Performance-enhanced collagen sponge and method of making same

Abstract

The application provides a performance enhanced collagen sponge and a preparation method thereof. First, a collagen gel is prepared through an improved dialysis process, then the collagen gel is compounded with glycerol to form a collagen solution, then the solution is treated by using an irradiation cross-linking technology, and finally the product is prepared by irradiation sterilization after vacuum freeze-drying. The product of the application is directly dialyzed without acid dissolution, and the subsequent dialysis is performed by using a low-concentration phosphate buffer solution, so that the pH of the dialysis solution can be quickly adjusted to neutral, and the production cycle of the dialysis process is greatly shortened. In addition, by using the low-concentration phosphate buffer solution dialysis combined with the irradiation cross-linking process, the rehydration of the product is fast under the premise of ensuring the mechanical strength of the product, the formation of a 'dry core' in the material is avoided, the cell growth into the material is facilitated, and a certain amount of glycerol is added in the collagen solution before irradiation cross-linking, so that the collagen can be prevented from shrinking and deforming after irradiation cross-linking, and the size change of the product can be reduced.

Description

Technical Field

[0001] This invention falls under the category of medical products and relates to a performance-enhanced collagen sponge and its preparation method. This product can be used in fields such as plastic surgery, dentistry, and general surgery, and is mainly used for hemostasis and regenerative repair of soft tissue injuries. Background Technology

[0002] Collagen is an important bioregenerative material with low immunogenicity, biodegradability, and tissue repair-promoting properties. It has been processed into various forms, including powders, sponges, and films, for the repair of soft and hard tissue injuries. Collagen sponges are particularly widely used in plastic surgery, general surgery, dermatology, and trauma orthopedics, offering hemostatic and regenerative repair benefits. Currently, collagen sponges are mainly extracted from animal tissues such as bovine Achilles tendons, bovine hide, and porcine skin, or humanized collagen is prepared using genetic engineering technology. The collagen is then formulated into a solution of a certain concentration and freeze-dried under vacuum to create a porous sponge-like structure. This porous structure facilitates the absorption of blood or exudate and provides a three-dimensional scaffold for tissue regeneration and repair.

[0003] As a scaffold for tissue regeneration and repair, collagen sponges require a certain level of mechanical support, meaning they must provide space for cell and tissue ingrowth within a short period after implantation. However, natural collagen sponges have poor mechanical strength and degrade too quickly. While cross-linking with glutaraldehyde or carbodiimide cross-linking agents (EDC / NHS) can improve mechanical strength, it reduces the material's biocompatibility and hydrophilicity, especially for thicker collagen sponges such as plugs or cylinders, which are generally used for filling and repairing extraction sockets. Cross-linked products absorb water very slowly after implantation, allowing blood or bodily fluids to seep into the material. This can lead to insufficient blood supply, difficulty in cell and tissue ingrowth, and even granuloma formation after a certain period, ultimately resulting in implantation failure. Therefore, there is an urgent clinical need for a collagen sponge with high mechanical strength, good support, rapid rehydration, good biocompatibility, and easy cell and tissue ingrowth to solve these technical problems.

[0004] In view of this, the present invention is hereby proposed. Summary of the Invention

[0005] This invention provides a performance-enhanced collagen sponge and its preparation method. An optimized dialysis process is used to prepare a collagen gel matrix, which is then compounded with glycerol to form a homogeneous solution system. Subsequently, irradiation is used to initiate a molecular cross-linking reaction. Finally, freeze-drying technology is combined to successfully prepare a collagen sponge material with enhanced mechanical properties and water absorption characteristics. This invention can quickly adjust the pH of the dialysis solution to neutral, significantly shortening the production cycle of the dialysis process. While ensuring the mechanical strength of the product, it rehydrates quickly, avoids the formation of a "dry core" inside the material, and facilitates cell ingrowth into the material. It can also prevent collagen shrinkage and deformation after irradiation cross-linking, reducing changes in product size. It has broad application prospects and is conducive to widespread application.

[0006] This invention provides a method for preparing a performance-enhanced collagen sponge, comprising the following steps:

[0007] S1: Dialysis, the collagen raw material is dialyzed using water for injection and phosphate buffer until the pH of the solution reaches above 6.0;

[0008] S2: Crosslinking. Take the collagen sample after S1 dialysis, mix it with glycerol to prepare a collagen solution, the collagen solution contains 0.1-2% glycerol and 0.5-1% collagen, stir thoroughly and evenly, dispense into freeze-drying molds, and crosslink by irradiation.

[0009] S3: Freeze-drying. The gel-like sample obtained by cross-linking S2 is freeze-dried, packaged, and then sterilized by irradiation to prepare a performance-enhanced collagen sponge.

[0010] Preferably, in S1, the collagen raw material is extracted from animal tissues such as bovine Achilles tendon, cowhide, pigskin, or fish skin, or is recombinant human collagen.

[0011] Preferably, in S1, the phosphate buffer is obtained by diluting commercially available PBS solution 10-100 times, and the dialysis method of the phosphate buffer is: dialysis for 1-4 days, with the solution changed every 6-12 hours.

[0012] Preferably, in step S2, the irradiation is cobalt-60 irradiation or electron beam irradiation, and the irradiation dose is 2-10 kGy.

[0013] Preferably, in step S2, the freeze-drying mold is a cube, frustum, or cone shape, used to make plug-shaped collagen sponges.

[0014] Preferably, in step S3, the irradiation sterilization is performed using cobalt-60 irradiation sterilization, electron beam irradiation sterilization, or ethylene oxide sterilization.

[0015] The present invention also provides a performance-enhanced collagen sponge prepared by the above preparation method.

[0016] The present invention provides a performance-enhanced collagen sponge and its preparation method, which has the following beneficial effects.

[0017] 1. Traditional collagen preparation methods typically involve dissolving the extracted and salted-out collagen in dilute acid before dialysis. The product of this invention is directly dialyzed without acid dissolution, and is subsequently dialyzed using a low-concentration phosphate buffer solution, which can quickly adjust the pH of the dialysis solution to neutral, greatly shortening the production cycle of the dialysis process.

[0018] 2. To improve the mechanical strength of products, commercially available collagen sponges generally use chemical cross-linking processes, but this can easily lead to water being unable to penetrate the material and forming a "dry core," resulting in poor biocompatibility. In contrast, the product of this invention uses a low-concentration phosphate buffer solution for dialysis, combined with an irradiation cross-linking process. While ensuring the mechanical strength of the product, it rehydrates quickly and avoids the formation of a "dry core" inside the material, which is conducive to cell ingrowth into the material.

[0019] 3. Adding a certain amount of glycerol to the collagen solution formulation before irradiation crosslinking in this invention can prevent collagen from shrinking and deforming after irradiation crosslinking and reduce changes in product size. Attached Figure Description

[0020] Figure 1 This is a photograph of the actual appearance (sheet-shaped) of the collagen sponge of the present invention.

[0021] Figure 2 This is a photograph of the actual appearance (plug-like) of the collagen sponge product of the present invention.

[0022] Figure 3 H&E staining photographs of rats in Comparative Example 2 after subcutaneous implantation 4 weeks later;

[0023] Figure 4 This is an H&E staining photograph of rats that received the sample in Example 1 after 4 weeks of subcutaneous implantation. Detailed Implementation

[0024] The present invention will be further described below with reference to specific embodiments and accompanying drawings to help understand the content of the present invention.

[0025] This invention provides a method for preparing a performance-enhanced collagen sponge, comprising the following steps:

[0026] S1: Dialysis. The collagen raw material is dialyzed using water for injection and phosphate buffer until the pH of the solution reaches 6.0 or higher. The collagen raw material is extracted from animal tissues such as bovine Achilles tendon, bovine hide, pig skin, or fish skin, or is recombinant human collagen. The phosphate buffer is obtained by diluting commercially available PBS solution 10-100 times. The dialysis method with phosphate buffer is: dialysis for 1-4 days, with the solution changed every 6-12 hours.

[0027] S2: Cross-linking. Take the collagen sample after S1 dialysis, and prepare a collagen solution by mixing it with glycerol. The collagen solution contains 0.1-2% glycerol and 0.5-1% collagen. Stir thoroughly until homogeneous, dispense into freeze-drying molds, and cross-link using irradiation. The irradiation is cobalt-60 irradiation or electron beam irradiation, with an irradiation dose of 2-10 kGy. The freeze-drying mold is cubic, frustum, or conical in shape, used to make plug-like collagen sponges.

[0028] S3: Freeze-drying. The gel-like sample obtained by cross-linking in S2 is freeze-dried, packaged, and then sterilized by irradiation to prepare a performance-enhanced collagen sponge. The irradiation sterilization is performed by cobalt-60 irradiation sterilization, electron beam irradiation sterilization, or ethylene oxide sterilization.

[0029] The present invention also provides a performance-enhanced collagen sponge prepared by the above preparation method.

[0030] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; and the materials and reagents used can be purchased commercially unless otherwise specified.

[0031] Example 1: A method for preparing bovine collagen sponge

[0032] Using bovine Achilles tendon as raw material, type I collagen was extracted by acid extraction combined with pepsin digestion according to the conventional method described in the literature. After salting out, collagen precipitate was obtained, and then collagen sponge was prepared using the following steps:

[0033] S1: Dialysis. The collagen precipitate obtained after salting out is placed in a dialysis bag and dialyzed with water for injection for 3 days, changing the solution every 6 hours. Then, dialyze with 0.01× phosphate buffer (i.e., commercially available PBS solution diluted 100 times) for 4 days, changing the solution every 12 hours. At the end of dialysis, the pH of the solution in the dialysis bag should reach above 6.0.

[0034] S2: Cross-linking, i.e., taking the collagen sample after dialysis in the previous step and preparing a collagen solution (mass fraction) according to the following formula:

[0035] Glycerin 0.1%

[0036] Collagen 0.5%

[0037] Water 99.4%

[0038] After thorough mixing, the mixture is dispensed into freeze-drying molds and then cross-linked by cobalt-60 irradiation at a dose of 2 kGy. If the freeze-drying molds are designed in the shape of a cube, frustum, or cone, plug-like collagen sponges can be prepared.

[0039] S3: Freeze-drying, which involves placing the gel-like sample obtained by cross-linking in the previous step into a vacuum freeze dryer for freeze-drying until it is completely freeze-dried.

[0040] Cut the freeze-dried sponge sample from the previous step into the required size and specifications, package it, and then sterilize it with cobalt-60 irradiation to obtain the performance-enhanced collagen sponge. See also: sheet-shaped collagen sponge and plug-shaped collagen sponge. Figure 1 and Figure 2 .

[0041] Example 2: A method for preparing porcine collagen sponge

[0042] Using pigskin as raw material, type I collagen was extracted by acid extraction combined with pepsin digestion according to the conventional method described in the literature. After salting out, collagen precipitate was obtained, and then collagen sponge was prepared using the following steps:

[0043] S1: Dialysis. The collagen precipitate obtained after salting out is placed in a dialysis bag and dialyzed with water for injection for 7 days, changing the solution every 12 hours. Then, dialyze with 0.1× phosphate buffer (i.e., commercially available PBS solution diluted 10 times) for 1 day, changing the solution every 6 hours. At the end of dialysis, the pH of the solution in the dialysis bag should reach above 6.0.

[0044] S2: Cross-linking, i.e., taking the collagen sample after dialysis in the previous step and preparing a collagen solution (mass fraction) according to the following formula:

[0045] 2% glycerol

[0046] 1% Collagen

[0047] Water 97%

[0048] After thorough mixing, the mixture is dispensed into freeze-drying molds and then cross-linked using electron beam irradiation at a dose of 10 kGy.

[0049] S3: Freeze-drying, which involves placing the gel-like sample obtained by cross-linking in the previous step into a vacuum freeze dryer for freeze-drying until it is completely freeze-dried.

[0050] Cut the freeze-dried sponge sample from the previous step into the required size and specifications, package it, and sterilize it with ethylene oxide to obtain the finished porcine collagen sponge.

[0051] Example 3: A method for preparing recombinant human collagen sponge

[0052] Using commercially available recombinant human type III collagen as raw material, a collagen solution was prepared using dilute acid. After salting out, collagen precipitate was obtained. Then, collagen sponges were prepared using the following steps:

[0053] S1: Dialysis. The collagen precipitate obtained after salting out is placed in a dialysis bag and dialyzed with water for injection for 5 days, changing the solution every 8 hours. Then, dialyze with 0.05× phosphate buffer (i.e., commercially available PBS solution diluted 20 times) for 3 days, changing the solution every 8 hours. At the end of dialysis, the pH of the solution in the dialysis bag should reach above 6.0.

[0054] S2: Cross-linking, i.e., taking the collagen sample after dialysis in the previous step and preparing a collagen solution (mass fraction) according to the following formula:

[0055] 1% glycerol

[0056] Collagen 0.7%

[0057] Water 98.3%

[0058] After thorough mixing, the mixture is dispensed into freeze-drying molds and then cross-linked by cobalt-60 irradiation at a dose of 6 kGy.

[0059] S3: Freeze-drying, which involves placing the gel-like sample obtained by cross-linking in the previous step into a vacuum freeze dryer for freeze-drying until it is completely freeze-dried.

[0060] Cut the freeze-dried sponge sample from the previous step into the required size and specifications, package it, and then sterilize it by electron beam irradiation to obtain the finished recombinant human collagen sponge.

[0061] Comparative Example 1: Preparation and Performance Comparison of Collagen Sponges Using Different Dialysis Processes

[0062] Preparation and pH test of Comparative Example 1: Collagen sponges were prepared using a traditional dialysis process. The collagen precipitate obtained after salting out was placed in a dialysis bag and dialyzed with 0.6% acetic acid solution for 3 days to fully dissolve the collagen precipitate. Then, the sample was dialyzed with water for injection, with the solution changed every 8 hours. The pH of the sample in the dialysis bag was monitored daily. The results showed that the average pH of the sample after 7 and 14 days of dialysis was 3.6±0.3 and 5.1±0.2, respectively. Samples were taken at 7 and 14 days of dialysis, and performance-enhanced collagen sponges were prepared according to the methods described in steps S2 and S3 of Example 1. The pH of the products was determined according to the method described in the industry standard "YY / T 1511-2017 Collagen Sponges". The results showed that the pH of the collagen sponges prepared by sampling at 7 and 14 days of dialysis was 3.8±0.2 and 5.1±0.3, respectively. The sample prepared at 7 days of dialysis did not meet the pH requirement of 4.0-7.0 in "YY / T 1511-2017 Collagen Sponges".

[0063] Acidity / alkalinity test of the samples in Example 1: The collagen precipitate was dialyzed using the dialysis method described in step S1 of Example 1. Three batches of samples were processed, and the acidity / alkalinity of the samples in the dialysis bag was measured at the end of dialysis. The results showed that the average pH value of the three batches of samples at the end of dialysis was 6.7±0.2. The acidity / alkalinity of the finished product was determined according to the method described in the industry standard "YY / T 1511-2017 Collagen Sponge", and the result was 6.3±0.2, which meets the requirement of 4.0-7.0 in "YY / T 1511-2017 Collagen Sponge".

[0064] The above tests show that, compared with the traditional method of first dissolving with acid and then dialysis, the present invention allows the collagen precipitate after salting out to be directly dialyzed without being dissolved with acid, and the subsequent use of low-concentration phosphate buffer for dialysis can quickly adjust the pH of the dialysis solution to neutral, greatly shortening the production cycle of the dialysis process.

[0065] Comparative Example 2: Preparation of Chemically Cross-linked Collagen Sponge

[0066] Using the dialysis and freeze-drying steps described in Example 1, the irradiation crosslinking was replaced with chemical crosslinking, i.e., bovine Achilles tendon was used as raw material, and type I collagen was extracted using the conventional acid method described in the literature. After salting out, collagen precipitate was obtained, and then collagen sponge was prepared using the following steps:

[0067] S1: Dialysis, that is, the collagen precipitate obtained after salting out is placed into a dialysis bag, first dialyzed with water for injection for 3 days, changing the medium every 6 hours; then dialyzed with 0.01× phosphate buffer (that is, commercially available PBS solution diluted 100 times) for 4 days, changing the medium every 12 hours.

[0068] S2: Freeze-drying, which involves adding water to the gel-like sample obtained by dialysis in the previous step to prepare a 0.5% collagen solution, stirring thoroughly, dispensing it into freeze-drying molds, and placing it in a vacuum freeze dryer for freeze-drying until completely freeze-dried.

[0069] S3: Chemical cross-linking. Take the collagen sponge sample from the previous freeze-drying step and place it in a cross-linking solution containing 0.019% 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 0.0058% N-hydroxysuccinimide for 24 hours at 4°C. Then, take out the sample and wash it thoroughly with PBS solution to remove any cross-linking agent residue. Place it in a vacuum freeze dryer for freeze-drying again. After freeze-drying, cut it into the required size and specifications, package it, and sterilize it with cobalt-60 irradiation to obtain the chemically cross-linked collagen sponge product.

[0070] Comparative Example 3: Preparation of Collagen Sponge Without Added Glycerin

[0071] The collagen solution formulation in the "crosslinking" step of Example 1 was adjusted to contain only 0.5% collagen, i.e., glycerol was removed. The remaining steps were the same as in Example 1, and the appearance of the samples during preparation was observed. The results showed that compared to the collagen sponge prepared in Example 1, the sample in Comparative Example 3 exhibited significant shrinkage and deformation after irradiation crosslinking, releasing a large amount of water. After freeze-drying, the sample surface appeared uneven. Therefore, it can be concluded that adding glycerol can reduce the degree of deformation of the sample after irradiation crosslinking to a certain extent.

[0072] Performance Testing: Comparison of Rehydration Performance and Subcutaneous Implantation Test

[0073] Rehydration performance test: Take the samples prepared in Example 1, Comparative Example 1 (dialysis for 14 days), and Comparative Example 2, cut them into 1cm×1cm squares, measure the initial thickness of the samples, place them in physiological saline, and observe them until the samples are completely rehydrated and become translucent (the unrehydrated particles are white). Record the time required for complete rehydration. Then, take out the samples, gently press and squeeze out the water from the material, and rehydrate them again. Repeat this process 50 times. Finally, place the samples in physiological saline to fully rehydrate, take out the samples to test the thickness, and calculate the thickness change rate according to the following formula:

[0074] Thickness change rate = (thickness before rehydration - thickness after rehydration) / thickness before rehydration

[0075] Experimental results showed that the rehydration times of the samples prepared in Example 1 and Comparative Example 1 were 50 s and 820 s, respectively, while the sample in Comparative Example 2 could not be completely wetted even after soaking in physiological saline for 24 hours, and a "dry core" phenomenon existed inside the material. This indicates that the present invention uses low-concentration phosphate buffer for dialysis, combined with irradiation crosslinking, which can achieve faster rehydration and avoid the formation of a "dry core" inside the material compared with chemical crosslinking. In addition, after 50 cycles of compression, the thickness change rates of the samples prepared in Example 1, Comparative Example 1 (dialyzed for 14 days) and Comparative Example 2 were 5.3%, 16.7%, and 4.9%, respectively. This indicates that the present invention uses low-concentration phosphate buffer for dialysis, which can improve the mechanical support and resilience of the material, reduce the material deformation after external compression (5.3% vs. 16.7%), and the combination of irradiation crosslinking can achieve a mechanical property improvement effect comparable to that of EDC / NHS chemical crosslinking (5.3% vs. 4.9%).

[0076] Animal experiment with subcutaneous implantation in rats: Following the method described in "GB / T16886.6-2022 Biological Evaluation of Medical Devices Part 6: Local Reaction Test after Implantation", the local reaction of the samples prepared in Example 1 and Comparative Example 2 after subcutaneous implantation in the back of SD rats was evaluated. The results showed that 4 weeks after subcutaneous implantation in rats, the sample group of Comparative Example 2 showed very few cells infiltrating into the material, with only a small number of cells adhering to the surface or superficial layer of the material. Figure 3 As shown, this may be due to chemical cross-linking leading to increased hydrophobicity, slower water absorption and easy "drying" of the material, and decreased biocompatibility; while the material of Example 1 sample showed more fibroblast infiltration and angiogenesis, such as Figure 4 As shown, this indicates that compared to chemically cross-linked collagen sponges, the product of this invention is more conducive to cell and tissue ingrowth into the material, and more easily integrates with autologous tissue to achieve tissue remodeling. Furthermore, anatomical examination revealed that the material was still present at the implantation site 8 weeks after implantation, indicating that the irradiation cross-linking process can delay the degradation of the collagen sponge in vivo.

[0077] This invention employs an optimized dialysis process to prepare a collagen gel matrix, which is then combined with glycerol to form a homogeneous solution system. Irradiation is then used to initiate a molecular cross-linking reaction, and finally, freeze-drying technology is combined to successfully prepare a collagen sponge material with enhanced mechanical properties and water absorption characteristics. The product of this invention is directly dialyzed without acid dissolution, and subsequent dialysis using a low-concentration phosphate buffer solution allows for rapid adjustment of the dialysis solution's pH to neutral, significantly shortening the production cycle of the dialysis process. Furthermore, the use of low-concentration phosphate buffer dialysis, combined with the irradiation cross-linking process, ensures rapid rehydration while maintaining the product's mechanical strength, preventing the formation of a "dry core" within the material and facilitating cell ingrowth. Adding a certain amount of glycerol to the collagen solution formulation before irradiation cross-linking prevents collagen shrinkage and deformation after irradiation cross-linking, reducing changes in product size.

[0078] This article uses specific examples to illustrate the inventive concept in detail. The description of the above embodiments is only for the purpose of helping to understand the core idea of ​​the present invention. It should be noted that any obvious modifications, equivalent substitutions or other improvements made by those skilled in the art without departing from the inventive concept should be included within the protection scope of the present invention.

Claims

1. A method for preparing a performance-enhancing collagen sponge, characterized in that, Includes the following steps: S1: Dialysis. The collagen raw material is dialyzed using water for injection and phosphate buffer until the pH of the solution reaches 6.0 or higher. The phosphate buffer is obtained by diluting commercially available PBS solution 10-100 times. The dialysis method with phosphate buffer is: dialysis for 1-4 days, with the solution changed every 6-12 hours. S2: Crosslinking. Take the collagen sample after S1 dialysis, mix it with glycerol to prepare a collagen solution, the collagen solution contains 0.1-2% glycerol and 0.5-1% collagen, stir thoroughly and evenly, dispense into freeze-drying molds, and crosslink by irradiation. S3: Freeze-drying. The gel-like sample obtained by cross-linking S2 is freeze-dried, packaged, and then sterilized by irradiation to prepare a performance-enhanced collagen sponge.

2. The method for preparing a performance-enhancing collagen sponge according to claim 1, characterized in that, In S1, the collagen raw material is extracted from animal tissues such as bovine Achilles tendon, cowhide, pigskin, or fish skin, or is recombinant human collagen.

3. The method for preparing a performance-enhancing collagen sponge according to claim 2, characterized in that, In S2, the irradiation is cobalt-60 irradiation or electron beam irradiation, and the irradiation dose is 2-10 kGy.

4. The method for preparing a performance-enhancing collagen sponge according to claim 3, characterized in that, In S2, the freeze-drying mold is a cube, frustum, or cone shape, used to make plug-shaped collagen sponges.

5. The method for preparing a performance-enhancing collagen sponge according to claim 4, characterized in that, In S3, irradiation sterilization is performed using cobalt-60 irradiation sterilization, electron beam irradiation sterilization, or ethylene oxide sterilization.

6. A performance-enhanced collagen sponge prepared by any one of the preparation methods described in claims 1-5.

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