Composite biomaterials with controlled release of active ingredient, preparation process and uses

Abstract

The invention relates to a composite biomaterial based on collagen, on at least one hydrophobic organic polymer and on at least one active ingredient, the process for preparing same, a dressing comprising such a composite biomaterial, an abdominal wall reinforcement comprising such a composite biomaterial, and also the uses of said composite biomateral, especially in the therapeutic field.

Claims

1. Synthetic composite biomaterial comprising: collagen, at least one organic polymer and at least one active ingredient, wherein: the organic polymer is biodegradable, biocompatible, hydrophobic and has a glass transition temperature of less than or equal to 50 C. and a mean molar mass ranging from 5 to 120 kDa, the collagen is in the form of striated fibrils, in which the periodicity of the striations is 67 nm, the collagen/organic polymer weight ratio ranges from 10/1 to 1/3, the active ingredient is a hydrophobic active ingredient chosen from anti-inflammatories, antibiotics, compounds promoting tissue repair or wound healing, and a mixture thereof.

2. Biomaterial according to claim 1, wherein said biomaterial is in the form of a composite hydrogel and comprises from 70 to 95% by weight of water relative to the total weight of the composite biomaterial.

3. Biomaterial according to claim 2, wherein said biomaterial has an elasticity ranging from 50 000 Pa to 100 000 Pa.

4. Biomaterial according to claim 2, wherein said biomaterial has an elongation at break ranging from 40 to 75%.

5. Biomaterial according to claim 1, wherein said biomaterial is in the form of a dry composite material and comprises at most 10% by weight of water relative to the total weight of the composite biomaterial.

6. Biomaterial according to claim 1, wherein the organic polymer is chosen from aliphatic polyesters, polyethylene glycols, polyanhydrides and poly(ortho-esters).

7. Biomaterial according to claim 1, wherein the organic polymer is an aliphatic polyester chosen from a polyglycolide, a polylactide, a copolymer of glycolide and lactide, a polylactone and a polyhydroxyalkanoate.

8. Biomaterial according to claim 1, wherein the organic polymer is in the form of nanodomains having a mean size of less than or equal to 700 nm.

9. Process for preparing a composite biomaterial as defined in claim 1, comprising at least the following steps: i) preparing a hydrogel of collagen in the form of striated fibrils in which the periodicity of the striations is 67 nm, the concentration of collagen in the hydrogel being at least 10 mg/ml, ii) dehydrating the hydrogel as prepared in step i) by incubating said hydrogel in several successive mixed solutions having an increasing proportion of organic solvent, said aqueous and organic solvents being miscible, followed by a final incubation in a pure solution of said organic solvent, iii) bringing the dehydrated hydrogel from step ii) into contact with an impregnation solution comprising at least one organic polymer and at least one active ingredient as defined in any one of claims 1 to 8, the volume of impregnation solution/volume of dehydrated hydrogel volume ratio being greater than or equal to 3, iv) rinsing the impregnated hydrogel from step iii) with an organic solvent, then with an aqueous solvent.

10. Process according to claim 9, wherein step i) is carried out according to the following sub-steps: i-1) preparing a solution of acid-soluble collagen, the collagen content of which varies from 1 to 5 mg/ml, i-2) evaporating the solution from step i-1) in air, and i-3) bringing the solution from step i-2) into contact with a base.

11. Process according to claim 9, wherein step ii) is carried out by incubating the hydrogel from step i) in a mixed organic solvent/aqueous solvent solution in which the content of organic solvent is from 20 to 30% by volume, then in a mixed organic solvent/aqueous solvent solution in which the content of organic solvent is from 40 to 50% by volume, then in a mixed organic solvent/aqueous solvent solution in which the content of organic solvent is from 60 to 70% by volume, then in a mixed organic solvent/aqueous solvent solution in which the content of organic solvent is from 80 to 95% by volume, then in a pure solution of said organic solvent.

12. Process according to claim 9, wherein the incubation of step ii) in each of the solutions lasts from 30 min to 2 h.

13. Process according to claim 9, wherein the concentration of organic polymer of the impregnation solution ranges from 20 to 500 mg/ml.

14. Process according to claim 9, wherein said process also comprises a step v) of lyophilization of the composite biomaterial of step iv).

15. Composite biomaterial as defined in claim 1, for medical use thereof.

16. Composite biomaterial as defined in claim 1, for use thereof in the treatment of chronic wounds.

17. Composite biomaterial as defined in claim 1, for use thereof in the preventative treatment of infections after cardiac or colorectal surgery.

18. Composite biomaterial as defined in claim 1, for use thereof in the treatment of hernias of the abdominal wall or eventrations.

19. Therapeutic dressing comprising: an internal layer and an external layer of a secondary dressing chosen from an adhesive, a compress, a bandage and a mixture thereof, wherein the internal layer comprises a composite biomaterial as defined in claim 1.

20. Abdominal wall reinforcement, wherein said abdominal wall reinforcement comprises a composite biomaterial as defined in claim 1.

Description

EXAMPLES

[0119] The starting materials used in the examples are listed below: [0120] PLGA 7-17 KDa, 719897-5G, Sigma, 50:50 (Lactide:Glycolide), [0121] PLGA 24-38 KDa, 719870-5G, Sigma, 50:50 (Lactide:Glycolide), [0122] PLGA 30-60 KDa, P2191-5G, Sigma, 50:50 (Lactide:Glycolide), [0123] PLA 10 KDa, 764620-5G, Sigma [0124] PLA 30 KDa, 767344-5G, Sigma [0125] PCL 14 KDa, 440752-5G, Sigma, [0126] rat tail tendons, Gibco A1048301, [0127] spironolactone, S3378-1G, Sigma, [0128] dexamethasone, D4902-1G, Sigma, and [0129] aldosterone, A9477-25MG, Sigma.

[0130] Unless otherwise indicated, all the materials were used as received from the manufacturers.

[0131] The prepared or commercial materials were characterized by transmission electron microscopy (TEM), hydroxyproline assay, measurement of contact angle, measurement of elongation at break, measurement of breaking strength, measurement of elasticity, measurement of toxicity, measurement for monitoring the release of the active ingredient by UV spectroscopy and measurement of the biological activity of the spironolactone.

[0132] The analysis by transmission electron microscopy (TEM) was carried out using an apparatus sold under the trade name Technai Spirit G2, by FEI.

[0133] The contact angle was measured using a tensiometer sold under the trade name DSA 30 by KRUSS. This measurement was carried out according to the following Wilhelmy method: a solution of organic polymer with a concentration of approximately 160 mg/ml dissolved in tetrahydrofuran (THF) is deposited on a clean and dry glass slide. Drying is carried out in order to form a film of the organic polymer at the surface of the glass slide. A drop of water is then deposited on the glass slide covered with organic polymer. This operation is carried out at approximately 25 C. The tensiometer measures the surface tension between the water and the glass slide covered with organic polymer and calculates the resulting contact angle with the water. The larger the contact angle, the more the organic polymer is hydrophobic.

[0134] The elasticity was determined at 25 C. using an apparatus sold under the trade name Electroforce 3220 by Bose.

[0135] The shear modulus was determined at 25 C. using an apparatus sold under the trade name MCR 301 by Anton Paar.

[0136] The elongation at break was determined at 25 C. using an apparatus sold under the trade name Electroforce 3220 by Bose.

[0137] The breaking strength was determined at 25 C. using an apparatus sold under the trade name Electroforce 3220 by Bose.

[0138] The hydroxyproline assay makes it possible to determine the concentration of collagen in the different solutions or materials. Hydroxyproline is an amino acid that is heavily present in the collagen polypeptide chain. The assay was carried out in the following manner: the collagen solution to be tested was hydrolysed under acid conditions (HCl 6 M) at approximately 108 C. The hydroxyproline was thus released, then the resulting mixture was dried. The hydroxyproline was oxidized by Chloramine T then complexed with dimethylamino-4-benzaldehyde (DMBA) to give a coloured product. The concentration was determined by spectrophotometric measurement at 557 nm by comparison with a calibration range.

[0139] The toxicity of the materials was measured as follows: primary human dermal fibroblasts were seeded onto a culture plate. The composite hydrogels were then incubated with the fibroblasts for 1 or 6 days. The cell viability was measured by a metabolic test (AlamarBlue Assay) and compared with that of control fibroblasts (without addition of composite hydrogel).

[0140] The monitoring of the release of the active ingredient was measured by UV spectroscopy using an apparatus sold under the trade name Uvikon XL, by NorthStar Scientific.

[0141] The biological activity of the spironolactone was measured in the following manner: H9C2 cells are myoblasts genetically modified by the incorporation of the reporter gene encoding luciferase downstream of the promoter having sequences binding the complex of aldosterone plus its receptor. When aldosterone was added, luciferase was expressed and detected by bioluminescence. When the spironolactone released by the tested hydrogels is active, its incubation with H9C2s inhibits aldosterone and makes the bioluminescence signal zero. This inhibition reports the biological activity of the spironolactone on H9C2s incubated with aldosterone. The spironolactone activity was measured directly on the spironolactone releasing liquid originating from the different types of hydrogels tested.

Example 1: Preparation of a Synthetic Composite Biomaterial in Accordance with the First Subject of the Invention

[0142] 1.1 Extraction of the Collagen

[0143] The collagen was extracted from rat tail tendons, known to be very rich in type I collagen. For this purpose, rat tendons were rinsed with phosphate-buffered saline (PBS), centrifuging them for approximately 5 min at approximately 4 C. and at 3000 G (G is the unit of measurement of the centrifugation speed and corresponds to acceleration due to gravity) until the solution becomes clear, devoid of cells and of blood. They were then rinsed with an approximately 4 M solution of NaCl in order to destroy all the remaining cells. After another rinse in PBS to eliminate all traces of NaCl, the washed tendons were mixed with a solution of approximately 0.5 M acetic acid for approximately 24 h, then the resultant mixture was centrifuged at approximately 3000 G for approximately 20 min. The resulting mixture then comprised triple helices of collagen I in the presence of other proteins. The collagen I was selectively precipitated by dropwise addition of an approximately 4 M solution of NaCl to the resulting solution to obtain a final NaCl concentration of 0.7 M. The resulting mixture was then centrifuged at 3000 G and the precipitate obtained was dissolved in 0.5 M acetic acid to form a solution comprising essentially collagen I. This solution was dialysed against the same solvent in order to completely eliminate the NaCl (4 baths of approximately 24 h) and centrifuged at approximately 21 000 G for approximately 3 hours to eliminate the final colloidal aggregates.

[0144] The collagen solution prepared in this way was stored at approximately 4 C. in order to conserve its triple helix structure. The assay of hydroxyproline in this solution made it possible to determine its concentration of collagen, which was approximately 5 mg/ml.

[0145] 1.2 Preparation of a Concentrated Collagen I Hydrogel

[0146] The solution of collagen at approximately 5 mg/ml as prepared above was dissolved in an approximately 0.5 M acetic acid solution. The solution was evaporated in the air under a sterile fume hood until a final collagen concentration of approximately 40 mg/ml was achieved. The evaporation of the solvent was monitored by weighing. The final concentration of the solution was then confirmed by hydroxyproline assay.

[0147] The solution of collagen at approximately 40 mg/ml obtained in this way was introduced into a mould, then the mould/collagen solution assembly was centrifuged in order to flatten out irregularities. The resulting mould was then placed under ammonia vapours for approximately 12 h, to enable gelling and fibrillogenesis of the collagen I. The hydrogel obtained was rinsed several times in sterile PBS baths (i.e. which had undergone moist sterilization in an autoclave) to eliminate the ammonia and bring the pH to 7. The pH was monitored before each washing. The pure collagen hydrogel obtained is denoted M.sub.A-H.

[0148] 1.3 Dehydration of a Concentrated Collagen I Hydrogel

[0149] The collagen I hydrogel as obtained above was dehydrated gradually by incubation in several successive mixed THF/water baths: a THF/water bath having a concentration by volume of THF of 30%, a THF/water bath having a concentration by volume of THF of 50%, a THF/water bath having a concentration by volume of THF of 70%, a THF/water bath having a concentration by volume of THF of 95% and finally a pure THF bath. Incubation in each of the baths lasted approximately 1 h.

[0150] Following the dehydration step, a dehydrated pure collagen hydrogel M.sub.A-HD was obtained.

[0151] The dehydrated pure collagen hydrogel M.sub.A-HD can be rinsed with PBS and lyophilized to give a dry pure collagen material M.sub.A-S then M.sub.A-S can be rehydrated with PBS to re-form a pure collagen hydrogel denoted M.sub.A-HR.

[0152] 1.4 Preparation of the Synthetic Composite Biomaterial M.sub.1

[0153] A solution comprising 160 mg/ml of PLGA 30-60 kDa and 10.sup.2 M (i.e. 4.16 mg/ml) of spironolactone was prepared by dissolving the spironolactone in THF then by dissolving the PLGA in the preceding mixture.

[0154] The dehydrated pure collagen hydrogel M.sub.A-HD as prepared above was impregnated for approximately 12 h with the solution comprising the PLGA and the spironolactone, the volume of the solution being at least 5 times greater than that of the dehydrated pure collagen hydrogel.

[0155] After impregnation, the composite hydrogel obtained was rinsed 3 times for approximately 30 seconds with pure THF to eliminate the excess PLGA, then 3 times for approximately 30 minutes with sterile PBS to fix the polymer within the fibrillar collagen network and form the composite biomaterial of the invention M.sub.1-H in the form of a composite hydrogel.

[0156] A dry form of said composite biomaterial was also obtained. For this purpose, the composite biomaterial M.sub.1-H was submerged in liquid nitrogen for approximately 10 min in order to avoid the formation of ice crystals, then lyophilized for approximately 24 h (temperature of less than 40 C., vacuum at 100 Barr), to form the composite biomaterial of the invention M.sub.1-5 in the form of a dry composite material.

[0157] The dry composite material was then rehydrated by addition of a phosphate-buffered saline to form a material M.sub.1-HR in the form of a composite hydrogel.

[0158] The collagen/organic polymer weight ratio in said biocomposite material was 1/1.

[0159] FIG. 1 shows a transmission electron micrograph of a pure collagen hydrogel M.sub.A-H as obtained in example 1.2 (FIG. 1a) which does not form part of the invention, and a micrograph of the composite biomaterial M.sub.1-HR in the form of a composite hydrogel in accordance with the invention, obtained in example 1.4 (FIG. 1b).

[0160] FIG. 1 shows a homogeneous composite biomaterial in which the fibrillar and striated structure of the collagen has been preserved. Moreover, microscopic domains of PLGA polymer cannot be discerned, which means that the polymer is uniformly distributed within the collagen.

[0161] The table below summarizes the values of the contact angles of the different organic polymers used in example 1 and the examples below:

TABLE-US-00001 PLGA PLGA PLGA PCL 7-17 kDa 24-38 kDa 30-60 kDa 14 kDa Contact angle 68.73 70.03 71.03 109.33 () Sum of the mean 0.78 0.49 0.51 1.49 deviations ()

Example 2: Preparation of Other Synthetic Composite Biomaterials in Accordance with the First Subject of the Invention

[0162] Composite biomaterials M.sub.2-HR, M.sub.3-HR and M.sub.4-HR were prepared by impregnating the dehydrated pure collagen hydrogel M.sub.A-no as obtained in example 1.3 above with the following respective impregnation solutions: [0163] a solution comprising approximately 10.sup.2 M of spironolactone and approximately 160 mg/ml of PLGA 7-17 kDa, [0164] a solution comprising approximately 10.sup.2 M of spironolactone and approximately 160 mg/ml of PLGA 24-38 kDa, and [0165] a solution comprising approximately 10.sup.2 M of spironolactone and approximately 160 mg/ml of PCL 14 kDa.

[0166] The steps of impregnation, rinsing, lyophilization and rehydration are identical to those described in example 1.4 above.

[0167] FIG. 2 represents the diameter (in mm) of the composite biomaterials in the form of composite hydrogels in accordance with the invention M.sub.1-HR, M.sub.2-HR, M.sub.3-HR and M.sub.4-HR and, by way of comparison, the diameter of a pure collagen hydrogel not in accordance with the invention M.sub.A-H as obtained in example 1.2 and of a pure collagen hydrogel M.sub.A-HR as obtained in example 1.3.

[0168] FIG. 2 shows that the presence in the hydrogel of an active ingredient and a hydrophobic organic polymer as defined in the invention has only very little influence on the diameter of the hydrogel obtained, or no influence at all. The composite hydrogels therefore have a good capacity to be hydrated, close to that of pure collagen, and do not retract.

[0169] FIG. 3 represents the mass of organic polymer (in mg) present in the composite biomaterials in the form of dry composite materials in accordance with the invention M.sub.1-S, M.sub.2-S, M.sub.3-S and M.sub.4-S.

[0170] FIG. 3 shows that the composite biomaterial of the invention may incorporate a greater amount of organic polymer when the PLGA 7-17 kDa is used (M.sub.2-S). The capacities of integration of the organic polymers tested are nonetheless suitable and are greater than approximately 15 mg, and preferably range approximately from 20 to 31 mg for a mass of collagen of the order of 25 mg.

[0171] FIG. 4 represents the swelling by volume of the composite biomaterials in the form of composite hydrogels in accordance with the invention M.sub.1-HR, M.sub.2-HR, M.sub.3-HR and M.sub.4-HR and, by way of comparison, of a pure collagen hydrogel M.sub.A-HR as obtained in example 1.3 (as % relative to the initial volume of the pure collagen hydrogel M.sub.A-H).

[0172] FIG. 4 shows a good capacity for swelling of the composite hydrogels in accordance with the invention (approximately 55% to 80%), even similar to that of a pure collagen hydrogel when PLGA 7-17 kDa is used.

[0173] FIG. 5 represents the elongation at break of the composite biomaterials in the form of composite hydrogels in accordance with the invention M.sub.1-HR, M.sub.2-HR, M.sub.3-HR and M.sub.4-HR and, by way of comparison, that of a pure collagen hydrogel M.sub.A-HR as obtained in example 1.3 (as % relative to the initial length of each of the materials tested).

[0174] FIG. 6 represents the breaking strength (in MPa) of the composite biomaterials in the form of composite hydrogels in accordance with the invention M.sub.1-HR, M.sub.2-HR, M.sub.3-HR and M.sub.4-HR and, by way of comparison, that of a pure collagen hydrogel M.sub.A-HR as obtained in example 1.3.

[0175] FIG. 7 represents the elasticity (in Pa) of the composite biomaterials (Young's modulus) in the form of composite hydrogels in accordance with the invention M.sub.1-HR, M.sub.2-HR, M.sub.3-HR and M.sub.4-HR and, by way of comparison, that of a pure collagen hydrogel M.sub.A-HR as obtained in example 1.3.

[0176] FIG. 8 represents the shear modulus (in Pa) at different frequencies (approximately 1 Hz and 10 Hz) of the composite biomaterial in the form of a composite hydrogel in accordance with the invention M.sub.1-HR, and, by way of comparison, that of a pure collagen hydrogel M.sub.A-HR as obtained in example 1.3 and that of a collagen hydrogel impregnated by spironolactone (i.e. without organic polymer) M.sub.B-HR obtained from the dehydrated pure collagen material M.sub.A-HD as obtained in example 1.3 which has been impregnated with a solution comprising approximately 10.sup.2 M of spironolactone in THF and rinsed, lyophilized and rehydrated in accordance with example 1.4 above. Thus, a significant improvement in the mechanical strength of the composite biomaterial of the invention is observed compared to a pure collagen hydrogel or a pure collagen hydrogel comprising spironolactone.

[0177] FIG. 9 represents the profile of release of spironolactone of the composite biomaterials in the form of dry composite materials in accordance with the invention M.sub.1-S (curve with triangles), M.sub.1-S1 (curve with diamonds) and M.sub.1-S2 (curve with squares). The different curves show the cumulative amount of spironolactone released (in nmol) as a function of time (in hours).

[0178] It is worth noting that the biomaterials initially used in this experiment are in the form of dry composite materials. However, they are instantly rehydrated during the measurement for monitoring the release of the active ingredient. The same applies for the measurements of the toxicity of the biomaterials and the biological activity of spironolactone, as described below.

[0179] The composite biomaterials M.sub.1-S1 and M.sub.1-S2 were prepared as in example 1 except in terms of the concentration of PLGA in the impregnation solution which was approximately 40 mg/ml for M.sub.1-S1 and approximately 80 mg/ml for M.sub.1-S2 (Instead of approximately 160 mg/ml for M.sub.1-S).

[0180] It is observed that a concentration of approximately 160 mg/ml is preferred for promoting the release of a constant dose of spironolactone over time and for obtaining better control of the release of spironolactone. It is worth noting that the three composite biomaterials in accordance with the invention only released approximately from 30 to 60% of spironolactone in 400 h (approximately 2 weeks).

[0181] FIG. 10 represents the profile of release of spironolactone of the composite biomaterials of the invention M.sub.1-S (curve with crosses), M.sub.1-S (curve with diamonds), M.sub.3-S (curve with squares) and M.sub.4-S (curve with triangles). The different curves show the daily dose of spironolactone released (as nanomol per day) as a function of time (in days).

[0182] FIG. 11 represents the toxicity of the composite biomaterial of the invention M.sub.1-S on cells (fibroblasts) and by way of comparison: [0183] the toxicity of a dry collagen material impregnated with spironolactone M.sub.B-S obtained from the dehydrated pure collagen material M.sub.A-HD as obtained in example 1.3 which was impregnated with a solution comprising approximately 10.sup.2 M of spironolactone in THF, rinsed and lyophilized in accordance with example 1.4 above, [0184] the toxicity of a dry collagen material impregnated with PLGA M.sub.C-S obtained from the dehydrated pure collagen material M.sub.A-HD as obtained in example 1.3 which was impregnated with a solution comprising approximately 160 mg/ml of PLGA 30-60 kDa in THF, rinsed and lyophilized in accordance with example 1.4, and [0185] the toxicity of a dry pure collagen material M.sub.A-S as obtained in example 1.3.

[0186] Moreover, C represents a negative control (well containing just the cells without hydrogel). The different charts show the cell viability of the materials M.sub.1-S, M.sub.B-S, M.sub.C-S and M.sub.A-S as defined above (in %) at 1 day and at 6 days, relative to the negative control C.

[0187] On the sixth day, all the points are above 100% since the cells have proliferated and the study is cumulative. It is observed that the spironolactone has a slight toxic effect on the cells since the two triangles are slightly below the control (circles). Indeed, on the first day, only 74% of the cells survived. However, this does not prevent cell proliferation (the straight lines are increasing). FIG. 11 also shows the non-toxicity associated with the hydrophobic organic polymer PLGA since the squares are at the same level as the control (circles).

[0188] FIG. 12 represents the biological activity of the spironolactone released from a hydrogel after 15 days of incubation in PBS (with changing of the buffer every day); when the following are used:

[0189] (1) a mixture of a composite biomaterial in accordance with the invention M.sub.1-S with aldosterone at approximately 10.sup.8 M, and by way of comparison:

[0190] (2) a mixture of a material M.sub.B-S with aldosterone at approximately 10.sup.8 M,

[0191] (3) a mixture of a material M.sub.C-S with aldosterone at approximately 10.sup.8 M,

[0192] (4) a mixture of a material M.sub.A-S with aldosterone at approximately 10.sup.8 M,

[0193] (5) a negative control (well containing the culture medium without aldosterone),

[0194] (6) aldosterone at approximately 10.sup.8 M, and

[0195] (7) a mixture of aldosterone at approximately 10.sup.8 M and spironolactone at approximately 10.sup.6 M.

[0196] FIG. 12 shows the production of luciferase (in relative light units or RLU) depending on the type of medium used. It is observed that the luciferase activity is greater under the conditions (6) than under the conditions (5), meaning that the aldosterone has indeed bound to the mineralocorticoid receptor and caused strong transcription. When spironolactone at approximately 10.sup.6 M (conditions (7)) is added, the effect of the aldosterone is inhibited and the receptor is not activated. The conditions (1) using the composite biomaterial M.sub.1-S in accordance with the invention show comparable activity to that obtained under the conditions (7), indicating that spironolactone has indeed been released by the composite biomaterial M.sub.1-S, while retaining the activity thereof. When there is no PLGA (conditions (2)), the collagen does not retain the spironolactone and the remaining amount of spironolactone is inactive or of relatively low concentration to act against aldosterone. Finally, the two final conditions (3) and (4) correspond to controls without spironolactone and are at the same height as that associated with the conditions (6) containing solely aldosterone at 10.sup.8 M; there is therefore no effect of the collagen hydrogel, nor of the collagen hydrogel comprising PLGA.

Example 3: Preparation of Other Synthetic Composite Biomaterials in Accordance with the First Subject of the Invention

[0197] Composite biomaterials M.sub.5-HR and M.sub.6-HR were prepared by impregnating the dehydrated pure collagen hydrogel M.sub.A-HD as obtained in example 1.3 above with the following respective impregnation solutions: [0198] a solution comprising approximately 10.sup.2 M of spironolactone and approximately 160 mg/ml of PLA 10 kDa, and [0199] a solution comprising approximately 10.sup.2 M of spironolactone and approximately 160 mg/ml of PLA 30 kDa.

[0200] The steps of impregnation, rinsing, lyophilization and rehydration are identical to those described in example 1.4 above.

[0201] FIG. 13 shows the load of spironolactone (in g) of the composite biomaterials in the form of dry composite materials in accordance with the invention M.sub.1-S, M.sub.2-S, M.sub.3-S, M.sub.4-S, M.sub.5-S and M.sub.6-S, per mg of composite biomaterial, and by way of comparison the load of spironolactone (in g) of the dry pure collagen material M.sub.A-S per mg of said material.

[0202] Other composite biomaterials M.sub.7-HR and M.sub.8-HR were prepared by impregnating the dehydrated pure collagen hydrogel M.sub.A-HD as obtained in example 1.3 above with the following respective impregnation solutions: [0203] a solution comprising approximately 5.310.sup.2 M (i.e. 21 mg/ml) of dexamethasone and approximately 160 mg/ml of PLGA 7-17 kDa, and [0204] a solution comprising approximately 3.210.sup.2 M (i.e. 12.6 mg/ml) of dexamethasone and approximately 160 mg/ml of PLGA 7-17 kDa.

[0205] The steps of impregnation, rinsing, lyophilization and rehydration are identical to those described in example 1.4 above.

[0206] FIG. 14 shows the profile of release of dexamethasone of the composite biomaterials in the form of dry composite materials in accordance with the invention M.sub.7-S (FIG. 14a)) and M.sub.8-S (FIG. 14b)). The different curves show the cumulative amount of dexamethasone released (in g) as a function of time (in days).