WO2017028625A1

WO2017028625A1 - Pleural/meningeal patch and preparation method therefor

Info

Publication number: WO2017028625A1
Application number: PCT/CN2016/087456
Authority: WO
Inventors: 吴奕光; 冯奕清; 黎剑辉; 江长兵; 吴灿光
Original assignee: 深圳市阳光之路生物材料科技有限公司
Priority date: 2015-08-19
Filing date: 2016-06-28
Publication date: 2017-02-23
Also published as: CN105079875A

Abstract

Disclosed are a pleural/meningeal patch and a preparation method therefor. The preparation method comprises the steps of: S1, dissolving squid cartilage chitosan into colloidal fluid, the deacetylation degree of the squid cartilage chitosan being greater than 60%, and the molecular weight of the squid cartilage chitosan being greater than 0.5*106; and S2, coating with the colloidal fluid to obtain the pleural/meningeal patch. The pleural/meningeal patch prepared by the method is capable of effectively promoting the regeneration and repair of pleura and dura of a human body, and is finally degraded and absorbed in the body, thereby having a higher clinical application value.

Description

Pleural/meningeal patch and preparation method thereof

Technical field

The invention relates to the technical field of biological materials, in particular to a pleural/meningeal patch and a preparation method thereof.

Background technique

The dura mater and pleura are important functional membrane tissues between the brain and the skull, the inner surface of the chest wall and the upper surface of the chest and the surface of the lung, respectively, with similar biological characteristics and functions. Dural and pleural defects are mainly caused by tumor resection, trauma, inflammation, viral infection and radiation damage. Improper repair of the dura mater often causes cerebrospinal fluid leakage, meningeal tissue adhesion and scar formation of brain tissue. In severe cases, epilepsy can be caused. The chest wall defect not only affects the appearance, but also has different degrees of thoracic internal organ damage, and the area is large. Chest wall defects often cause abnormal breathing, interfere with normal breathing cycle function, and even lead to death. Therefore, studying the ideal dural/pleural repair materials to meet clinical needs has always been a concern of brain surgery and thoracic surgery.

The pleural and dura mater alternatives have undergone four processes of autologous materials, foreign materials, biomaterials, and synthetic materials, but all have advantages and disadvantages. In the domestic patent "a hard brain / meningeal bio-patch material and its preparation method" (application number 201410373258.6), the membrane structure of cattle is used as a material, and the material is sequentially placed in a surfactant solution, a lye The peroxide solution, the glycidyl ether epoxy cross-linking agent solution having a mass percentage concentration of 1-5%, and the phosphate buffer solution are immersed, and the dura mater material is obtained after drying radiation, and the material is likely to cause adhesion. It is not easy to store and disinfect, and may even have an immune response. In the domestic patent "Preparation method of aliphatic polyester-chitosan composite fiber tissue repair scaffold" (Application No. 200810051315), chitosan is disclosed as a partial deacetylated product of chitin, which has a polysaccharide with mucopolysaccharide. (GAG) has a similar chemical structure and has many excellent properties for tissue engineering scaffolding materials. However, like other natural macromolecules, chitosan has poor mechanical properties in supporting tissue cells. As natural macromolecules and synthetic polyester composites are gradually used in tissue engineering, the mixing of chitosan with synthetic polyester with good mechanical properties can effectively improve the mechanical properties of the materials. The fly in the ointment is that this method sacrifices many amino groups and hydroxyl groups on chitosan, and these groups are active groups with improved cell adhesion. In the domestic patent "a three-dimensional patch for nerve regeneration and dural repair and preparation method" (Application No. 201310230908.7), the patch of three-dimensional structure is proposed to repair the human dura mater, and the support layer is woven from the regenerated bacterial cellulose with high modulus, high strength and good shape retention ability, and the intermediate layer is made of reinforced antibacterial chitosan. Membrane composition, the outermost layer is composed of anti-adhesive cross-linked sodium hyaluronate with strong moisturizing, water permeability and gas permeable properties; the gap existing in the patch woven structure can phagot the bacteria causing infection, and the material preparation is complicated and the cost is extremely high. . In the domestic patent "a composite artificial dura mater and its preparation method" (application No. 201410627515.4), it is proposed to combine chitosan with vinyl silicone rubber, hydroxy silicone oil, polyethylene glycol, meteorological white carbon and vulcanizing agent. -25, polylactic acid, tricalcium phosphate and polytetrafluoroethylene are mixed at high temperature, and the prepared rubber mixture is mixed with gasoline to form a slurry, which is spun and knitted to obtain an artificial dura mater. The mechanical properties of the material are obtained. Preferably, but its biocompatibility is poor and non-absorbable.

Summary of the invention

The object of the present invention is to provide a pleural/meningeal patch and a preparation method thereof, which solve the technical problems that the prior art has a complicated preparation process, biocompatibility and mechanical properties.

To this end, the method for preparing a pleural/meningeal patch proposed by the present invention comprises the following steps:

S1, dissolving squid cartilage chitosan into a glue; wherein the squid cartilage chitosan has a degree of deacetylation greater than 60% and a molecular weight greater than 0.5×10 ⁶ ;

S2. Applying the film to the glue to obtain the pleural/meningeal patch.

Preferably, the coating film in the step S2 is a spin coating film.

Preferably, the rotational speed of the spin coating film is 2000 to 3000 rad/min, and the time is 9 to 12 s.

Preferably, in step S1, the squid cartilage chitosan is dissolved in a solvent at a ratio of solid to liquid of 1.0:100 to 5.0:100 g/ml to obtain the gum solution.

Preferably, the solvent is acetic acid having a mass percent concentration of 2%.

Preferably, before the coating of the glue liquid in the step S2, the glue liquid is filtered and defoamed.

Preferably, the preparation of the salmon cartilage chitosan in step S1 comprises the following steps:

S11. The squid cartilage β-chitin is dissolved in a NaOH solution having a mass percentage concentration of 30% to 40% at a ratio of solid to liquid of 5.0:100 to 25:100 g/ml, and stirred at 50 to 80 ° C for 1 to 6 hours. After filtration, the first filter residue is obtained;

S12. The first filter residue is washed to neutral and then dried to obtain a first salmon cartilage chitosan as the salmon cartilage chitosan of step S1.

Preferably, after step S12, the following steps are further included:

S13, the first squid cartilage chitosan obtained in step S12 is used as a first intermediate product, and the first intermediate product is dissolved in a mass percentage concentration at a ratio of solid to liquid ratio of 5.0:100 to 25:100 g/ml. 30% ~ 40% of the NaOH solution, stirred at 50 ~ 80 ° C for 1 ~ 6h and then filtered to obtain a second filter residue;

S14. The second filter residue is washed to neutrality and then dried to obtain a second salmon cartilage chitosan having a different degree of deacetylation from the first intermediate product as the salmon cartilage chitosan of step S1.

Preferably, after step S12 or step S14, the following steps are further included:

S15, the first squid cartilage chitosan obtained in step S12 or the second squid cartilage chitosan obtained in step S14 is used as a second intermediate product at a solid-liquid ratio of 5.0:100 to 25:100 g/ml. Proportion, the second intermediate product is dissolved in a hydrochloric acid solution having a mass percentage concentration of 1% to 5% at room temperature, and sonicated at 60 to 80 ° C for 2 to 8 hours to obtain a mixed solution;

S16, adding 0.1~1.0 mol/L NaOH solution to the mixture to adjust the mixture to neutrality, and suction filtration to obtain a third filter residue;

S17. The third filter residue is washed to neutrality and then dried to obtain a third salmon cartilage chitosan having a molecular weight different from that of the second intermediate product, as the salmon cartilage chitosan of step S1.

The present invention also provides a pleural/meningeal patch which is prepared using any of the above-described methods for preparing a pleural/meningeal patch.

The biomaterial chitosan has good antibacterial property, film forming property, biodegradability and biocompatibility, and has the characteristics of rapidly promoting wound healing and preventing tissue adhesion. Therefore, the chitosan film has a pleural and dura mater patch. Potential application value. However, commercially available chitosan is generally prepared from shrimp and crab shell α-chitin, which is prepared by strong alkali and high temperature deacetylation method. Its molecular weight is generally low, about 0.1×10 ⁶ , the prepared chitosan Membrane mechanical properties are poor, can not be used as a pleural and dura mater patch in clinical. The invention provides a preparation method for preparing a novel degradable pleural or dura mater and a preparation method thereof, which is prepared by preparing a squid cartilage chitosan with a specific molecular weight and a degree of deacetylation as a raw material, and only comprises a cartilage-containing cartilage. The solid content of chitosan has good dry and wet mechanical properties, and the film thickness of the film is more uniform by the method of spin coating, which eliminates the pores of the film patch and improves its anti-seepage performance. In addition, as a medical biomaterial, the patch has good antibacterial properties, biocompatibility, biodegradability, and no inflammatory reaction to surrounding tissues, and is used for temporarily replacing the pleura and dura mater of human body defects, thereby effectively promoting the human body. The regenerative repair of the pleura and dura mater is finally degraded and absorbed in the body, so it has high clinical application value.

DRAWINGS

1 is a comparison diagram of thickness uniformity of a cast film and a spin coating film according to a first embodiment of the present invention;

2 is a graph showing the in vitro enzymatic residual rate of a patch according to a first embodiment of the present invention;

3 is a view showing the inflammation of the patch implanted in the body according to the first embodiment of the present invention;

Figure 4 is a diagram showing the enzymatic hydrolysis of a patch according to a first embodiment of the present invention in mice;

Figure 5 is a graph showing changes in MDA in serum of mice after patch implantation according to a first embodiment of the present invention; wherein, the first blank column indicates MDA content in serum of normal mice, EG-13, EG-24, EG -49, EG-60 showed the MDA content in the serum of mice on the 13th, 24th, 49th and 60th day of the experimental group, respectively, CG-13, CG-24, CG-49, CG-60 respectively MDA content in the serum of mice on the 13th, 24th, 49th, and 60th day of the group.

detailed description

The present invention will be further described in detail below in conjunction with the specific embodiments and with reference to the accompanying drawings. It is to be understood that the following description is only illustrative, and is not intended to limit the scope of the invention.

The preparation method of a pleural/meningeal patch proposed by the invention comprises the following steps:

S1, dissolving the squid cartilage chitosan or the squid cartilage chitosan powder having a degree of deacetylation of more than 60% and a molecular weight of more than 0.5×10 ⁶ into a glue according to a ratio of solid to liquid ratio of 1:100 to 5:100 g/ml;

S2, filtering the glue, defoaming, and then rotating the coating to obtain a pleural/meningeal patch.

Wherein, the preparation of the squid cartilage chitosan in step S1 comprises:

S11, according to the ratio of solid-liquid ratio of 5.0:100 to 25:100g/ml, the squid cartilage β-chitin or squid cartilage chitosan is dissolved in a 3% by mass concentration of 30% to 40% NaOH solution, at 50-80 After stirring at ° C for 1 to 6 hours, it is filtered to obtain a filter residue;

S12. The filter residue obtained in the step S11 is washed to neutrality and then dried to obtain the squid cartilage chitosan used in the step S1.

The degree of deacetylation of squid cartilage chitosan was determined by elemental analysis, and the viscosity average molecular weight of squid cartilage chitosan was determined by a dilute solution viscosity method to obtain a series of squid cartilage chitosan as shown in Table 1 below. It can be seen from Table 1 that using different deacetylation conditions (such as alkali concentration, time, temperature), the obtained chimon cartilage chitosan has similar molecular weights and different degrees of deacetylation.

Table 1

among them,

--chitin: salmon cartilage β-chitin;

2+1 ^a : deacetylation twice, the first 2h, the second 1h;

2+1+1 ^b : three times of deacetylation, the first 2h, the second 1h, the third 1h;

1+1+1+1+1 ^c : deacetylation five times, each time 1h;

CS-75 ^d : CS-75 was sonicated at 70 ° C for 2 h;

Nil ^e : Not completely dissolved, the viscosity average molecular weight could not be determined.

Further, the preparation of the squid cartilage chitosan further comprises, after step S12:

S13. The squid cartilage chitosan obtained in step S12 is used as an intermediate product, and the intermediate product is dissolved at a concentration of 1% to 5% by weight of hydrochloric acid at a ratio of a solid-liquid ratio of 5.0:100 to 25:100 g/ml. In the solution, ultrasonic treatment at 60 ~ 80 ° C for 2 ~ 8h, to obtain a mixed solution;

S14, adding 0.1~1.0mol/L NaOH solution to the mixture to adjust the mixture to neutrality, and suction filtration to obtain filter residue;

S15. The filter residue obtained in the step S14 is washed to neutrality and then dried to obtain a squid cartilage chitosan having a molecular weight different from that of the intermediate product, which is used as the squid cartilage chitosan used in the step S1.

The degree of deacetylation of squid cartilage chitosan was determined by elemental analysis. The viscosity average molecular weight of squid cartilage chitosan was determined by dilute solution viscosity method, and a series of squid cartilage chitosan as shown in Table 2 below were obtained. As can be seen from Table 2, by changing the sonication time and/or temperature, the obtained cartilage chitosan has a similar degree of deacetylation and a different molecular weight.

Table 2

among them,

--chitin: salmon cartilage β-chitin;

CS-150: DD (80.00 ± 0.65)%, MW (1.52 ± 0.25) × 10 ⁶ carp cartilage chitosan.

The invention is described in detail below by means of preferred embodiments.

Embodiment 1:

This embodiment provides a method for preparing a pleural/meningeal patch, comprising the following steps:

S1, 1.3g of squid cartilage chitosan (DD=75%, MW=1.52×10 ⁶ ) powder is dissolved in 100mL, 2% by mass concentration of acetic acid solution, and configured as a glue;

S2, rotating the coating film with filtered and defoamed glue (r=2000 rad/min, t=9 s) to obtain a chitosan film;

S3. The chitosan film is dried in a blast drying oven at 60 ° C, washed with a dilute alkali solution and deionized water, and again dried in a blast drying oven at 60 ° C to obtain a pleural/meningeal patch.

Wherein, the preparation of the squid cartilage chitosan powder in step S1 comprises:

S11, in a solid-liquid ratio of 10:100 (g / ml) ratio, the salmon cartilage β-chitin is dissolved in a mass percentage concentration of 35% NaOH solution, stirred at 80 ° C for 2h and filtered to obtain filter residue;

S12. The filter residue was washed with deionized water until neutral, and then dried and ground in a blast drying oven at 60 ° C to obtain a squid cartilage chitosan (DD=75%, MW=1.52×10 ⁶ ) powder.

Since the viscosity of the glue disposed in step S1 is large, the glue of the cast coating film is difficult to spread evenly on the substrate, and the spin coating process is a combination of the viscous force between the glue and the substrate and the centrifugal force caused by the rotation. The action causes the glue to flow on the flat substrate to form a film, and the formed liquid film is flat and has less undulation. Therefore, the squid cartilage chitosan film prepared by the spin coating method has a more uniform thickness and no pores in the film, and the anti-seepage performance is greatly improved. See FIG. 1 for casting film formation and rotation according to the first embodiment of the present invention. A comparison of the uniformity of the film thickness; wherein the upper image is a cast film, and the lower image is a spin film. The pleural/meningeal patch prepared in this example has a thickness of 0.10 mm, a wet tensile strength of 11.45±0.12 MPa, an elongation at break of 209.64±21.21%, and a swelling ratio of 239%. The specific mechanical properties are shown in Table 3 below. Show:

table 3

2 is a graph showing the in vitro enzymatic residual rate of the patch according to the first embodiment of the present invention. The in vitro enzymatic performance test shows that the patch residual rate decreases with time.

The biocompatibility, biodegradability and surrounding tissue inflammation of the patch were evaluated by subcutaneous implantation of a patch test in mice as an experimental group. The mice were not placed in the mice, and the sutures were simply sutured as the control group, and the mice were not treated as the normal group. Macroscopic observations revealed that the wounds in the mice slowly healed, and only two months later the wound had a linear scar. HE staining showed that there was an inflammatory reaction in the early stage of wound healing in the experimental group and the control group, and then the inflammatory reaction gradually disappeared. Masson staining light microscopy revealed that wound tissue in the experimental group and the control group contained a large number of fibroblasts and a large amount of collagen deposition. 3 is a diagram showing the inflammation of the implanted patch in vivo according to the first embodiment of the present invention. According to the ratio of COX-2/GAPDH, the inflammatory reaction of the wound tissue occurs in the first 24 days after patch implantation. The most intense time is because the wound is not completely healed and the incompatibility caused by the patch in the body causes inflammation; after 24 days, the patch implant material is completely fused with the tissue, and the wound is basically healed, and the inflammatory reaction is gradually reduced; At 60 days, the COX-2/GAPDH ratio was smaller than that of the normal group, indicating that the inflammatory response slowly disappeared. 4 is a diagram showing the enzymatic hydrolysis of a patch according to a specific embodiment 1 of the present invention. According to the curve of the residual rate in the patch over time, it can be seen that the patch is slowly enzymatically absorbed in the body, and the first 13 after implantation. Days, the patch degraded slowly, and then the mouse self-healing ability strengthened, the wound slowly healed, make up Tablet absorption also accelerated; 60 days after patch implantation, the patch residual rate was 58%. 5 is a graph showing the change of MDA in the serum of mice after patch implantation according to the first embodiment of the present invention. By comparing the serum MDA content in the experimental group, the control group and the normal group, the patch is implanted. The MDA content of the experimental group and the control group at 13, 39, and 60 days was higher than that of the normal group, and the MDA content of the control group and the experimental group were basically equal, indicating that the implantation of the patch did not cause additional damage to the mouse tissue cells. Injury, the patch has good biocompatibility.

The above comprehensive performance test and animal experiment results show that the pleural and dura mater patches prepared by the spin coating method have good dry and wet mechanical properties, more uniform thickness, and eliminate the pores of the membrane. Improve its barrier properties. In addition, the patch has good biocompatibility, biodegradability, and no inflammatory response to surrounding tissues.

Embodiment 2:

S1, 1.5g of squid cartilage chitosan (DD=95%, MW=1.15×10 ⁶ ) powder is dissolved in 100mL, 2% by mass concentration of acetic acid solution, and configured as a glue;

S2, rotating the coating film with filtered and defoamed glue (r=2500 rad/min, t=10 s) to obtain a chitosan film;

S11, the squid cartilage chitosan (DD=75%, MW=1.52×10 ⁶ ) powder prepared in Example 1 was dissolved in a mass percentage concentration 30 at a ratio of solid to liquid ratio of 5:100 (g/ml). In a % NaOH solution, after stirring at 50 ° C for 4 h, the filter residue was obtained by filtration;

S12. The filter residue was washed with deionized water until neutral, and then dried and ground in a blast drying oven at 60 ° C to obtain a chirp cartilage chitosan (DD=95%, MW=1.15×10 ⁶ ) powder.

The pleural/meningeal patch prepared in this example has a thickness of 0.12 mm, a wet tensile strength of 9.3 ± 0.27 MPa, an elongation at break of 200.58 ± 10.25%, and a swelling ratio of 156%.

Embodiment 3:

S1, 2g of squid cartilage chitosan (DD=75%, MW=0.9×10 ⁶ ) powder is dissolved in 100mL, 2% by mass concentration of acetic acid solution, and configured as a glue;

S2, rotating the coating film with filtered and defoamed glue (r=2200 rad/min, t=11 s) to obtain a chitosan film;

S11, the squid cartilage chitosan (DD=75%, MW=1.52×10 ⁶ ) powder prepared in Example 1 was dissolved in the mass at room temperature according to a ratio of solid to liquid ratio of 10:100 (g/ml). In a hydrochloric acid solution having a percentage concentration of 1%, it was ultrasonically treated at 60 ° C for 4 hours in an ultrasonic cleaner to obtain a mixed solution;

S12, adding 1 mol/L NaOH solution to the mixture to adjust the mixture to neutrality, and suction filtration to obtain filter residue;

S13. The filter residue is washed with deionized water and absolute ethanol until neutral, and then dried and ground in a blast drying oven at 60 ° C to obtain a chirp cartilage chitosan (DD=75%, MW=0.9×10 ⁶ ) powder. .

The pleural/meningeal patch prepared in this example has a thickness of 0.18 mm, a wet tensile strength of 8.29 ± 0.35 MPa, an elongation at break of 189.66 ± 36.72%, and a swelling ratio of 235%.

Embodiment 4:

S1, 1.5g of squid cartilage chitosan (DD=83%, MW=1.12×10 ⁶ ) powder is dissolved in 100mL, 2% by mass concentration of acetic acid solution, and configured as a glue;

S2, rotating the coating film with filtered and defoamed glue (r=3000 rad/min, t=12 s) to obtain a chitosan film;

S3, the chitosan film is dried in a blast drying oven at 60 ° C, washed with a dilute alkali solution and deionized water, and again dried in a blast drying oven at 60 ° C to obtain a pleural/meningeal patch. .

S11, in a solid-liquid ratio of 20:100 (g / ml) ratio, the salmon cartilage β-chitin is dissolved in a 40% by mass concentration of NaOH solution, stirred at 70 ° C for 4h and filtered to obtain filter residue;

S12. The filter residue was washed with deionized water until neutral, and then dried and ground in a blast drying oven at 60 ° C to obtain a chirp cartilage chitosan (DD=83%, MW=1.12×10 ⁶ ) powder.

The pleural/meningeal patch prepared in this example has a thickness of 0.13 mm, a wet tensile strength of 7.43±1.24 MPa, an elongation at break of 154.24±7.48%, and a swelling ratio of 234%.

Those skilled in the art will recognize that many variations are possible in the above description, and thus the embodiments are only used to describe one or more specific embodiments.

While the invention has been described and described with reference to the embodiments of the embodiments In addition, many modifications may be made to adapt a particular situation to the teachings of the invention, without departing from the inventive concept. Therefore, the invention is not limited to the specific embodiments disclosed herein, but the invention may also include all embodiments and equivalents thereof.

Claims

A method for preparing a pleural/meningeal patch, comprising the steps of:

S1, dissolving squid cartilage chitosan into a glue; wherein the squid cartilage chitosan has a degree of deacetylation greater than 60% and a molecular weight greater than 0.5×10 6 ;

S2. Applying the film to the glue to obtain the pleural/meningeal patch.
The method of producing a pleural/meningeal patch according to claim 1, wherein the coating film in step S2 is a spin coating film.
The method for preparing a pleural/meningeal patch according to claim 2, wherein the rotational speed of the spin coating film is 2000 to 3000 rad/min, and the time is 9 to 12 s.
The method for preparing a pleural/meningeal patch according to claim 1, wherein in step S1, the squid cartilage chitosan is dissolved in a solvent at a ratio of solid to liquid of 1.0:100 to 5.0:100 g/ml. The glue was obtained.
The method of preparing a pleural/meningeal patch according to claim 4, wherein the solvent is acetic acid having a mass concentration of 2%.
The method for preparing a pleural/meningeal patch according to claim 1, wherein in the step S2, before the coating of the glue solution, the glue is filtered and defoamed.
The method for preparing a pleural/meningeal patch according to claim 1, wherein the preparation of the squid cartilage chitosan in step S1 comprises the following steps:

S11. The squid cartilage β-chitin is dissolved in a NaOH solution having a mass percentage concentration of 30% to 40% at a ratio of solid to liquid of 5.0:100 to 25:100 g/ml, and stirred at 50 to 80 ° C for 1 to 6 hours. After filtration, the first filter residue is obtained;

S12. The first filter residue is washed to neutral and then dried to obtain a first salmon cartilage chitosan as the salmon cartilage chitosan of step S1.
The method for preparing a pleural/meningeal patch according to claim 7, further comprising the following steps after step S12:

S13, the first squid cartilage chitosan obtained in step S12 is used as a first intermediate product, and the first intermediate product is dissolved in a mass percentage concentration at a ratio of solid to liquid ratio of 5.0:100 to 25:100 g/ml. 30% ~ 40% of the NaOH solution, stirred at 50 ~ 80 ° C for 1 ~ 6h and then filtered to obtain a second filter residue;

S14, washing the second filter residue to neutral and drying, to obtain deacetylation with the first intermediate product. Different degrees of the second squid cartilage chitosan, as the step S1 of the squid cartilage chitosan.
The method for preparing a pleural/meningeal patch according to claim 7 or 8, further comprising the following steps after step S12 or step S14:

S15, the first squid cartilage chitosan obtained in step S12 or the second squid cartilage chitosan obtained in step S14 is used as a second intermediate product at a solid-liquid ratio of 5.0:100 to 25:100 g/ml. Proportion, the second intermediate product is dissolved in a hydrochloric acid solution having a mass percentage concentration of 1% to 5% at room temperature, and sonicated at 60 to 80 ° C for 2 to 8 hours to obtain a mixed solution;

S16, adding 0.1~1.0 mol/L NaOH solution to the mixture to adjust the mixture to neutrality, and suction filtration to obtain a third filter residue;

S17. The third filter residue is washed to neutrality and then dried to obtain a third salmon cartilage chitosan having a molecular weight different from that of the second intermediate product, as the salmon cartilage chitosan of step S1.
A pleural/meningeal patch produced by the method for producing a pleural/meningeal patch according to any one of claims 1 to 9.