Lattis (matrice de σollagène) : préparation de tissu conjonctif non minéralisé équivalent. 1.4. Culture in Lattis (σollagen matrix): preparation of equivalent non-mineralized connective tissue.
Les lattis sont constitués avec le même collagène I que celui utilisé pour former les films collagéniques . Après neutralisation de la solution acide de collagène (3mg/lattis) , le gel contenant les cellules et qui est en cours de polymérisation est versé dans une boîte de pétri de 5cm de diamètre. HE800 est ajouté au collagène avant l'addition des cellules à raison de 150μg, 300μg ou 600μg par lattis (respectivement 5%, 10%, et 20% de la quantité totale de collagène) 1.4.1) Préparation de la solution stock The laths are made with the same collagen I as that used to form the collagen films. After neutralization of the acid collagen solution (3 mg / lattice), the gel containing the cells and which is being polymerized is poured into a petri dish 5 cm in diameter. HE800 is added to the collagen before the addition of the cells at a rate of 150 μg, 300 μg or 600 μg per lattice (respectively 5%, 10%, and 20% of the total amount of collagen) 1.4.1) Preparation of the stock solution
Pour 50 ml de solution stock ajouter 10ml de Sérum de Veau Fœtal  For 50 ml of stock solution add 10ml of Fetal Calf Serum
1.4.2) Préparation des lattis :  1.4.2) Preparation of laths:
Toutes les étapes de préparation du lattis se font dans la glace.  All stages of lath preparation are done in the ice.
-Remuer puis verser dans la boîte de Pétri puis laisser pendant 5 min à 37 0C le lattis. -Stir then pour into the Petri dish then leave the lattice for 5 min at 37 ° C.
-Après Ih les boîtes sont légèrement remuées afin de décoller les lattis des rebords.  -After Ih the boxes are slightly stirred in order to take off the laths from the edges.
-Les milieux de culture sont changés chaque semaine.  -The culture media are changed weekly.
1.4.3) Caractérisation des lattis (des matrices de collagène) : 1.4.3) Characterization of laths (collagen matrices):
A différents temps de culture (11 et 40 jours) les lattis sont récupérés fixés dans du paraformaldéhyde puis préparés en vue de l'inclusion en paraffine. Des coupes de 7μm d'épaisseur sont alors effectuées au microtome. Les colorations spécifiques de ces coupes permettent d' observer  At different culture times (11 and 40 days) the lattices are recovered fixed in paraformaldehyde and then prepared for inclusion in paraffin. 7μm thick sections are then made in a microtome. The specific colorings of these sections allow us to observe
FEUHLE m ιπû?vx:^tn et d'étudier la structure, la cellularité des tissus conjonctifs reconstruits. Certains des paramètres mis en évidence peuvent par la suite être étudiés par analyse d'images et ainsi être quantifiés. La qualité de la fibrillation collagénique est observée après coloration au rouge Sirius, la cellularité des tissus conjonctifs équivalents a pu être estimée par analyse d' images après coloration des coupes à l' hémalun-éosine . FEUHLE m ιπû? Vx: ^ tn and to study the structure and cellularity of the reconstructed connective tissues. Some of the highlighted parameters can then be studied by image analysis and thus be quantified. The quality of collagen fibrillation is observed after staining with Sirius red, the cellularity of equivalent connective tissues could be estimated by image analysis after staining of the hemalun-eosin sections.
1.4.4) Détermination du nombre de fibroblastes contenus dans les lattis : 1.4.4) Determination of the number of fibroblasts contained in the laths:
La coloration par l' hémalun-éosine permet de distinguer les cellules de la matrice qui les environne. En effet l'hémalun colore les noyaux des cellules en bleu noir, tandis que l'éosine colore en rouge plus ou moins intense les cytoplasmes et les structures extracellulaires (éosinophile) . Le contraste ainsi créé permet de distinguer chaque cellule sous un microscope équipé d'une caméra CDD reliée à un analyseur d'images semi-automatique. Les cellules se trouvant dans les champs définis par le grandissement microscopique sont alors comptées dans les lattis à 11 et 40 jours. Une dizaine de champs par coupe a été analysée. Deux groupes de cellules peuvent ainsi être différenciés selon leur situation géographique : les cellules se trouvant à l'intérieur du lattis (matrice de collagène) d'une part et les cellules se trouvant à la périphérie du lattis d'autre part. Pour calculer la cellularité des tissus conjonctifs équivalents chaque lattis est considéré cylindrique, la périphérie du lattis se définissant comme une couronne de lOμm d'épaisseur (équivalente au diamètre de deux assises cellulaires) représentant 2% du volume total du lattis.  Staining with hemalun-eosin makes it possible to distinguish the cells of the matrix which surrounds them. In fact, hemalun colors the nuclei of cells in black blue, while eosin colors more or less intense red cytoplasms and extracellular structures (eosinophils). The contrast thus created makes it possible to distinguish each cell under a microscope equipped with a CDD camera connected to a semi-automatic image analyzer. The cells found in the fields defined by the microscopic magnification are then counted in the lattices at 11 and 40 days. A dozen fields per cut were analyzed. Two groups of cells can thus be differentiated according to their geographic location: the cells located inside the lattice (collagen matrix) on the one hand and the cells located on the periphery of the lattice on the other hand. To calculate the cellularity of the equivalent connective tissues, each lattice is considered to be cylindrical, the periphery of the lattice being defined as a crown of 10 μm in thickness (equivalent to the diameter of two cellular foundations) representing 2% of the total volume of the lattice.
FEPfLLE PE (EKLE 26) 1.5. Immuno-détection indirecte de l'oc-actine des muscles lisses . FEPfLLE PE (EKLE 26) 1.5. Indirect immuno-detection of oc-actin in smooth muscles.
Les cellules fixées sont re-perméabilisées dans l'éthanol à 70% (20min) , puis réhydratées dans du PBS (10 min) . Les peroxydases endogènes sont bloquées avec une solution méthanol (30%), H2O2 (0,3%). Cette opération est suivie d'un rinçage au PBS (2min) puis d'un blocage des sites antigéniques non spécifiques par une solution PBS/lait écrémé 1% (Ih). Les cultures sont alors incubées avec un anticorps primaire (IgG de souris) dirigé contre l'α-actine humaine (1/30; 50min) puis rincées au PBS (3 x 10 min) . Les cellules sont alors incubées, dans le noir pendant 60 min avec un anticorps anti-IgG de souris biotinylé (1/200), rincées au PBS (3 x 10 min) puis incubées avec de la streptavidine couplée à la peroxydase (1/200 ). The fixed cells are re-permeabilized in 70% ethanol (20 min), then rehydrated in PBS (10 min). Endogenous peroxidases are blocked with a methanol solution (30%), H 2 O 2 (0.3%). This operation is followed by rinsing with PBS (2 min) and then blocking of the non-specific antigenic sites with a PBS / 1% skimmed milk solution (1 h). The cultures are then incubated with a primary antibody (mouse IgG) directed against human α-actin (1/30; 50 min) and then rinsed with PBS (3 x 10 min). The cells are then incubated in the dark for 60 min with a biotinylated anti-mouse IgG antibody (1/200), rinsed with PBS (3 x 10 min) and then incubated with streptavidin coupled to peroxidase (1/200 ).
Après rinçage (PBS 3x 10 min), la révélation de l'activité peroxydasique avec la 3-3' diaminobenzidine s'effectue dans un tampon Tris/HCl (10OmM, pH 7,2-7,4) contenant 0,1% de H2O2 (15 min, à l'obscurité). L'activité peroxydasique fait apparaître un matériel fibrillaire brun (correspondant aux microfilaments d'α-actine) dans le cytoplasme des cellules positives . After rinsing (PBS 3 × 10 min), the revelation of the peroxidase activity with 3-3 ′ diaminobenzidine is carried out in a Tris / HCl buffer (10OmM, pH 7.2-7.4) containing 0.1% of H 2 O 2 (15 min, in the dark). The peroxidase activity reveals a brown fibrillar material (corresponding to the microfilaments of α-actin) in the cytoplasm of positive cells.
Les produits utilisés proviennent de la firme DAKO. Les expériences contrôles concernant l' immunodétection de l'α- actine des muscles lisses ont été réalisées en omettant l'anticorps primaire et/ou en utilisant un anticorps secondaire d'une autre espèce animale que celle ayant permis l'obtention de l'anticorps primaire.  The products used come from the firm DAKO. The control experiments concerning the immunodetection of the α-actin of smooth muscles were carried out by omitting the primary antibody and / or by using a secondary antibody of another animal species than that which made it possible to obtain the antibody. primary.
2. Résultats et Discussion. 2. Results and Discussion.
FEUILLE BE im?UC:∑lEÏÏ (itèSLE 2δ) 2.1. Prolifération des fibroblastes gingivaux cultivés sur film de HE800. SHEET BE im? UC: ∑lEÏÏ (itèSLE 2δ) 2.1. Proliferation of gingival fibroblasts cultured on HE800 film.
Les surfaces de culture ont été traitées par le HE800, afin de former un film polysaccharidique au fond des boîtes. On observe, lors premiers jours de culture (Jours 2 et 4) que le nombre de cellules ensemencées dans les boîtes enduites par le HE800 est largement inférieur au nombre de celles des boîtes témoins (Tableau I). Au dernier jour de l'expérimentation, par contre on remarque une inversion de ces résultats (Tableaux I et II) . Les courbes présentées montrent que les cellules cultivées sur film d'HEδOO observent un temps de latence avant l'entrée en phase exponentielle de croissance, plus long que celui exprimé par les cellules cultivées sur plastique. D'autre part, alors que le nombre de cellules des cultures témoins atteint un plateau les jours les plus tardifs de l'expérience (cf. Tableau III), les cultures sur film HE800 continuent à proliférer. Les observations effectuées lors de la culture ou après fixation des cellules permettent d' avancer des hypothèses quant aux comportements cellulaires exprimés dans les différentes conditions de culture :  The culture surfaces were treated with HE800, in order to form a polysaccharide film at the bottom of the dishes. It is observed, during the first days of culture (Days 2 and 4) that the number of cells seeded in the boxes coated with HE800 is much lower than the number of those in the control boxes (Table I). On the last day of the experiment, on the other hand, we notice an inversion of these results (Tables I and II). The curves presented show that the cells cultured on HEδOO film observe a lag time before entering the exponential growth phase, longer than that expressed by the cells cultured on plastic. On the other hand, while the number of cells in the control cultures reached a plateau on the later days of the experiment (cf. Table III), the cultures on HE800 film continue to proliferate. The observations made during the culture or after fixing of the cells make it possible to advance hypotheses as to the cellular behaviors expressed in the different culture conditions:
Les cultures sur film d'HEδOO se caractérisent lors des premiers jours de cultures par la présence de nombreuses cellules qui n'adhèrent pas au support. Cette non-adhésion peut expliquer le retard à la prolifération observée lors des comptages cellulaires dans ces cultures. The cultures on HEδOO film are characterized during the first days of cultures by the presence of numerous cells which do not adhere to the support. This non-adhesion may explain the delay in proliferation observed during cell counts in these cultures.
Les cellules témoins sont réparties de manière uniforme dans la boîte, sans orientation particulière, alors que les cellules ensemencées sur film HE800 s'organisent en cordons au centre de la boîte. Ces résultats montrent l'incidence d'HEδOO sur l'adhésion cellulaire. En effet les regroupements cellulaires qui sont observés habituellement, dans les  The control cells are distributed uniformly in the box, without any particular orientation, while the cells sown on HE800 film are organized in cords in the center of the box. These results show the incidence of HEδOO on cell adhesion. Indeed, the cellular groupings which are usually observed in
FEOILiE K KEϋSPUC∑jV-SKT fRS€LE 26] cultures gingivales, n'ont pas d'orientation spécifique. Après les 2 premiers jours de culture ces cordons cellulaires commencent à former une structure centrale circulaire, allant en se densifiant exclusivement vers le centre (prolifération centripète) . On peut aussi observer de nombreuses cellules à la périphérie de la boîte mais sans orientation particulière. Certaines cellules peuvent être présentes dans les zones séparant les regroupements cellulaires, elles sont isolées et semblent beaucoup plus étirées en longueur que les autres cellules des boîtes HE800 ou même témoin. FEOILiE K KEϋSPUC∑jV-SKT fRS € LE 26] gingival cultures, have no specific orientation. After the first 2 days of culture, these cellular cords begin to form a circular central structure, densifying exclusively towards the center (centripetal proliferation). We can also observe many cells at the periphery of the box but without any particular orientation. Some cells may be present in the areas separating the cell groups, they are isolated and seem to be much more stretched in length than the other cells in the HE800 or even control boxes.
Le marquage immuno-cytochimique concernant l'α-actine des muscles lisses montre :  The immuno-cytochemical labeling concerning smooth muscle α-actin shows:
- Dans les témoins, de nombreuses cellules positives côtoient des cellules n'exprimant pas ces microfilaments.  - In the controls, numerous positive cells rub shoulders with cells not expressing these microfilaments.
-Dans les cultures en présence d'HE800, les cellules présentes dans les formations circulaires centrales n'expriment pas l'α-actine des muscles lisses par contre, des cellules exprimant cette isoforme de l'actine peuvent être trouvées en périphérie de la boîte. -In cultures in the presence of HE800, the cells present in the central circular formations do not express the α-actin of smooth muscles on the other hand, cells expressing this actin isoform can be found on the periphery of the box .
Ces résultats sont le reflet d'une sélection des souches fibroblastiques, en effet certaines cellules peuvent ne pas exprimer naturellement les récepteurs membranaires nécessaires à leur adhésion sur le film d'HEδOO. Parmi les sous-populations fibroblastiques non adhérentes, on retrouve celles qui expriment l'α-actine des muscles lisses c'est-à- dire les myofibroblastes . Dans les expériences contrôles (omission de l'anticorps primaire ou utilisation d'un anticorps secondaire inapproprié) aucune positivité n'a été observée .  These results reflect a selection of fibroblast strains, indeed certain cells may not naturally express the membrane receptors necessary for their adhesion to the HEδOO film. Among the nonadherent fibroblastic subpopulations, there are those which express the α-actin of smooth muscles, that is to say the myofibroblasts. In the control experiments (omission of the primary antibody or use of an inappropriate secondary antibody) no positivity was observed.
(RÈGLE 26) Tableau I : Variation du nombre de cellules par puits au cours de la culture (RULE 26) Table I: Variation in the number of cells per well during the culture
Tableau II: Pourcentages de prolifération Tableau III : Temps de doublement (heures) Table II: Percentages of proliferation Table III: Doubling time (hours)
Td Td Td 0-2 2-4 4-7Td Td Td 0-2 2-4 4-7
Témoin 84, 68 29 58 333.2 HE800 9522, 64 25 50 90 Witness 84, 68 29 58 333.2 HE800 9522, 64 25 50 90
2.2. Structuration du collagène de type I en présence 2.2. Structuring of type I collagen in the presence
d'HE800. of HE800.
2.2.1) Premières constatations 2.2.1) First observations
Afin de réaliser les films comprenant à la fois du collagène et de l' exopolysaccharide HE800, des solutions d'HE800 et de collagène I sont préalablement mélangées avant dépôt sur les boîtes de culture. De manière surprenante, il a été constaté que l'addition de l' exopolysacharide bactérien à la solution collagénique provoque l'apparition d'un agglomérat dense de couleur blanche. Cet agglomérat a pu être étalé sur une lame histologique puis colorée au rouge Sirius, colorant spécifique des collagènes. Ces lames histologiques montrent qu'effectivement le matériel étalé sur la lame est coloré par le rouge Sirius. De plus l'observation d'un matériel déviant dans différentes directions la lumière polarisée montre bien la présence de collagène sous sa forme fibrillaire.  In order to make the films comprising both collagen and exopolysaccharide HE800, solutions of HE800 and collagen I are previously mixed before depositing on the culture dishes. Surprisingly, it has been found that the addition of the bacterial exopolysacharide to the collagen solution causes the appearance of a dense white agglomerate. This agglomerate could be spread on a histological slide and then stained with Sirius red, a specific dye for collagens. These histological slides show that indeed the material spread on the slide is colored by Sirius red. In addition, the observation of material deviating in different directions with polarized light clearly shows the presence of collagen in its fibrillar form.
2.2.2) Organisation de filins composites collagène/HE800 : 2.2.2) Organization of collagen / HE800 composite cables:
Les différents films déposés sont composés de :  The various films deposited are composed of:
PHfmE D2 nz^?us:::z::i (PÎGLΞ - (1) collagène (40μg) PHfmE D2 nz ^? Us ::: z :: i (PÎGLΞ - (1) collagen (40μg)
- (2) collagène (40μg) + HE800 (50μg)  - (2) collagen (40μg) + HE800 (50μg)
- (3) collagène (40μg) + HE800 (200μg)  - (3) collagen (40μg) + HE800 (200μg)
L'observation au microscope du fond des boîtes montre pour les films (3) l'apparition d'un réseau dense composé de longs filaments. Les films (2) comportent quelques fibres beaucoup plus courtes, tandis que les films (1) n'en comportent pratiquement pas. Les 3 films se colorent au rouge Sirius, mais seul le film (3) montre un réseau fibrillaire déviant la lumière polarisée. Observation under the microscope of the bottom of the boxes shows for the films (3) the appearance of a dense network composed of long filaments. The films (2) have some much shorter fibers, while the films (1) have practically none. The 3 films are colored Sirius red, but only the film (3) shows a fibrillar network deflecting polarized light.
Ces résultats indiquent que HE800 promeut la formation de fibres collagéniques, mais aussi permet une meilleure résistance de la trame collagénique devant des facteurs physiques tels que la température et les contraintes mécaniques .  These results indicate that HE800 promotes the formation of collagen fibers, but also allows better resistance of the collagen frame in the face of physical factors such as temperature and mechanical stresses.
2.3. Tissu conjonctif non minéralisé: Microscopie photonique et électronique 2.3. Non-mineralized connective tissue: Light and electron microscopy
Les cellules sont cultivées dans une matrice collagénique (modèle de culture tridimensionnelle) afin de mimer au mieux les interactions cellules/matrice observées dans le tissu conjonctif. Ces lattis ou tissus conjonctifs équivalents sont composés de collagène I seul (Témoins) ou de collagène I et d'HE800 en différentes proportions (quantité d'EPS = 20, 10 et 5% par rapport à la quantité de collagène contenu dans le lattis, c'est-à-dire respectivement 300, 150 et 75μg) .  The cells are cultured in a collagen matrix (three-dimensional culture model) in order to best mimic the cell / matrix interactions observed in the connective tissue. These lattices or equivalent connective tissues are composed of collagen I alone (Witnesses) or collagen I and HE800 in different proportions (amount of EPS = 20, 10 and 5% relative to the amount of collagen contained in the lattice, i.e. 300, 150 and 75μg respectively).
2.3.1) Rétraction des lattis : 2.3.1) Lattice retraction:
Le premier paramètre étudié est la vitesse de rétraction des lattis : Les courbes de rétraction des lattis témoins et des lattis comprenant de 1ΗE800 sont similaires. Malgré ces similarités, on remarque que les lattis HE800 ont une vitesse  The first parameter studied is the lattice retraction speed: The retraction curves for control laths and lattices comprising 1ΗE800 are similar. Despite these similarities, we notice that the HE800 laths have a speed
^kI!.1 de rétraction plus lente que les lattis témoins lors des jours précoces de culture. Après le lleme jour la rétraction des lattis est presque totale. ^ kI !. 1 slower retraction than control lattices during the early days of culture. After the 11th day the lattice retraction is almost complete.
2.3.2) Nombre de fibroblastes contenus dans les lattis : 2.3.2) Number of fibroblasts contained in the laths:
Le nombre de cellules présentes dans chaque lattis varie, aux deux temps de culture entre 180 000 et 250 000 cellules. Le nombre de cellules à la périphérie représente 2 à 12% du nombre total de cellules. Ces données sont compatibles avec ce qui a été décrit dans la littérature pour ce modèle de culture. Les résultats des tableaux IV, V et VI montrent le nombre de cellules par unité volumétrique (mm3) présentes dans la totalité du lattis et dans ses différentes régions . Les densités cellulaires volumétriques totales des lattis après 11 et 40 jours sont comprises entre 3200 et 5900 cellules/mm3 (cf. tableau IV). Ces valeurs sont comparables à celles retrouvées dans un tissu conjonctif humain normal comme cela à été décrit précédemment (Miller et al., Exp Dermatol. 2003 Aug; 12 (4) : 403-11) . La cellularité physiologique des lattis témoins et des lattis comprenant du HE800 atteste donc de la validité du modèle de culture utilisé et de la compatibilité d'HE800 avec ce modèle physiologique . The number of cells present in each lattice varies, at the two culture times, between 180,000 and 250,000 cells. The number of cells at the periphery represents 2 to 12% of the total number of cells. These data are compatible with what has been described in the literature for this culture model. The results of Tables IV, V and VI show the number of cells per volumetric unit (mm 3 ) present in the entire lattice and in its different regions. The total volumetric cell densities of the laths after 11 and 40 days are between 3200 and 5900 cells / mm 3 (see Table IV). These values are comparable to those found in normal human connective tissue as previously described (Miller et al., Exp Dermatol. 2003 Aug; 12 (4): 403-11). The physiological cellularity of the control lattices and of the lattices comprising HE800 therefore attests to the validity of the culture model used and to the compatibility of HE800 with this physiological model.
La densité cellulaire totale (cf. tableau IV) des lattis témoins ne varie pas quelque soit le temps de culture. Au lleme jour de culture, la densité cellulaire totale des lattis HE800 est inférieure de 25 à 40% à celles des lattis témoin. Au 40eme jour de culture les densités cellulaires des lattis témoins et des lattis HE800 sont équivalentes. Les variations de densités cellulaires observées à l'intérieur des lattis (cf. tableau V) reproduisent exactement celles de la totalité du lattis. L'organisation topologique des cellules de la The total cell density (see Table IV) of the control lattices does not vary regardless of the culture time. On the 11th day of culture, the total cell density of the HE800 lattices is 25 to 40% lower than those of the control lattices. On the 40 th day of culture, the cell densities of the control lattices and of the HE800 lattices are equivalent. The variations in cell densities observed inside the laths (cf. Table V) reproduce exactly those of the entire lath. The topological organization of the cells of the
LE DI k(.lκU) LiΛ>%,~. tau -.SU S I-US. Sm Â.\ couronne périphérique (tableau VI) diverge par contre totalement de celles des tableaux IV et V : LE DI k ( .lκU) LiΛ>%, ~. tau -.SU S I-US. Sm Â. \ However, the peripheral crown (Table VI) diverges completely from that of Tables IV and V:
- A 11 jours de culture les densités cellulaires de la couronne périphérique sont 4 fois supérieures à celles de l'intérieur du lattis et ne présentent que peu de variation entre les tissus conjonctifs équivalents témoins et les tissus conjonctifs équivalents comprenant HE800 (tableau VI) . - At 11 days of culture, the cell densities of the peripheral crown are 4 times greater than those of the interior of the lath and show little variation between the equivalent connective connective tissues and the equivalent connective tissues comprising HE800 (Table VI).
- A 40 jours de culture, une forte diminution de la cellularité périphérique est observée. Si cette diminution n'est que de 40% pour les lattis témoins, elle atteint 100 à 250% pour les lattis contenant du HE800. La densité cellulaire des lattis témoin reste 3 fois plus élevée à la périphérie qu'à l'intérieur (tableau IV et VI) par contre ces densités sont comparables pour les lattis HE800. - At 40 days of culture, a sharp decrease in peripheral cellularity is observed. If this reduction is only 40% for the control laths, it reaches 100 to 250% for the laths containing HE800. The cell density of the control lattices remains 3 times higher at the periphery than inside (table IV and VI) on the other hand these densities are comparable for the HE800 laths.
La cellularité globale des lattis HE800 est plus faible que celle des lattis témoins aux jours précoces de culture puis deviennent équivalentes au jours tardifs de culture. Ces variations qui se répercutent sur les densités cellulaires des régions internes peuvent s'expliquer par une stimulation de la prolifération cellulaire, ou une migration massive de cellules périphériques vers l'intérieur.  The overall cellularity of the HE800 lattices is lower than that of the control lattices in the early days of culture and then becomes equivalent to the late days of culture. These variations which have repercussions on the cell densities of the internal regions can be explained by a stimulation of cell proliferation, or a massive migration of peripheral cells towards the interior.
En effet à la périphérie des lattis, le nombre de cellules diminue au cours du temps de culture, cette diminution est particulièrement accentuée dans les lattis comprenant du HE800 (diminution par 2 à 3,5 fois du nombre de cellules). Cette diminution peut s'expliquer par, une perte d'adhésion des cellules périphérique qui se détache de la matrice extracellulaire et/ou une migration massive de ces cellules à l'intérieur. Cela explique les gains globaux de cellularité, au cours du temps, dans les lattis contenant HE800.  Indeed, at the periphery of the lattices, the number of cells decreases during the culture time, this reduction is particularly accentuated in the lattices comprising HE800 (decrease by 2 to 3.5 times in the number of cells). This decrease can be explained by a loss of adhesion of the peripheral cells which detaches from the extracellular matrix and / or a massive migration of these cells inside. This explains the overall gains in cellularity, over time, in lattices containing HE800.
LUI LLE Ue, umuti^L^auikli l (.CcCa LE £Ό) Tableau IV : Densité cellulaire dans la totalité du lattis nombre de cellule par mm3 (entre parenthèse : diamètre des lattis en mm) . HIM LLE EU, umuti ^ L ^ auikli l (.CcCa LE £ Ό) Table IV: Cell density in the entire lattice number of cells per mm 3 (in brackets: lattice diameter in mm).
Tableau V : Densité cellulaire à l'intérieur du lattis nombre de cellule par mm3. Table V: Cell density inside the lattice cell number per mm 3 .
Tableau VI : Densité cellulaire à la périphérie du lattis : Table VI: Cell density at the periphery of the lath:
nombre de cellule par mm3 number of cells per mm 3
Conclusion : HE800 favorise la prolifération des fibroblastes dermiques dans la matrice extracellulaire et/ou favorise leur mobilisation c'est-à-dire la sélection, la migration et la pénétration massive des cellules périphériques . 2.4.3) Etat de la matrice collagénique : Conclusion: HE800 promotes the proliferation of dermal fibroblasts in the extracellular matrix and / or promotes their mobilization, that is to say the selection, migration and massive penetration of peripheral cells. 2.4.3) State of the collagen matrix:
2.4.3.1) Microscopie photonique 2.4.3.1) Light microscopy
La coloration au rouge Sirius (coloration de Junquera) permet de colorer spécifiquement les collagènes, dans la peau par exemple ses collagènes apparaissent sous la forme d'une structure filamenteuse lâche, colorée en rouge.  The Sirius red coloring (Junquera coloring) makes it possible to specifically color the collagens, in the skin for example its collagens appear in the form of a loose filamentary structure, colored in red.
Les colorations au rouge Sirius des coupes histologiques après observation en lumière transmise et lumière polarisée montrent que l'addition d'HE800 lors de la formation du lattis permet la formation d'une matrice beaucoup plus dense et à des temps beaucoup plus courts que dans les lattis témoins . The Sirius red staining of the histological sections after observation in transmitted light and polarized light shows that the addition of HE800 during the formation of the lattice allows the formation of a much denser matrix and at much shorter times than in the lattis witnesses.
Pour exemple : la densité de la matrice collagénique témoin après 40 jours de culture est équivalente à celle observée dans les matrices collagéniques constituées en présence de HE800 à 11 jours de culture. Cet effet sur la densité est beaucoup plus important aux doses les plus faibles (10%, 5%) .  For example: the density of the control collagen matrix after 40 days of culture is equivalent to that observed in the collagen matrices formed in the presence of HE800 at 11 days of culture. This effect on density is much greater at the lowest doses (10%, 5%).
2.4.3.2) Microscopie électronique 2.4.3.2) Electron microscopy
La microscopie électronique a été pratiquée sur des tissus conjonctifs équivalents cultivés pendant 11 jours. Un bon état ultrastructural des cellules a été observé, que ce soit dans les témoins ou que ce soit dans les lattis formés en présence des différentes concentrations d'HEδOO.  Electron microscopy was performed on equivalent connective tissue cultured for 11 days. A good ultrastructural state of the cells was observed, either in the controls or in the laths formed in the presence of the different concentrations of HEδOO.
Aucunes fibres collagéniques n'a pu être observées dans les lattis témoins, les lattis comprenant 20% de HE800 permettent d'observer quelques éléments fibrillaires pris dans un gel constitué de l' exopolysaccharide. Les lattis comprenant 10% et 5 % d' exopolysaccharides sont très différents, en effets de nombreuses fibres collagéniques sont présentes, elles sont réparties dans tout le lattis et présentent, pour certaines, No collagen fibers could be observed in the control laths, the laths comprising 20% of HE800 make it possible to observe some fibrillar elements taken in a gel consisting of the exopolysaccharide. The laths comprising 10% and 5% of exopolysaccharides are very different, in effect many collagen fibers are present, they are distributed throughout the lattice and present, for some,
FFIIIISF PT PFMPï/srr fil» rFB"a* (RÈGLE 2S) une striation périodique. Ces fibres ou fibrilles collagéniques sont piégées dans le gel constitué par 1ΗE800. FFIIIISF PT PFMPï / srr wire " rFB" a * (RULE 2S) periodic striation. These collagen fibers or fibrils are trapped in the gel constituted by 1ΗE800.
Conclusion : I/HE800 accélère la fibrillation collagénique et favorise la constitution d'une matrice extracellulaire. Conclusion: I / HE800 accelerates collagen fibrillation and promotes the constitution of an extracellular matrix.

Claims

REVENDICATIONS
1) Utilisation d'un polysaccharide ou d'un sel de ce polysaccharide de masse molaire moyenne en poids comprise entre 500 000 et 2 000 000 g/mol, préférentiellement comprise entre 700 000 et 900 000 g/mol, caractérisé par une séquence osidique répétitive linéaire comprenant les 4 résidus osidiques suivants : 1) Use of a polysaccharide or a salt of this polysaccharide with a weight-average molar mass of between 500,000 and 2,000,000 g / mol, preferably between 700,000 and 900,000 g / mol, characterized by an osidic sequence linear repetitive comprising the following 4 oid residues:
[(-3)-D GlcNac β (1-4) D GIcA β (1-4) D GIcA β (1-4) D GalNac α (1-)] pour la fabrication d'une composition, d'un médicament ou d'un dispositif médical ayant une activité régénératrice ou protectrice sur les tissus conjonctifs non minéralisés du parodonte.  [(-3) -D GlcNac β (1-4) D GIcA β (1-4) D GIcA β (1-4) D GalNac α (1-)] for the manufacture of a composition, of a medicament or of a medical device having a regenerative or protective activity on the non-mineralized connective tissues of the periodontium.
2) Utilisation selon la revendication 1 caractérisée en ce que ledit polysaccharide est un polysaccharide excrété par l'espèce Vibrio diabolicus ou un dérivé de ce polysaccharide .  2) Use according to claim 1 characterized in that said polysaccharide is a polysaccharide excreted by the species Vibrio diabolicus or a derivative of this polysaccharide.
3) Utilisation du polysaccharide tel que défini dans la revendication 1 ou 2 pour la fabrication d'une composition, d'un médicament ou d'un dispositif médical pour traiter une pathologie buccale dudit tissu conjonctif.  3) Use of the polysaccharide as defined in claim 1 or 2 for the manufacture of a composition, a medicament or a medical device for treating an oral pathology of said connective tissue.
4) Utilisation selon la revendication 3 caractérisée en ce que la pathologie buccale est liée à un état inflammatoire ou à un état traumatique.  4) Use according to claim 3 characterized in that the oral pathology is linked to an inflammatory state or to a traumatic state.
5) Utilisation selon la revendication 3 ou 4 caractérisée en ce que la pathologie buccale est choisie dans le groupe consistant en la parodontite, la gingivite, la fibrose gingivale, la récession gingivale, l'aphte, l'aphtose buccale récidivante, les maladies aphteuses et les maladies bulleuses .  5) Use according to claim 3 or 4 characterized in that the oral pathology is chosen from the group consisting of periodontitis, gingivitis, gingival fibrosis, gingival recession, canker sore, recurrent oral aphthosis, foot and mouth disease and bullous diseases.
6) Utilisation selon l'une quelconque des revendications 1 à 5, caractérisée en ce que ladite composition ou ledit médicament est pour une administration topique au niveau du parodonte. 6) Use according to any one of claims 1 to 5, characterized in that said composition or said drug is for topical administration in the periodontium.
7) Utilisation selon l'une quelconque des revendications 1 à 6, caractérisée en ce que ladite composition ou ledit médicament est une composition topique à usage buccal sous la forme d'un gel, d'une solution, d'une émulsion ou d'un spray.  7) Use according to any one of claims 1 to 6, characterized in that said composition or said drug is a topical composition for oral use in the form of a gel, a solution, an emulsion or a spray.
8) Utilisation selon l'une quelconque des revendications 1 à 6, caractérisée en ce que ladite composition ou ledit médicament est une pâte dentifrice, un bain de bouche, un spray ou une colle pour denture.  8) Use according to any one of claims 1 to 6, characterized in that said composition or said drug is a toothpaste, a mouthwash, a spray or a glue for toothing.
9) Utilisation selon l'une quelconque des revendications 1 à 6, caractérisée en ce que ledit dispositif médical est un pansement buccal.  9) Use according to any one of claims 1 to 6, characterized in that said medical device is an oral dressing.
10) Utilisation selon l'une quelconque des revendications 1 à 6, caractérisée en ce que ledit dispositif médical comprend une matrice de collagène.  10) Use according to any one of claims 1 to 6, characterized in that said medical device comprises a collagen matrix.
11) Utilisation selon la revendication 10, caractérisée en ce que ladite matrice de collagène comprend un collagène choisi dans le groupe constitué des collagènes de type I, III, V ou d'un mélange de ceux-ci.  11) Use according to claim 10, characterized in that said collagen matrix comprises a collagen selected from the group consisting of type I, III, V collagens or a mixture of these.
12) Utilisation selon l'une quelconque des revendications 10 ou 11, caractérisée en ce que ladite matrice de collagène comprend un collagène de type I.  12) Use according to any one of claims 10 or 11, characterized in that said collagen matrix comprises a type I collagen.
13) Utilisation selon l'une quelconque des revendications 10 à 12, caractérisée en ce que ladite matrice comprend en outre un ou plusieurs facteurs de croissance qui favorisent la colonisation de la matrice par les fibroblastes gingivaux et la reconstitution du tissu conjonctif.  13) Use according to any one of claims 10 to 12, characterized in that said matrix further comprises one or more growth factors which promote colonization of the matrix by gingival fibroblasts and the reconstitution of connective tissue.
14) Utilisation selon l'une quelconque des revendications 10 à 13, caractérisée en ce que ladite matrice comprend un ou plusieurs facteurs de croissance choisis dans le groupe constitué par TGF-beta, PDGF, FGF, VEGF, BMPs, CTGF. 14) Use according to any one of claims 10 to 13, characterized in that said matrix comprises one or more growth factors chosen from the group consisting of TGF-beta, PDGF, FGF, VEGF, BMPs, CTGF.
15) Utilisation selon l'une quelconque des revendications 10 à 14, caractérisée en ce que ladite matrice comprend en outre des fibroblastes gingivaux.  15) Use according to any one of claims 10 to 14, characterized in that said matrix further comprises gingival fibroblasts.
16) Utilisation selon l'une quelconque des revendications 10 à 15, caractérisée en ce que ledit dispositif médical est un dispositif résorbable ou un implant .  16) Use according to any one of claims 10 to 15, characterized in that said medical device is an absorbable device or an implant.
17) Utilisation selon l'une quelconque des revendications 10 à 16, caractérisée en ce que ledit dispositif médical est un substitut de gencive.  17) Use according to any one of claims 10 to 16, characterized in that said medical device is a gum substitute.
EP06841874A 2005-12-07 2006-12-06 Use of a polysaccharide which is excreted by the vibrio dlabolicus species for the regeneration and protection of the periodontium Withdrawn EP1959970A1 (en)

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