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
  • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
  • A61L27/24—Collagen
• • 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
  • A61L27/18—Macromolecular materials obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
• • 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
  • A61L2430/00—Materials or treatment for tissue regeneration
  • A61L2430/40—Preparation and treatment of biological tissue for implantation, e.g. decellularisation, cross-linking

Definitions

• the present invention relates to a process for the preparation of dense fibrillar matrices of collagen, the matrices thus obtained and their uses for tissue repair.
• the first biomaterials based on collagen led to industrial applications concerned skin substitutes.
• the products marketed have been proposed as implants for burn victims or dressings for chronic wounds and ulcers.
• the first dermal substitute sold as Integra ® is an acellular sponge of collagen and glutaraldehyde-bonded proteoglycans. Associated with a sheet of keratinocytes, marketed under the name of Epicel ®, the equivalent dermis becomes a skin substitute.
• the substitutes include polymers of synthetic origin or integrate cells and their success will depend on cell / matrix interrelations.
• the only systems where fibroblasts are found with contacts in 3D are the collagen hydrogels developed in the United States. These hydrogels associated with sheets of keratinocytes then become cutaneous repair materials (Apligraf ®). The major disadvantage of these biomaterials lies in their poor mechanical properties.
• collagen-based materials are derived from two manufacturing processes, leading to either sponges or hydrogels. These basic materials are then treated to enhance the quality of commercial products by association with chemical molecules (consolidation), cells (fibroblasts) or protective films (silicone).
• Collagen sponges obtained by lyophilization of acid-soluble collagen solutions are porous three-dimensional systems. The size of the pores, from 15 to 150 ⁇ in diameter, varies according to the freezing criteria preceding lyophilization. The sponge structure allows seeded fibroblasts to proliferate by fully colonizing the substrate and then secreting fibrillar collagen.
• the applications, in cell or gene therapy, of these systems are related to their biocompatible and biodegradable support properties.
• the collagen hydrogels are obtained by precipitating the collagen monomers into fibrils in an acid-soluble solution.
• the weakly concentrated fibrillar gel ⁇ 1 mg / ml
• a significant contraction of the gel is then observed in the following days ( ⁇ 3% of initial surface after 14 days of culture) and results in what is called an equivalent dermis.
• This reconstituted tissue becomes an equivalent skin when it is secondarily seeded by cells of the epidemic, the keratinocytes, which proliferate and cover the structure.
• hydrogels have been the subject of numerous studies in terms of remodeling (synthesis of collagen and metalloproteinases) and cell proliferation. While hydrogels are interesting cell culture models, they have proven insufficient as substrates for tissue repair. The mechanical properties of the hydrogels were improved by increasing their concentration by compression (see ⁇ 11.4), resulting in easily dehydrated and friable films, their thickness of the order of 40 ⁇ is too fine for many applications.
• the inventors have developed techniques for working with collagen at high concentrations, controlling the establishment of a concentration-related supramolecular order and stabilizing during a sol / gel transition to result in dense fibrillar matrices. .
• the inventors have also shown that matrices containing 40 mg / ml of collagen exhibit particularly advantageous properties in terms of their cellular and mechanical responses. They are colonized in vitro by fibroblasts seeded on their surface following the hydrolysis of collagen via metalloproteases.
• the application WO 2010/004182 in the name of the inventors relates to the mineralization of these dense fibrillar matrices of collagen in a higher concentration range (from 80 mg / ml) for an application in the field of bone substitutes.
• Evaporation Process The initial acidic solutions of collagen, whose initial concentration is less than 5 mg / mL, are placed in a crystallizer (or other open wide container) under a hood in a sterile medium. The solution is allowed to evaporate to the desired concentration (Helary et al., Biomaterials, 2005).
• Method - Acidic solutions of collagen with an initial concentration of less than 5 mg / mL are placed in a dialysis coil.
• the porosity of the membrane is set so that the solvent, the salts and the molecules whose molecular weight is smaller than the pore size diffuse through the wall but not the collagen.
• the assembly is immersed in a solution of polymer (generally polyethylene glycol or PEG) whose concentration is equal to or slightly greater than that which one wants to achieve (Gobeaux et al., J. Mol Biol 2008) .
• polymer generally polyethylene glycol or PEG
• the collagen fibrils are not homogeneous in size.
• the concentrated solution of recovered collagen is not macroscopically homogeneous (see FIG. 1B), so the mechanical properties of the final material are not constant for the entire material.
• Method - This method proposes the compression of hydrated collagen gels of collagen to obtain dense matrices.
• the fibrilar networks remain isotropic but the mechanical properties are much higher than those of the initial gel.
• the viability of cells inserted into such matrices, in the form of wound sheets, is verified (Brown et al., 2005).
• the inventors have thus developed a method making it possible to obtain dense fibrous matrices of collagen obtained at concentrations 5 to 60 times higher than conventional hydrogels, or even more and containing no additives intended to reinforce the mechanical properties but only of Pure collagen reconstituted in the form of fibrils and dense fibril networks, which avoids the phenomena of inflammation, toxicity and rejection.
• the matrices obtained are colonizable by connective tissue cells and their mechanical properties are close to those of biological tissues.
• the subject of the present invention is a process for the preparation of a homogeneous material based on coUagene starting by the concentration of a solution of acid-soluble coUagene, said process comprising:
• the permeable element is chosen with a pore size of molecular weight (MW) lower than that of coUagene on the one hand and the outer polymer on the other hand. Keeping the coUagene solution separated from the polymer solution by the permeable element in contact allows the solvent to diffuse internal coUagene solution to the outer polymer solution. This makes it possible to obtain the formation of a more concentrated solution of homogeneous coUagene inside the permeable element or on the surface of the permeable element by this selective transfer of solvent.
• the osmotic pressure of the polymer solution is greater than that of the coUagene solution.
• Continuous injection by controlled means may be carried out by any system known to those skilled in the art capable of allowing this type of injection, in particular an electric syringe pump or a pump.
• the injection speed is adapted so that the pressure force on both sides of the membrane is identical; and the flow is adapted depending on the polymer and its viscosity so that the skilled person finds the speed avoiding the retraction of the membrane or swelling until piercing.
• the speed depends on the type of polymer used and its molecular weight, which is greater than the pores of the membrane. Adaptation of speed is a matter of general knowledge of man job. By way of example with PEG with a molecular weight of 35 KDa, a speed of between 1 and 15 ⁇ / min can be used.
• the injection can be interrupted to reach the equilibrium (that is to say when the concentration of collagen is then homogeneous everywhere in the mold) faster therefore to decrease the concentration gradient faster and resumed in order to elaborate a multi system -controlled concentration and modular organization. It is also possible to interrupt the injection without waiting for the equilibrium which will allow to have a continuous gradient in concentration.
• FIG. 1 An example of a device that can be used to implement the method of the invention is illustrated in FIG.
• the permeable element is a dialysis cell and said method comprises the following steps:
• the term pure collagen solution is understood to mean a solution containing no compound added solely for the purpose of reinforcing the properties final mechanical properties of the material; it is especially free of crosslinking agents such as aldehydes and synthetic reinforcing polymers such as polyesters or natural ones such as chitosan.
• additives such as molecules of interest such as agents for modifying its porosity or coUagen surface charges can be added to the material.
• the coUagene used in the present invention may be of natural or recombinant origin. It may come in particular from the skin or tendons from which it is extracted by the acidic or enzymatic route according to techniques known to those skilled in the art.
• the dialysis cell at least one of whose ends is closed by a dialysis membrane, may also be a mold consisting entirely of a dialysis membrane.
• the coUagene matrix will be made on the whole surface of the mold not only on the membrane placed at the end.
• the product can therefore be formulated according to the thickness and the concentration in the form of film, flexible membrane, tube or custom mold.
• the acid solutions of coUagene used in step a) are prepared by techniques known to those skilled in the art from natural or recombinant coUagene.
• the aqueous solvent advantageously acetic acid at a concentration between 17 and 500 mM can contain various additives such as inorganic salts, other types of coUagene or glycosaminoglycans such as sulfated heparin.
• the concentration of coUagene of the initial solution used in step a) is between 0.01 and 5 mg / ml, advantageously between 0.5 and 3 mg / ml.
• the polymer used may be any acid-soluble polymer whose molecular weight is greater than the pore size of the permeable element. It is in particular chosen from the group comprising Dextran® and polyethylene glycol (PEG).
• the polymer is polyethylene glycol whose molecular weight is greater than that of coUagene, thus greater than 3000 Da.
• the concentration of the polymer solution can vary according to the case and its adjustment is within the reach of the skilled person. If the volume of polymer solution is very large then the volume of solvent from the coUagene solution is negligible and negligibly dilutes the polymer solution. In this case, the initial polymer concentration will be equal to the final coUagene concentration. If the volume of polymer solution is not large and the initial polymer concentration equal to the final collagen concentration, so as not to slow down the diffusion the polymer solution will be changed regularly. If the volume of polymer solution is not large and the initial polymer concentration is greater than the final collagen concentration, then the initial polymer concentration is calculated such that after dilution with the volume of solvent from the collagen solution, it is equal to the desired final concentration of collagen.
• the method may further comprise, before or after step d), a step of forming fibrils in the pure collagen material.
• the formation of these fibrils makes it possible to mimic the fibrillar structure of the collagen of the biological tissues.
• the formation of the fibrils is carried out by any technique known to those skilled in the art, in particular by neutralization of the solution based on homogeneous pure collagen by a gas phase (basic) or liquid (basic or neutral). It can be done either in situ by replacing the polymer with the gaseous or liquid phase, which enables a "one-pot" preparation of the fibrillar collagen, or by immersion of the collagen material in a gaseous or liquid phase.
• the method of the invention can also be implemented to manufacture multi-layer products by interrupting the injection.
• the injection can be interrupted to reach equilibrium faster and resumed to make different layers not continuous in concentration. It is also possible to stop the injection and not to wait for equilibrium to have different continuous layers in concentration.
• the invention also relates to a homogeneous material based on pure collagen obtainable by a method as defined above.
• the product obtained according to the invention is easily handled without tears and suturable without additional crosslinking. It is homogeneous and does not show any variation in concentrations.
• materials are obtained having a concentration of about 5 to about 1000 mg / mL, especially 5, 10, 20, 30, 40 and 250 mg / mL.
• the elastic modulus is approximately 974 ⁇ 239 Pa and the viscous modulus approximately 145 ⁇ 40 Pa.
• the elastic modulus is approximately 2809 ⁇ 336 Pa and the viscous modulus approximately 263 ⁇ 48 Pa.
• the elastic modulus is approximately 9254 ⁇ 1032 Pa and the viscous modulus of about 786 ⁇ 46 Pa.
• the values of the elastic and viscous modules are measured at 1 Hz and 2% deformation.
• the material according to the invention can therefore be used as a tissue substitute, especially as a wall reinforcement, for the manufacture of prostheses, as a soft tissue substitute or as a filling material.
• the invention therefore also relates to implantable medical devices characterized in that they comprise a material according to the invention.
• FIG. 1 represents A: a solution of acid-soluble collagen at a concentration of less than 5 mg / ml; B: concentrated solution of collagen after dialysis; C: concentrated solution of collagen according to the method of the invention.
• FIG. 2 represents a collagen (acid-soluble) (final concentration -200 mg / ml) obtained by dialysis which is macroscopically inhomogeneous (FIG. 2A). After neutralization, the size of the fibers observed by scanning electron microscopy is inhomogeneous (FIGS. 2B to 2D). The initial concentration of acid-soluble collagen is about 3 mg / ml.
• Figure 3 shows an example of a device used according to the invention.
• the whole volume of the dialysis cell is homogeneously filled and concentrated
• the injection is stopped for a time T so that the whole of the solution present in the cell concentrates homogeneous way.
• FIG. 4 represents a collagen (acid-soluble) (final concentration -250 mg / ml) obtained according to the technique of the invention which is macroscopically homogeneous (FIG. 4A). After neutralization, the fiber size observed by scanning electron microscopy is homogeneous (FIGS. 4B to 4D) and is organized (cholesteric geometry) with a large scale structure homogeneity (FIG. B). The initial concentration of acid-soluble collagen is about 1 mg / mL.
• Figure 5 illustrates the implantation of dense matrices at 300 mg / mL intramuscularly (A) and subcutaneously (B).
• Figure 6 illustrates the macroscopic appearance of dense matrices at 20 mg / mL (MD Figure 6A) and 40 mg / mL (MD 40 Figure 6B) 15 days after intramuscular implantation.
• Figure 7 shows the macroscopic appearance after 15 and 30 days of implantation of a 300 mg / mL dense matrix (MD 300).
• Figure 8 shows the microscopic appearance of dense matrices at 20 mg / mL (MD Figure 8A) and 40 mg / mL (MD 40 Figure 8B) 15 days after subcutaneous implantation.
• FIG. 9 represents the microscopic appearance after 15 days of implantation of a dense matrix at 300 mg / ml (MD300) subcutaneously at a magnification x 4.
• the arrows represent the cells that migrate / co Ionize to the inside the gel
• Figure 10 shows the microscopic appearance after 15 days of implantation of a dense matrix at 300 mg / ml (MD300) subcutaneously at 10x magnification.
• the arrows represent the cells that migrate inside the gel
• Figure 11 shows the microscopic appearance after 15 days of implantation of a dense matrix at 300 mg / ml (MD300) intramuscularly at x4 magnification.
• the arrows represent the cells that migrate inside the gel
• Figure 12 shows the microscopic appearance after 15 days of implantation of a dense matrix at 300 mg / ml (MD300) intramuscularly at 10x magnification.
• the arrows represent the cells that migrate inside the gel.
• Type I collagen is prepared from tails of young Wistar rats, according to the following procedure. Rat tail tendons are excised in a sterile laminar flow hood, then washed in phosphate buffered saline containing 137 mM NaCl, 2.68 mM KCl, 8.07 mM Na 2 HPO 4 , and 1, 47 mM NaH 2 PO 4 , to remove cells and traces of blood. The tendons are then soaked in 4M NaCl solution to remove the remaining intact cells and precipitate a portion of the high molecular weight proteins. After a further washing with the saline buffer solution, the tendons are dissolved in an aqueous solution of 500 mM acetic acid.
• the solution thus obtained is clarified by centrifugation at 41000 g for 2 h.
• Proteins other than type I collagen are selectively precipitated in 300 mM aqueous NaCl solution and then removed by centrifugation at 41000 g for 3 h.
• Collagen is recovered from the supernatant by precipitation in aqueous 600 mM NaCl followed by centrifugation at 3000 g for 45 min.
• the pellets thus obtained are dissolved in a 500 mM aqueous solution of acetic acid and then carefully dialyzed in the same solvent. to completely eliminate NaCl.
• the solutions are maintained at 4 ° C. and centrifuged at 41000 g for 4 h before being used.
• the fibrillogenesis is performed by immersing the collagen material under ammonia vapor for 24 hours.
• the collagen (acid-soluble) obtained by dialysis is macroscopically inhomogeneous (FIG. 2A) and the size of the fibers is inhomogeneous (FIGS. 2B to 2D).
• the friable collagen obtained by the process according to the invention is macroscopically homogeneous (FIG. 4A) and the size of the fibers is also homogeneous (FIGS. 4B to 4D).
• Wistar rats Wistar rats (Wi / Wi, Charles-Rivers France) are anesthetized with sodium pentobarbital solution (30 mg / kg, Centravet France). disinfected and a laparotomy is performed on the midline. Two pockets are made on both sides of the median line: one in subcutaneous, the other in intramuscular. The dense matrices of collagen concentrations ranging from 40 to 300 mg / mL are then implanted. The muscle pocket is closed, then the skin (Vicryl ® 4/0) See Figure 5. Fifteen, thirty or sixty days after implantation, the rats are euthanized with an excess of sodium penthobarbital and the MD are explanted and fixed in 4% paraformaldehyde (Merck France). The samples are then included in paraffin.
• the dies are easily handled without tears.
• the implantation of the dense matrices does not cause a reaction to severe foreign bodies after 15 days of implantation of matrices at 20 and 40 mg / mL (FIGS. 6A and 6B).
• the colonization rate of the implants by the cells in vivo depends on the collagen concentration. The higher the concentration, the slower the speed. The thicker the matrix, the longer the colonization at the core of the material will be.
• the dense matrix at 20 mg / ml (MD20) is colonized by fibroblast cells (FIG. 8A).
• the dense matrix at 40 mg / mL (MD40)
• Subcutaneous and generally for concentrations of 300 mg / ml (MD 300)
• cell colonization is more towards the cutaneous face than towards the muscular fascia.
• the MD300 is surrounded by a thin capsule.
• a few cells are observed and begin to migrate inside the gel ( Figures 9 and 10).
• the applications can therefore be extremely variable depending on the type of dense matrix selected and the implantation site.

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• 2010
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