Method for preparing collagen sponge by using oxidized sodium carboxymethyl cellulose

Abstract

The invention relates to a method for preparing collagen sponge by using oxidized sodium carboxymethyl cellulose. Comprising the following steps: (1) preparing an oxidized sodium carboxymethyl cellulose solution; (2) adding collagen into acetic acid, and dialyzing for 4-5 days to obtain a collagen solution; and (3) adding the oxidized sodium carboxymethyl cellulose solution into the collagen solution, carrying out cross-linking reaction at 0-4 DEG C for 20-30 hours, and freeze-drying to obtain the collagen sponge. OCMC is adopted as a cross-linking agent for preparing the collagen sponge for the first time, the preparation method of the collagen sponge and the optimal adding amount of oxidized sodium carboxymethyl cellulose are determined, and the prepared collagen sponge is higher in mechanical strength, better in water absorption performance and excellent in hemostatic effect in clinical use. The hemolysis rate of the collagen is far lower than 5%, and the collagen has an obvious blood coagulation promoting effect, does not generate cytotoxicity, does not influence the safety of the collagen as a natural biological material, and can be used for preparing a medical material for hemostasis.

Description

Technical Field

[0001] The invention relates to a method for preparing collagen sponge by utilizing oxidized sodium carboxymethyl cellulose, and belongs to the technical field of medical biomaterials. Background Art

[0002] Collagen is the main protein in the extracellular matrix, accounting for about one-third of the total protein in mammals. The most important of these is type I collagen, which is the structural basis of many connective tissues (such as skin, bones and tendons). It has functions such as supporting tissues and providing stability, and is involved in various biological pathways, playing an important role in the evolution of multicellular life. In addition, collagen has become a potential source of material for tissue engineering scaffolds and medical dressings due to its biological properties such as promoting wound healing, promoting tissue regeneration, good biocompatibility and degradability. Compared with terrestrial animal tissues such as pig skin and cowhide, collagen from marine organisms is safer, less immunogenic and less religious. Therefore, marine-derived collagen can be used as a good matrix for biomedical materials.

[0003] Although collagen materials have broad potential for biomedical applications, their practical applications are limited by poor thermal stability, low mechanical strength, and susceptibility to enzymatic degradation. Therefore, researchers in this field are committed to using chemically synthesized substances or natural ingredients as cross-linking agents to modify the physical and chemical properties of collagen sponges to improve their application performance.

[0004] Cross-linking refers to the process by which linear or branched polymer chains are linked by covalent bonds to form a network of polymers. The principle of chemical cross-linking is to introduce exogenous cross-links between and within collagen fibers through the interaction between the cross-linking agent and collagen. This chemical reaction between the carboxyl and amino groups in the collagen and the cross-linking agent increases its molecular weight, improves its mechanical properties, and enhances its resistance to degradation. Commonly used cross-linking agents include chemical cross-linkers and natural cross-linkers. However, while hemostatic sponges prepared using chemical cross-linkers have improved hemostatic properties, they also face challenges such as reduced biosafety and residual cytotoxicity. Therefore, natural cross-linkers such as sodium alginate, polyphenols, and β-(1,3)-glucan have been widely studied in recent years due to their lower toxicity and improved biocompatibility.

[0005] Oxidized sodium carboxymethyl cellulose (OCMC) is the product of sodium carboxymethyl cellulose (CMC-Na) oxidized with sodium periodate. Two aldehyde groups form on its molecular chain, which can cross-link with free primary amine groups in collagen molecules, enhancing the mechanical strength of collagen. In recent years, research on the use of OCMC as a cross-linking agent in the preparation of biomaterials has increased, but a method for preparing collagen sponges using OCMC as a cross-linking agent remains lacking. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention provides a method for preparing a collagen sponge using sodium oxidized carboxymethyl cellulose. This method, for the first time, utilizes OCMC as a cross-linking agent to prepare a collagen sponge. The method and the optimal dosage of sodium oxidized carboxymethyl cellulose are determined, and the physicochemical properties and hemostatic activity of the resulting product are analyzed, laying the foundation for the development of novel collagen sponges.

[0007] The technical solutions of the present invention are as follows:

[0008] A method for preparing collagen sponge using oxidized sodium carboxymethyl cellulose comprises the following steps:

[0009] (1) Prepare a sodium carboxymethyl cellulose oxide solution with a concentration of 15-45 mg / L;

[0010] (2) adding collagen to acetic acid, mixing well, and dialyzing for 4-5 days to obtain a collagen solution;

[0011] (3) Adding the sodium carboxymethyl cellulose oxide solution prepared in step (1) to the collagen solution obtained in step (2), mixing evenly, and cross-linking reaction at 0-4° C. for 20-30 hours. After freeze-drying, a collagen sponge is obtained.

[0012] Preferably, according to the present invention, in step (1), the concentration of the sodium carboxymethyl cellulose oxide solution is 25-35 mg / L.

[0013] Further preferably, the concentration of the sodium carboxymethyl cellulose oxide solution is 30 mg / L.

[0014] Preferably, according to the present invention, in step (2), the collagen is cod skin collagen.

[0015] Preferably according to the present invention, in step (2), the concentration of acetic acid is 0.1 to 1M.

[0016] According to the preferred embodiment of the present invention, in step (2), the dialysis is specifically as follows: at 4°C, using a dialysis bag with a cutoff of 3 to 4 kDa, first dialyzing with a 0.1 M acetic acid solution for 24 hours, then dialyzing with ultrapure water for 3 to 5 days, and replacing the dialysate every 12 hours until the solution pH is 6.0 to 7.0.

[0017] Preferably, according to the present invention, in step (2), the concentration of the collagen solution is 15 to 25 mg / L.

[0018] Further preferably, the concentration of the collagen solution is 20 mg / L.

[0019] According to the preferred embodiment of the present invention, in step (3), the mass of the oxidized sodium carboxymethyl cellulose is 0.01 to 2.25% of the mass of the collagen.

[0020] More preferably, the mass of the oxidized sodium carboxymethyl cellulose is 0.1% of the mass of collagen.

[0021] Preferably, according to the present invention, in step (3), the concentration of collagen in the mixed solution is 15 mg / ml.

[0022] According to a preferred embodiment of the present invention, in step (3), the freeze drying is specifically: pre-freezing at -80°C for 3 to 4 hours, and then placing in a freeze dryer for freeze drying.

[0023] A collagen sponge is prepared according to the method.

[0024] Application of the above collagen sponge in the preparation of medical materials.

[0025] Preferably according to the present invention, the medical material is a hemostatic material.

[0026] The technical features and beneficial effects of the present invention are as follows:

[0027] 1. The present invention provides a method for preparing a collagen sponge using sodium oxidized carboxymethyl cellulose (OCMC). Compared with chemical crosslinkers, OCMC used in the present invention has low cytotoxicity, good biocompatibility, and is readily available and inexpensive, making it suitable for industrial production. Furthermore, crosslinking with OCMC does not alter the triple helix structure of collagen, ensuring that the collagen sponge retains its excellent mechanical properties.

[0028] 2. The present invention explores the process of collagen processing and cross-linking to establish a new preparation process for collagen sponge, and the prepared collagen sponge has better performance.

[0029] 3. The collagen sponge provided by the present invention has higher mechanical strength and better water absorption properties, and has excellent hemostatic effects in clinical use. Its hemolysis rate is far below 5%, and it has a significant blood coagulation effect. It is non-cytotoxic and does not affect the safety of collagen as a natural biomaterial. It can be used to prepare medical materials for hemostatic purposes. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The water absorption rate of the collagen sponge at different OCMC concentrations in Example 1.

[0031] Figure 2 is the tensile strength of the collagen sponge at different OCMC concentrations in Example 2.

[0032] Figure 3FTIR infrared spectra of the collagen sponge and cod skin collagen in Example 3.

[0033] Figure 4 The results of blood compatibility, coagulation index and in vitro coagulation time of the collagen sponge in Example 3 are as follows;

[0034] In the figure, A is the hemolysis rate, B is the coagulation index, and C is the in vitro coagulation time. DETAILED DESCRIPTION

[0035] The technical solution of the present invention will be further described below with reference to the embodiments, but the protection scope of the present invention is not limited thereto.

[0036] Sources of biological materials

[0037] Sodium carboxymethylcellulose was purchased from Shanghai Sangon Biotechnology Co., Ltd. (China). Glacial acetic acid, nitric acid, and anhydrous ethanol were purchased from Sinopharm Group (China). Sodium periodate was purchased from Shanghai MacLean Biochemical Technology Co., Ltd. (China).

[0038] Erica collagen sponge was purchased from Beijing Paisheng Biotechnology Co., Ltd. (China).

[0039] Collagen sponge was purchased from Wuxi Beidi Bioengineering Co., Ltd. (China).

[0040] Medical gauze dressings were purchased from Jiangxi Aibang Medical Instrument Co., Ltd. (China).

[0041] Sheep anticoagulated blood was purchased from Nanjing Senbega Biotechnology Co., Ltd. (China).

[0042] The OCMC used can be obtained according to the method described in Chinese patent document CN101250827B.

[0043] The cod skin collagen used can be obtained according to the method described in Chinese patent document CN118146353A.

[0044] Example 1

[0045] Since good exudate absorption is one of the key indicators for evaluating collagen sponges, this example prepared collagen sponges with varying OCMC contents to determine the amount of OCMC added. The water absorption rates were then analyzed to determine the effect of different OCMC concentrations on the water absorption performance of the collagen sponge products. The specific process is as follows.

[0046] A method for preparing collagen sponge using oxidized sodium carboxymethyl cellulose comprises the following steps:

[0047] (1) Weigh OCMC powder and dissolve it in ultrapure water to prepare an OCMC solution with a concentration of 30 mg / ml;

[0048] (2) Cod skin collagen was weighed and dissolved in 0.5 M acetic acid solution. The mixture was mixed well and dialyzed for 5 days to prepare a 20 mg / ml collagen solution.

[0049] (3) OCMC solution was added to the collagen solution to adjust the OCMC content to 0%, 0.1%, 0.25%, 0.5%, 0.75%, 1%, 1.25%, 1.5%, 1.75%, 2%, and 2.25% of the collagen mass, and the collagen concentration was 15 mg / ml. After mixing, the solution was cross-linked at 4°C for 24 h and freeze-dried to obtain collagen sponges with different OCMC concentrations.

[0050] The water absorption rate of collagen sponges with different OCMC concentrations was measured, and the effect of OCMC addition on the water absorption performance of collagen sponge products was analyzed. The results are as follows Figure 1 shown.

[0051] Depend on Figure 1 It can be seen that the water absorption rate of pure collagen sponge without adding OCMC is 2399±79.80%; when the OCMC content is less than 1.5%, the change in water absorption rate of collagen sponge under different OCMC contents is not significant, which is basically consistent with the pure collagen sponge; although the water absorption rate of collagen sponge with 1.75% OCMC content is reduced, it is still greater than 2000%; but when the OCMC content in the collagen sponge reaches 2%, its water absorption multiple decreases to 1589±70.40%.

[0052] This indicates that when low concentration of OCMC is added to collagen, the change in water absorption rate is small, and at this time, the addition of OCMC has no effect on reducing the water absorption rate of collagen.

[0053] Example 2

[0054] Good mechanical properties are one of the most important indicators for evaluating collagen sponges. Therefore, to determine the optimal OCMC dosage, this example reduced the OCMC dosage compared to Example 1. Collagen sponges with varying OCMC contents were prepared. The tensile strength of these sponges was then analyzed to determine the effect of varying OCMC concentrations on the mechanical properties of the collagen sponge products. The specific process is as follows.

[0055] A method for preparing collagen sponge using oxidized sodium carboxymethyl cellulose comprises the following steps:

[0056] (1) Weigh OCMC powder and dissolve it in ultrapure water to prepare an OCMC solution with a concentration of 30 mg / ml;

[0057] (2) Cod skin collagen was weighed and dissolved in 0.5 M acetic acid solution. The mixture was mixed well and dialyzed for 5 days to prepare a 20 mg / ml collagen solution.

[0058] (3) OCMC solution was added to the collagen solution to achieve 0%, 0.1%, 0.25%, 0.5%, 0.75%, 1%, 1.25%, 1.5%, and 1.75% of the collagen mass, and a collagen concentration of 15 mg / ml. After mixing, the solution was cross-linked at 4°C for 24 h and freeze-dried to obtain collagen sponges with different OCMC concentrations.

[0059] The tensile strength of collagen sponges with different OCMC concentrations was measured. The commercially available Erica collagen sponge and Kejibang collagen sponge were used as controls to analyze the effect of OCMC addition on the mechanical properties of collagen sponge products. The results are as follows: Figure 2 shown.

[0060] Depend on Figure 2 The collagen sponge with 0.1% OCMC exhibited the highest tensile strength (613.12 ± 78.6 kPa), significantly higher than both pure collagen without the addition of a crosslinker and two currently available collagen sponge products. Furthermore, considering that crosslinkers are exogenous chemical components, lowering the amount added can mitigate risks to the human body and reduce toxicity.

[0061] Therefore, considering the two indicators of water absorption and mechanical properties, the collagen sponge prepared with 0.1% OCMC addition has both good water absorption rate and tensile strength, which is the optimal addition amount.

[0062] Example 3

[0063] Hemocompatibility is a key property of biomedical materials. The hemocompatibility of collagen sponges can be characterized by their hemolysis rate. This rate refers to the ability of red blood cell walls to rupture and release hemoglobin upon contact with the material. The lower the hemolysis rate, the better the material's biocompatibility. A hemolysis rate exceeding 5% is generally considered to indicate a degree of cytotoxicity. In this example, various properties of collagen sponges were measured, as detailed below.

[0064] A method for preparing collagen sponge using oxidized sodium carboxymethyl cellulose comprises the following steps:

[0065] (1) Weigh OCMC powder and dissolve it in ultrapure water to prepare an OCMC solution with a concentration of 30 mg / ml.

[0066] (2) Cod skin collagen was weighed and dissolved in 0.5 M acetic acid solution. The mixture was mixed well and dialyzed for 5 days to prepare a 20 mg / ml collagen solution.

[0067] (3) Add OCMC solution to the collagen solution so that the mass of OCMC is 0.1% of the mass of collagen and the concentration of collagen is 15 mg / ml. After mixing evenly, cross-link the mixture at 4°C for 24 hours and freeze-dry to obtain a collagen sponge.

[0068] The FTIR infrared spectrum of the collagen sponge was measured, and its physicochemical properties and hemostatic activity (blood compatibility, coagulation index and in vitro coagulation time) were analyzed. The results are shown in Table 1. Figure 3 、 Figure 4 When analyzing the hemostatic activity (blood compatibility, coagulation index and in vitro coagulation time), medical gauze was used as a control, 1% Triton X-100 was used as a positive control, 1×PBS was used as a negative control, and sheep anticoagulant blood was used as a blank control.

[0069] Table 1. Analysis of physicochemical properties of cod skin collagen sponge

[0070]

[0071]

[0072] As can be seen from Table 1, the drying loss rate and ash content of the collagen sponge prepared in this embodiment are low, indicating that there are fewer impurities in the cross-linked sponge; the pH value is about 6.0, which is within the pH range of 4.0 to 6.0 of human skin and is not likely to cause discomfort to the human body. In addition, the drying loss rate, ash content and hydroxyproline content are not significantly different from those of pure collagen, indicating that the dialysis treatment method and the addition of OCMC do not cause significant changes in the physical properties of collagen. The total content of heavy metals is less than 10 mg / kg, which meets the requirements for the total amount of heavy metals in the pharmaceutical industry standard of the People's Republic of China "YY / T 1511-2017 Collagen Sponge", indicating that the types and contents of heavy metals in the prepared cod skin collagen sponge meet national standards and are within the safe range.

[0073] Depend on Figure 3 It can be seen that the characteristic absorption peaks of collagen did not change before and after OCMC cross-linking, and were still amide A, amide B, and amide I, II, and III bands, and the peak positions remained basically unchanged, indicating that the collagen sponge prepared by the present invention better maintained the collagen triple helix structure.

[0074] Depend on Figure 4As shown in Figure 1, the hemolysis rate of the collagen sponge is far below 5%, indicating that the collagen sponge prepared by the present invention has good blood compatibility. Furthermore, the in vitro coagulation index represents the ratio of uncoagulated blood to the total blood volume within a certain reaction time. The lower the value, the better the coagulation function of the material.

[0075] Depend on Figure 4 B shows that the coagulation index value of the collagen sponge decreased significantly when the coagulation reaction was carried out for 5-10 minutes, and after 10 minutes, the value was lower than that of the blank control and medical gauze, indicating that the collagen sponge has an obvious blood coagulation effect.

[0076] like Figure 4 C shows that the in vitro coagulation time of the collagen sponge is about 30% lower than that of the blank control, indicating that the cod skin collagen sponge has the effect of promoting in vitro coagulation.

[0077] In summary, the inventors of this application have proposed a method for preparing a collagen sponge using OCMC as a crosslinking agent. By optimizing the preparation steps and the amount of OCMC added, the resulting collagen sponge exhibits excellent water absorption and tensile strength. Furthermore, analyses of the crosslinked sponge's physicochemical properties, structure, biocompatibility, and hemostatic activity demonstrate the collagen sponge's structural integrity and superior hemostatic activity, laying a solid foundation for the development of collagen hemostatic dressings. This method also expands the development and utilization of collagen.

Claims

1. A method for preparing collagen sponge using oxidized sodium carboxymethyl cellulose, characterized in that: The steps are as follows: (1) Prepare a sodium carboxymethyl cellulose oxide solution with a concentration of 15-45 mg / L; (2) adding collagen to acetic acid, mixing well, and dialyzing for 4-5 days to obtain a collagen solution; (3) Adding the sodium carboxymethyl cellulose oxide solution prepared in step (1) to the collagen solution obtained in step (2), mixing evenly, and cross-linking reaction at 0-4° C. for 20-30 hours. After freeze-drying, a collagen sponge is obtained.

2. The method according to claim 1, wherein In step (1), the concentration of the sodium carboxymethyl cellulose oxide solution is 25-35 mg / L; Further preferably, the concentration of the sodium carboxymethyl cellulose oxide solution is 30 mg / L.

3. The method according to claim 1, wherein In step (2), the collagen is cod skin collagen.

4. The method according to claim 1, wherein In step (2), the concentration of acetic acid is 0.1 to 1 M; the dialysis is specifically as follows: at 4°C, using a dialysis bag with a cutoff of 3 to 4 kDa, first dialyzing with a 0.1 M acetic acid solution for 24 hours, then dialyzing with ultrapure water for 3 to 5 days, and replacing the dialysate every 12 hours until the solution pH is 6.0 to 7.

0.

5. The method according to claim 1, wherein In step (2), the concentration of the collagen solution is 15 to 25 mg / L; Further preferably, the concentration of the collagen solution is 20 mg / L.

6. The method according to claim 1, wherein In step (3), the mass of the oxidized carboxymethyl cellulose sodium is 0.01 to 2.25% of the mass of the collagen; More preferably, the mass of the oxidized sodium carboxymethyl cellulose is 0.1% of the mass of collagen.

7. The method according to claim 1, wherein In step (3), the concentration of collagen in the mixed solution is 15 mg / ml; the freeze-drying is specifically: pre-freezing at -80°C for 3 to 4 hours, and then placing it in a freeze dryer for freeze drying.

8. A collagen sponge, characterized in that It is prepared according to the method according to any one of claims 1 to 7.

9. Use of the collagen sponge according to claim 8 in the preparation of medical materials.

10. Use of the medical material as claimed in claim 8 as a hemostatic material.

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