Injectable collagen suspensions, the preparation method thereof, and the uses thereof, particularly for forming dense collagen matrices

Abstract

Disclosed is the preparation of injectable collagen suspensions, to the method for preparing the suspensions, and to the uses thereof, particularly for forming dense collagen matrices.

Claims

1. An injectable suspension consisting of a powdered composition in an aqueous solution formed of water and acid or of a saline phosphate buffer, wherein said powdered composition consists of spherical or spheroidal solid particles consisting of more than 90% by mass of non-denatured and uncrosslinked collagen, and less than 10% by mass of an aqueous solution formed of water and acid, wherein a total collagen concentration is in a range of 20 to 200 mg per ml of suspension, wherein a diameter of said spherical or spheroidal solid particles is in a range of 0.25 μm to 10 μm, wherein the suspension forms dense fibrillar collagen matrices in vivo by assembly of collagen molecules of which the intermolecular distance is less than 10 nm, after being contacted with a physiological medium.

2. A cosmetic composition containing a suspension according to claim 1, said cosmetic composition further comprising one or more additives selected from anaesthetics, hyaluronic acid, elastins, vitamins and platelet rich plasma, ions, precursors of a mineral phase, proteoglycans, glycosaminoglycan, and non-fibrillar collagen types.

3. A pharmaceutical composition containing a suspension according to claim 1.

4. An injectable suspension consisting of a powdered composition in an acid-soluble collagen acidic aqueous solution, wherein said powdered composition consists of spherical or spheroid solid particles consisting of more than 99% by mass of non-denatured and uncrosslinked collagen, and less than 1% by mass of an aqueous solution formed of water and acid, wherein a diameter of said spherical or spheroid solid particles is in a range of 0.25 μm to 10 μm, wherein the total collagen concentration is greater than 20 mg per ml of suspension, and wherein the suspension forms dense fibrillar collagen matrices in vivo by assembly of collagen molecules of which the intermolecular distance is less than 10 nm, after being contacted with a physiological medium.

5. An injectable suspension consisting of a powdered composition in an acid-soluble collagen acidic aqueous solution, wherein said powdered composition consists of spherical or spheroid solid particles consisting of more than 99% by mass of non-denatured and uncrosslinked collagen, and less than 1% by mass of an aqueous solution formed of water and acid, wherein a diameter of said spherical or spheroid solid particles is in a range of 0.25 μm to 10 μm, wherein the total collagen concentration is greater than 100 mg per ml of suspension, and wherein the suspension forms dense fibrillar collagen matrices in vivo by assembly of collagen molecules of which the intermolecular distance is less than 10 nm, after being contacted with a physiological medium.

6. The suspension according to claim 1, wherein the acid present in said powdered composition is acetic acid.

Description

DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows collagen microparticles obtained by spray drying a solution of acid-soluble collagen (7 mg per mL) in acetic acid (0.5M) at 36° C.

(2) FIG. 2 shows collagen microparticles obtained by spray drying a solution of acid-soluble collagen (2.1 mg per mL) in acetic acid (0.5M) at 36° C.

(3) FIG. 3 illustrates the differential scanning calorimetry of collagen powders obtained by spray drying a solution of acid-soluble solution (0.7 mg.Math.mL-1) in acetic acid (0.5M) at 36 and 55° C. The samples were analysed under conditions of controlled humidity so as to allow the identification of the denaturation processes of the collagen.

(4) FIG. 4 shows a collagen gel obtained by diffusion of ammonia vapours in a PDMS mould filled with a collagen coacervate at a concentration of 5.6 mg.Math.ml.sup.−1 in the presence of ATP.

(5) FIG. 5 is an image recorded by transmission electron microscopy of a cut obtained by ultramicrotomy of a sample synthesised from acid-soluble collagen at a concentration of 5.6 mg.Math.ml.sup.−1 after coacervation by ATP and fibrillogenesis induced by ammonia vapours. The sample was fixed in an aqueous solution of dehydrated glutaraldehyde, dried to the supercritical point, enclosed in an epoxy matrix and contrasted by uranyl acetate.

(6) FIG. 6 shows the viscosity of solutions of acid-soluble collagen and of collagen coacervates according to Example 8 as a function of the total collagen concentration.

(7) FIG. 7 shows the distribution of the diameter of the collagen coacervates 0.32 mg per mL according to Example 9, measured by dynamic light scattering.

(8) FIG. 8 is an image of a powdered composition according to the invention, recorded by polarised light microscopy.

(9) FIG. 9 shows different states of aggregation of a suspension comprising or consisting of a powdered composition according to the invention, in a culture medium Dulbecco 1X (DMEM), from t=0 to t=48H after contacting said suspension with the DMEM.

EXAMPLES

Example 1: Injectable Suspension of Collagen Microparticles (50 mg per mL) Obtained By Spraying and Dispersed in a Solution of Acid-Soluble Collagen (2 mg Per mL)

(10) 1.1 Obtaining a Solution of Acid-Soluble Collagen.

(11) A solution of type I collagen is prepared from Wistar rat tail tendons. After excision in a laminar flow cabinet, the tendons are washed in a sterile saline phosphate buffer solution. The tendons are then immersed in a solution of 4M NaCl in order to remove the remaining intact cells and precipitate some of the proteins of elevated molecular weight. After washing by the saline phosphate buffer solution, the tendons are solubilised in a sterile 500 mM acetic acid solution. The solution obtained is clarified by centrifugation at 41000 g for 2 hours. The proteins other than the collagen are precipitated selectively in an aqueous solution of 300 mM NaCl and removed by centrifugation at 41000 g for 3 hours. The collagen is recovered from the supernatant by precipitation in a solution of 600 mM NaCl followed by centrifugation at 3000 g for 45 minutes. The pellets obtained are solubilised in an aqueous solution of 500 mM acetic acid, then dialysed in the same solvent in order to remove the NaCl ions. The solution is held at 4° C. and centrifuged at 41000 g for 4 hours prior to use.
1.2 Preparation of a Dry Powder from Non-Denatured Collagen.
A collagen solution with a concentration of 7 mg per mL is diluted in ultra-pure water (resistivity greater than 18.2 MΩ per cm) to obtain a collagen solution at a concentration of 1 mg per mL dissolved in an aqueous solution of 71 mM acetic acid. The solution thus obtained is dried in an atomiser (“spray drier” Buchi B290). The rate of injection of the collagen solution, limited to 1 ml per minute, is controlled by an automatic syringe pump connected to the nozzle of the drying instrument. The temperature of the nozzle is held at 36° C., and the internal temperature of the system, measured between the drying column and the particle collection cyclone, is maintained throughout the process between 26 and 31° C. The flow of air responsible for the shearing of the droplets at the outlet of the nozzle is 1052 L per hour. The suction power, which controls the drying of the droplets between the outlet of the nozzle and the collector, is fixed at 75% of the maximum capacity of the drying system (30 m.sup.3 per hour). The particles thus formed are collected by a high-performance cyclone.
1.3 Preparation of a Suspension Obtained by Dispersion of the Collagen Powder in a Solution of Acid-Soluble Collagen
A fraction of the powder thus obtained of mass 5.0 mg is dispersed in 100 μL of solution of acid-soluble collagen at an initial concentration of 2 mg per mL. The final collagen concentration of the suspension is 52 mg per ml. The suspension obtained after initial manual stirring and 30 seconds of stirring under vortex (2400 revolutions per minute) can be aspirated by a syringe equipped with a 27 gauge needle. The suspension has a white colour and a homogeneous macroscopic appearance.

Example 2: Injectable Suspension of Collagen Microparticles (7 mg per mL) Obtained by Spray Drying and Dispersed in a Solution of Acid-Soluble Collagen (7 mg Per mL)

(12) 2.1 Preparation of a Dry Powder of Non-Denatured Collagen.

(13) A collagen solution is obtained in accordance with Example 1.1 with a concentration of 7 mg per mL. This solution is dried in an atomiser (“spray drier” Buchi B290). The rate of injection of the collagen solution, limited to 1 ml per minute, is controlled by an automatic syringe pump connected to the nozzle of the drying instrument. The temperature of the nozzle is held at 36° C., and the internal temperature of the system, measured between the drying column and the particle collection cyclone, is maintained throughout the process between 26 and 31° C. The flow of air responsible for the shearing of the droplets at the outlet of the nozzle is 1052 L per hour. The suction power, which controls the drying of the droplets between the outlet of the nozzle and the collector, is fixed at 75% of the maximum capacity of the drying system (30 m.sup.3 per hour). The particles thus formed are collected by a high-performance cyclone. (FIG. 1).
2.2 Preparation of a Suspension Obtained by Dispersion of the Collagen Powder in a Solution of Acid-Soluble Collagen.
A fraction of the powder thus obtained of mass 7.0 mg is dispersed in 1 mL of solution of acid-soluble collagen at an initial concentration of 7 mg per mL. The final collagen concentration of the suspension is 14 mg per ml. The suspension obtained after initial manual stirring and 30 seconds of stirring under vortex (2400 revolutions per minute) can be aspirated by a syringe equipped with a 27 gauge needle. The suspension has a white colour and a homogeneous macroscopic appearance.
2.3 Fibrillogenesis Induced by Exposure to Ammonia Vapours.
After injection of the suspension in the closed PDMS mould, the assembly is exposed to ammonia vapours to promote fibrillogenesis. After 24 hours, the gel thus formed is demoulded, with no significant change in volume.

Example 3: Injectable Suspension of Collagen Microparticles (7 mg Par mL) Obtained by Spraying and Dispersed in a Saline Phosphate Buffer

(14) 3.1 Preparation of a Suspension Obtained by Dispersion of a Collagen Powder in a Saline Phosphate Buffer.

(15) A dry collagen powder is obtained in accordance with Example 2.2. A fraction of the powder thus obtained of mass 7.0 mg is dispersed in 1 mL of saline phosphate buffer. The final collagen concentration of the suspension is 7 mg per ml. The suspension obtained after initial manual stirring and 30 seconds of stirring under vortex (2400 revolutions per minute) can be aspirated by a syringe equipped with a 27 gauge needle. The suspension has a white colour and a homogeneous macroscopic appearance. The suspension is then injected into a 70 μL cylindrical PDMS mould.
3.2 Fibrillogenesis Induced by Exposure to Ammonia Vapours.
After injection of the suspension in the closed PDMS mould, the assembly is exposed to ammonia vapours to promote fibrillogenesis. After 24 hours, the gel thus formed is demoulded, with no significant change in volume.

Example 4: Injectable Suspension of Collagen Microparticles (40 mg Par mL) Obtained by Spraying and Dispersed in a Saline Phosphate Buffer

(16) 4.1 Preparation of a Suspension Obtained by Dispersion of the Collagen Powder in a Saline Phosphate Buffer

(17) A suspension of collagen microparticles is obtained in accordance with Example 3.2. A fraction of the powder thus obtained of mass 40 mg is dispersed in 1 mL of saline phosphate buffer. The final collagen concentration of the suspension is 40 mg per ml.

(18) 4.2 After injection of the suspension in the closed PDMS mould, the assembly is exposed to ammonia vapours to promote fibrillogenesis. After 24 hours, the gel thus formed is demoulded, with no significant change in volume.

Example 5: Obtaining Collagen Microparticles by Spraying from a Solution of Acid-Soluble Collagen at a Concentration of 2.1 mg Per mL

(19) 5.1 Preparation of a Dry Powder of Non-Denatured Collagen.

(20) A collagen solution is obtained in accordance with Example 1.1 with a concentration of 7 mg per mL. This solution is diluted in 500 mM acetic acid to obtain a final concentration of 2.1 mg per mL. Said solution is dried in an atomiser (“spray drier” Buchi B290). The rate of injection of the collagen solution, limited to 1 ml per minute, is controlled by an automatic syringe pump connected to the nozzle of the drying instrument. The temperature of the nozzle is held at 36° C., and the internal temperature of the system, measured between the drying column and the particle collection cyclone, is maintained throughout the process between 28 and 32° C. The flow of air responsible for the shearing of the droplets at the outlet of the nozzle is 1052 L per hour. The suction power, which controls the drying of the droplets between the outlet of the nozzle and the collector, is fixed at 75% of the maximum capacity of the drying system (30 m.sup.3 per hour). The particles thus formed are collected by a high-performance cyclone. (FIG. 2).

Example 6: Analysis of Microparticles of Non-Denatured and Denatured Collagen Obtained by Spraying from a Solution of Acid-Soluble Collagen with a Concentration of 0.7 mg Par mL

(21) 6.1 Preparation of a Dry Powder of Non-Denatured Collagen According to the Invention.

(22) A collagen solution is obtained in accordance with Example 1.1 with a concentration of 7 mg per mL. This solution is diluted in 500 mM acetic acid to obtain a final concentration of 0.7 mg per mL. Said solution is dried in an atomiser (“spray drier” Buchi B290). The rate of injection of the collagen solution, limited to 1 ml per minute, is controlled by an automatic syringe pump connected to the nozzle of the drying instrument. The temperature of the nozzle is held at 36° C., and the internal temperature of the system, measured between the drying column and the particle collection cyclone, is maintained throughout the process between 28 and 32° C. The flow of air responsible for the shearing of the droplets at the outlet of the nozzle is 1052 L per hour. The suction power, which controls the drying of the droplets between the outlet of the nozzle and the collector, is fixed at 75% of the maximum capacity of the drying system (30 m.sup.3 per hour). The particles thus formed are collected by a high-performance cyclone.
6.2 Preparation of a dry powder of denatured collagen not forming part of the invention. The collagen solution of Example 6.1 is dried in accordance with Example 6.1, with the temperature of the nozzle being the sole difference, maintained at 55° C. (FIG. 3).

Example 7: Injectable Solution of Collagen Coacervates Obtained in the Presence of Disodium Salt of ATP

(23) 7.1 Obtaining an Injectable Solution of Coacervates

(24) 250 μL, of an aqueous solution of disodium salt of 50 mM adenosine triphosphate are added to 1 mL of a collagen solution with a concentration of 7 mg par mL obtained in accordance with Example 1.1. The mixture thus obtained is stirred under vortex at 2400 revolutions per minute for 30 seconds. The cloudy suspension thus obtained is injected into a 70 μL PDMS mould.
7.2 Fibrillogenesis of the Suspension of Coacervates Induced by Exposure to Ammonia Vapours.
After injection of the suspension into the closed PDMS mould, the assembly is exposed to ammonia vapours to promote fibrillogenesis. After 24 hours, the gel thus formed is demoulded, with no significant change in volume (FIGS. 4 and 5).

Example 8: Injectable Suspensions of Coacervates of Collagen Obtained in the Presence of Disodium Salt of ATP

(25) 8.1 Obtaining an Injectable Suspension of Coacervates of Concentration 28 mg Par mL

(26) 200 μL, of an aqueous solution of disodium salt of 50 mM adenosine triphosphate are added to 1 mL of a collagen solution with a concentration of 7 mg par mL obtained in accordance with Example 1.1. The mixture thus obtained is stirred under vortex at 2400 revolutions per minute for 30 seconds. The cloudy suspension thus obtained is centrifuged for 2 minutes at 1700 g. A volume of 950 μL of supernatant is removed, and the pellet thus obtained stirred under a vortex so as to obtain a coacervate at a final concentration of 28 mg per mL. The coacervate thus obtained can be injected into a PDMS mould.
8.2 Obtaining an injectable suspension of coacervates of concentration 14 mg Per mL
200 μL, of an aqueous solution of disodium salt of 50 mM adenosine triphosphate are added to 1 mL of a collagen solution with a concentration of 7 mg par mL obtained in accordance with Example 1.1. The mixture thus obtained is stirred under vortex at 2400 revolutions per minute for 30 seconds. The cloudy suspension thus obtained is centrifuged for 30 seconds at 1700 g. A volume of 700 μL of supernatant is removed, and the pellet thus obtained stirred under a vortex so as to obtain a coacervate at a final concentration of 14 mg per mL. The coacervate thus obtained can be injected into a PDMS mould. The suspension obtained also can be injected into a type A gelatin gel (300 Bloom) dissolved 10% by mass in phosphate buffer.
8.3 Obtaining an Injectable Suspension of Coacervates of Concentration 5.8 mg Per mL
200 μL of an aqueous solution of disodium salt of 50 mM adenosine triphosphate are added to 1 mL of a collagen solution with a concentration of 7 mg par mL obtained in accordance with Example 1.1. The mixture thus obtained is stirred under vortex at 2400 revolutions per minute for 30 seconds. The cloudy suspension thus obtained has a final concentration of 5.8 mg per mL.
8.4 Obtaining an Injectable Suspension of Coacervates having a Concentration of 2.9 mg Per mL
500 μL of 500 mM acetic acid and 200 μL of an aqueous solution of disodium salt of 50 mM adenosine triphosphate are added to 500 μL of a collagen solution with a concentration of 7 mg par mL obtained in accordance with Example 1.1. The mixture thus obtained is stirred under vortex at 2400 revolutions per minute for 30 seconds. The cloudy suspension thus obtained has a final concentration of 2.9 mg per mL.
8.5 Obtaining an Injectable Suspension of Coacervates of Concentration 1.46 mg Par mL
750 μL of 500 mM acetic acid and 200 μL of an aqueous solution of disodium salt of 50 mM adenosine triphosphate are added to 250 μL it of a collagen solution with a concentration of 7 mg par mL obtained in accordance with Example 1.1. The mixture thus obtained is stirred under vortex at 2400 revolutions per minute for 30 seconds. The cloudy suspension thus obtained has a final concentration of 1.46 mg per mL.

(27) 8.6 Analysis of the Viscosity of the Coacervates Obtained and of Non-Coacervated Solutions of Acid-Soluble Collagen

(28) The viscosity of the suspensions of coacervates obtained in accordance with Examples 8.1 to 8.5 was measured in an Anton Parr MCR 302 rheometer in rotational viscometer mode using a cone-plane geometry. The viscosity was determined at shear rates from 1 to 100 s.sup.−1. The viscosity of the suspensions of coacervate at a shear rate of 50 s.sup.−1 was determined as 1.7 to 253 mPa.Math.s for collagen concentrations from 1.45 to 28 mg per ml, respectively. The viscosity of solutions of acid-soluble collagen at concentrations from 1 to 7 mg per mL was also determined. The viscosity values obtained in accordance with the same analysis conditions vary between 16.8 mPa.Math.s for a collagen concentration of 1 mg per mL and 217 mPa.Math.s for a collagen concentration of 7 mg per mL. FIG. 6 combines the viscosity data as a function of the collagen concentration for the collagen solutions dissolved in 500 mM acetic acid and the collagen coacervates.

Example 9: Suspension of Collagen Coacervates Obtained in the Presence of Disodium Salt of ATP

(29) 9.1 Observation of an Injectable Suspension of Coacervates having a Concentration of 0.83 mg Per mL

(30) 100 μL of an aqueous solution of disodium salt of 50 mM adenosine triphosphate are added to 1 mL of a collagen solution with a concentration of 0.35 mg par mL obtained in accordance with Example 1.1. The mixture thus obtained is stirred under vortex at 2400 revolutions per minute for 30 seconds. The cloudy suspension thus obtained, with a final concentration of 0.32 mg per mL, is analysed by dynamic light scattering in a Zetasizer Nano S. apparatus. The diameter distribution of the particles determined in terms of intensity after 5 consecutive measurements is centred at 837 nm with a standard deviation of 21 nm (FIG. 7).

Example 10: Study in Vivo

(31) A gel obtained from a suspension according to the present invention (for example according to example 2.3, 4.2 or 7.2) is implanted in vivo in the small animal (wistar rat) sub-cutaneously.

(32) The following were evaluated:

(33) a) the integration of the implants (absence of inflammation and colonisation by host cells leading particularly to the presence of endothelial cells);

(34) b) limited absorption compared to reference implants (particularly diluted collagen and hyaluronic acid).

(35) The lack of change in the volume of the implanted gel, for example on 15, 30 and 60 days after implantation, indicates that the gel is stable, in particular with respect to collagenases.