A collagen implant, its preparation method and application
By employing a single homogenization, freeze-drying, sterilization, and cross-linking process, the problems of high sterilization difficulty, viral risk, and rapid degradation in existing collagen implants have been solved, achieving the preparation of collagen implants with high biosafety and injectability.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- BEIJING YH BIOMAX BIOLOGIC TECH
- Filing Date
- 2026-04-21
- Publication Date
- 2026-07-14
AI Technical Summary
Existing technologies for preparing collagen implants have several drawbacks, including high difficulty in sterilization and filtration, the risk of viral infections from animal-derived materials, long production cycles, high costs, and rapid collagen degradation.
Collagen implants were prepared using a method involving homogenization, freeze-drying, sterilization, homogenization with phosphate buffer, and cross-linking.
It improves the biocompatibility and mechanical strength of collagen implants, prolongs the duration of the filling effect, and ensures the sterility and injectability of the product.
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Abstract
Description
Technical Field
[0001] This application relates to the technical fields of clinical medicine and materials science, specifically to a collagen implant, its preparation method, and its application. Background Technology
[0002] Wrinkles are the most obvious and prominent sign of skin aging, a major factor affecting perceived age, and one of the most significant markers of aging.
[0003] The dermis is the connective tissue layer to which the epidermis attaches, approximately 1-2 mm thick. It is divided into papillary dermis and reticular dermis, mainly containing fibroblasts responsible for secreting collagen, elastin, glycosaminoglycans, proteoglycans, fibronectin, and other extracellular matrix proteins, providing support and elasticity for the skin. The density, thickness, and tissue density of dermal collagen are major contributors to the overall firmness and elasticity of the tissue. With age, dermal collagen and elastic fibers degrade, leading to a decline in skin support and elasticity, a significant factor in skin laxity and sagging, clinically manifested as wrinkles and drooping.
[0004] Collagen accounts for 70-80% of the dermis's mass. Type I and Type III collagen, forming fibrous structures, are responsible for the skin's strength and elasticity, and also contain other extracellular matrix proteins. Therefore, developing collagen implants for filling soft tissues such as mucous membranes and skin has broad application prospects.
[0005] Current technologies for preparing collagen implants involve purifying and filtering proteins (large molecules) from animal tissues to obtain sterile raw materials, which are then manufactured into implants using aseptic methods. However, collagen is a large molecular material, making sterilization and filtration processes challenging. Furthermore, animal-derived materials pose a risk of carrying viruses, typically requiring specialized processes for virus inactivation. Aseptic filtration alone cannot guarantee complete virus removal. While purifying collagen from virus-free animals and then filtering it for sterilization involves a long production cycle, complex processes, and high costs, collagen is also a biodegradable material, and uncrosslinked collagen degrades relatively quickly. Summary of the Invention
[0006] To address the aforementioned technical problems, this application provides a collagen implant, its preparation method, and its application.
[0007] This application provides a method for preparing a collagen implant, specifically including the following steps in sequence: First homogenization: Bovine Achilles tendon collagen was added to a solution with a pH of 4.0-8.0 to obtain a collagen slurry with a concentration of 0.5-1wt%, and homogenized at 4-20℃ and 6000-2000rpm for 1.5-4h; then filtered to obtain a first homogenized slurry; Freeze-drying: The homogenized slurry is freeze-dried and then sterilized to obtain sterile collagen raw material. The freeze-drying parameters are as follows: the slurry is cooled to below -20℃ and held for 1.5~3h; then heated to -10~0℃ and held for 15~40h; then heated to 20~35℃ at a heating rate of 0.5-3℃ / min and held for 0.5~2h. Secondary homogenization: Sterile collagen raw material was added to 0.01~0.3M phosphate buffer solution with pH 7.0~7.4 to obtain collagen slurry with a concentration of 1~7wt%, and homogenized at 15~25℃ and 6000-15000rpm for 1~10h. Crosslinking: Add 0.001~0.05wt% of crosslinking agent to the slurry, perform homogenization and crosslinking, and obtain the collagen implant by aseptic packaging.
[0008] The working principle of the preparation method of the collagen implant provided in this application is as follows.
[0009] The first stage involves homogenizing bovine Achilles tendon collagen in a solution with a pH of 4.0–8.0. This process disrupts the covalent cross-linking within the collagen fibers, causing the tightly packed collagen fiber bundles to swell and loosen. Next, homogenization physically depolymerizes and disperses the collagen. Under high shear forces, the swollen collagen fiber bundles are mechanically broken down, significantly reducing the collagen particle size and laying the foundation for a uniform implant. Finally, filtration removes undepolymerized coarse fibers, tissue residue, and insoluble impurities, yielding a homogeneous collagen homogenized slurry.
[0010] The second stage involves freeze-drying the primary homogenized slurry to remove moisture, resulting in a solid raw material that provides standardized feedstock for the next stage of secondary homogenization. This material is then sterilized to ensure biosafety.
[0011] The third stage: Secondary homogenization redisperses the sterile collagen raw material to prepare a highly homogeneous collagen gel suitable for injection. Phosphate buffer provides physiological pH and ionic strength, ensuring the biocompatibility of the collagen in the final application. Compared to the 0.5-1% collagen concentration of primary homogenization, the increased collagen concentration (1-7%) ensures that the final implant forms an injectable gel with appropriate viscoelasticity, sufficient mechanical strength (filling effect), and long retention time in the body.
[0012] The fourth stage: cross-linking enhances the collagen network through chemical methods, enabling it to resist rapid degradation by collagenase in vivo and prolonging the duration of the filling effect. By controlling the amount of cross-linking agent and the cross-linking time, the cross-linking density can be precisely controlled, thereby ultimately regulating the product's viscosity (injectability) and in vivo degradation rate.
[0013] Preferably, the bovine Achilles tendon collagen is bovine Achilles tendon type I collagen, with the following performance parameters: collagen content ≥98%, impurity protein content ≤1%, and fat content ≤1%.
[0014] Preferably, the method of homogenization is as follows: bovine Achilles tendon collagen is added to a solution with a pH of 4.0 to 7.0 to obtain a collagen slurry with a concentration of 0.5 to 1 wt%, and homogenized at 6 to 20°C and 8000 to 12000 rpm for 1 to 3 hours; then filtered to obtain a homogenized slurry.
[0015] Preferably, the freeze-drying parameters are as follows: the homogenized slurry is transferred to a freeze-drying mold with a thickness of 5-6 cm, and cooled to -4-4℃ and held for 20-60 min; then cooled to below -20℃ and held for 1.5-3 h; the temperature is raised to -4-0℃ and held for 15-40 h under a pressure ≤-0.3 mbar; then the temperature is raised to 20-35℃ at a heating rate of 0.5-3℃ / min and held for 0.5-4 h.
[0016] Preferably, the process parameters for freeze drying are as follows: transfer the homogenized slurry to a freeze-drying mold with a thickness of 5-6 cm, cool to 0-2℃ and hold for 20-60 min; cool to below -20℃ and hold for 1.5-3 h; heat to -1-0℃ and hold for 20-30 h under a pressure ≤ -0.3 mbar; then heat to 20-32℃ at a heating rate of 1-2℃ / min and hold for 0.5-2 h.
[0017] Preferably, the secondary homogenization method is as follows: add sterile collagen raw material to 0.1~0.2M phosphate buffer solution with pH 7.0~7.4 to obtain collagen slurry with a concentration of 3~5wt%, and homogenize at 15~25℃ and 8000-12000rpm for 3~8h.
[0018] Preferably, the crosslinking method is as follows: 0.001~0.05wt% of crosslinking agent is added to the slurry, and homogenous crosslinking is carried out at 15~25℃ and 8000-15000rpm for 8~36h; then aseptic filling is performed to obtain the collagen implant; the crosslinking agent is selected from glutaraldehyde or EDC / NHS.
[0019] Preferably, the crosslinking agent is a glutaraldehyde solution with a concentration of 0.1~5wt%.
[0020] This application also provides a collagen implant, which is prepared using the aforementioned preparation method.
[0021] This application also provides the use of the described collagen implant in the preparation of medical materials.
[0022] In summary, the technical solution of this application has the following effects: The collagen implant prepared in this application undergoes two homogenization processes: the first homogenization is performed under low-concentration conditions, primarily to remove large, difficult-to-disperse particles and obtain a more uniform collagen particle size, thus shortening the time required for the second homogenization. The second homogenization is performed at high concentrations, with the main purpose of uniformly mixing the higher concentration of collagen with phosphate buffer to obtain a homogeneous collagen slurry.
[0023] The collagen implant prepared in this application is obtained by freeze-drying the material after the first homogenization, followed by sterilization of the freeze-dried raw material to obtain sterile raw material. Compared with sterilization by filtration, collagen raw material sterilized by EO or irradiation has a higher level of sterility assurance. Moreover, compared with terminally sterilized collagen implants, sterilization of raw material can effectively avoid denaturation or degradation problems caused by temperature rise in the implant during irradiation.
[0024] The collagen implant prepared in this application has different homogenization concentrations during the first homogenization process. The main purpose is to control the collagen viscosity and obtain a collagen slurry with more uniform particles.
[0025] The collagen implant prepared in this application is freeze-dried after the first homogenization and sterilized to obtain sterile collagen raw material; this step makes the low-concentration collagen slurry into a solid, removes water, and sterilizes to obtain sterile raw material; and then undergoes a second homogenization to obtain a high-concentration collagen slurry.
[0026] The collagen implant prepared in this application is cross-linked, which can more effectively improve the retention time after implantation. Attached Figure Description
[0027] Figure 1 This is a staining image of the collagen implant in Example 1 after it was implanted into the dermis of a rabbit. Detailed Implementation
[0028] The present application will be further described in detail below with reference to embodiments, comparative examples and performance test results. These embodiments should not be construed as limiting the scope of protection claimed in this application.
[0029] Example
[0030] Example 1
[0031] Example 1 provides a method for preparing a collagen implant.
[0032] In this embodiment, the performance parameters of bovine Achilles tendon type I collagen are as follows: protein content is higher than 98%, impurity protein content is lower than 1%, fat content is not higher than 1%; it does not contain tryptophan, and hydroxyproline content is higher than 10%.
[0033] In this embodiment, the collagen production method is derived from patent CN110743044 B.
[0034] In this embodiment, the homogenization process uses an IKA disperser.
[0035] The preparation method of the collagen implant in this embodiment is as follows.
[0036] First homogenization: 7g of bovine Achilles tendon type I collagen was added to 1L of acidic solution with pH 6.5 to obtain a collagen slurry with a concentration of 0.7wt%, and homogenized at 20℃ and 10000rpm for 2h; then filtered to obtain a first homogenized slurry.
[0037] Freeze-drying: The homogenized slurry was freeze-dried under specific conditions. The freeze-dried collagen was packaged in dialysis bags and sterilized with ethylene oxide (EO) to obtain sterile collagen raw material. The freeze-drying parameters were as follows: the slurry was held at 0°C for 30 min; cooled to -40°C and held for 2 h; heated to -5°C and held at a pressure of -0.4 mbar for 24 h; and then heated to 25°C at a rate of 2°C / min and held for 1 h.
[0038] Secondary homogenization: 3g of sterile collagen raw material was added to 0.1M phosphate buffer solution with pH 7.2 to obtain a collagen slurry with a concentration of 3wt%, and homogenized at 20℃ and 10000rpm for 5h.
[0039] Crosslinking: Add 0.005wt% of crosslinking agent (5wt% glutaraldehyde solution) to the slurry and perform homogenization crosslinking at 20℃ and 10000rpm for 12h. The collagen implant is then obtained by aseptic packaging.
[0040] Examples 2-4 Examples 2-4 each provide a method for preparing a collagen implant.
[0041] The difference between the above embodiments and Embodiment 1 is that the process parameters for the first homogenization are different, as shown below.
[0042] In Example 2: In the single homogenization method, the collagen slurry concentration was 0.5wt%, and it was homogenized at 20℃ and 10000rpm for 3h.
[0043] In Example 3: In the single homogenization method, the collagen slurry concentration was 0.8 wt%, and it was homogenized at 20°C and 10,000 rpm for 6 hours.
[0044] In Example 4: In the single homogenization method, the collagen slurry concentration was 0.8 wt%, and it was homogenized at 20°C and 10,000 rpm for 7 hours.
[0045] All other process parameters in the above embodiments are the same as those in Embodiment 1.
[0046] Examples 5-6 Examples 5-6 provide a method for preparing a collagen implant.
[0047] The difference between the above embodiments and Embodiment 1 is that the freeze-drying process parameters are different, as shown below.
[0048] In Example 5, the freeze-drying parameters were as follows: the slurry was cooled to -40°C and held for 2 hours; then heated to -5°C and held at a pressure of -0.4 mbar for 24 hours; and then heated to 25°C at a rate of 2°C / min and held for 1 hour.
[0049] In Example 6, the freeze-drying parameters were as follows: the slurry was kept at 0°C for 30 min; cooled to -40°C and kept for 2 h; heated to -5°C and kept at a pressure of -0.4 mbar for 24 h; and then heated to 25°C at a heating rate of 2°C / min and kept for 1 h.
[0050] All other process parameters in the above embodiments are the same as those in Embodiment 1.
[0051] Examples 7-8 Examples 7-8 provide a method for preparing a collagen implant.
[0052] The specific difference between the above embodiments and Embodiment 1 is that the crosslinking methods are different, as detailed below.
[0053] In Example 7, the crosslinking method was to add 0.03 wt% of the crosslinking agent (the crosslinking agent is a 1 wt% glutaraldehyde solution) to the slurry and perform homogeneous crosslinking for 18 hours.
[0054] In Example 8, the crosslinking method was to add 0.01 wt% of the crosslinking agent (a 10 wt% glutaraldehyde solution) to the slurry and perform homogeneous crosslinking for 12 hours.
[0055] Performance testing Appearance and morphology: The appearance of the collagen implant prepared in the examples in the glass bottle was observed with the naked eye.
[0056] In vitro degradation time: Prepare collagenase solution (weigh 6.06g of tris(hydroxymethyl)aminomethane, dissolve in 450mL of purified water, adjust the pH to 7.4 with hydrochloric acid, and bring the volume to 500mL to prepare 0.1mol / L HCl-tris buffer).
[0057] Collagenase was added to HCl-tris buffer to prepare a collagenase solution of 0.025 mg / ml.
[0058] Weigh 1g of collagen injection using an analytical balance and add 10g of prepared 0.025mg / ml collagenase solution to a test tube. Incubate in a 37℃ water bath. After the set time points (1h, 2h...9h...12h), remove the sample, centrifuge, and use a pipette to transfer 1g of the supernatant to a vial at room temperature. Add 1ml of hydrochloric acid to the vial, seal, and hydrolyze in a 110℃ oven for 24h. The HYP content can be obtained by measuring the absorbance using a UV spectrophotometer via a colorimetric method. The HYP content stabilization time is the complete degradation time. Perform the experiment in triplicate.
[0059] Injectability (viscosity): The viscosity of the collagen implant was measured at 25°C using an RVDV-1T viscometer with a #29 rotor at 20 rpm.
[0060] Test results are shown in Table 1.
[0061] Table 1. Performance test results of collagen implants in the examples and comparative examples.
[0062] The test results in the table above show that the collagen implant prepared using the technical solution provided in this application is a uniform gel-like substance without flocculants or precipitates. Its in vitro degradation time is ≥5 hours, and its viscosity is within the range of 6000-23000 cp. This range ensures that the collagen implant product is neither too thin (easily diffuses, lacks support) nor too thick (difficult to inject). The above performance test results indicate that the collagen implant prepared in this application has a good appearance and excellent resistance to degradation and injectability.
[0063] Application Example 1 Rabbit intradermal implantation experiment: The collagen implant prepared in Example 1 was implanted into the dermis of rabbits.
[0064] After implantation, the degradation of the material can be assessed through visual observation and tactile examination: after complete collagen degradation, no obvious bulges are observed on the skin surface at the original implantation site, and the surface feels smooth and flat. Furthermore, tissue samples are taken from the implantation site and stained; the results are as follows... Figure 1 As shown in the image, the dermis in the implanted area is thickened and the structure is full, forming a noticeable skin bulge, further confirming that the collagen implant can effectively play a filling and supporting role in the dermis.
[0065] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.
Claims
1. A method for preparing a collagen implant, characterized in that, Specifically, the following steps are performed sequentially: First homogenization: Bovine Achilles tendon collagen was added to a solution with a pH of 4.0-8.0 to obtain a collagen slurry with a concentration of 0.5-1wt%, and homogenized at 4-20℃ and 6000-2000rpm for 1.5-4h; then filtered to obtain a first homogenized slurry; Freeze-drying: The homogenized slurry is freeze-dried and then sterilized to obtain sterile collagen raw material. The freeze-drying parameters are as follows: the slurry is cooled to below -20℃ and held for 1.5~3h; then heated to -10~0℃ and held for 15~40h; then heated to 20~35℃ at a heating rate of 0.5-3℃ / min and held for 0.5~2h. Secondary homogenization: Sterile collagen raw material was added to 0.01~0.3M phosphate buffer solution with pH 7.0~7.4 to obtain collagen slurry with a concentration of 1~7wt%, and homogenized at 15~25℃ and 6000-15000rpm for 1~10h. Crosslinking: Add 0.001~0.05wt% of crosslinking agent to the slurry, perform homogenization and crosslinking, and obtain the collagen implant by aseptic packaging.
2. The method for preparing the collagen implant according to claim 1, characterized in that, The bovine Achilles tendon collagen is bovine Achilles tendon type I collagen, with the following performance parameters: collagen content ≥98%, impurity protein content ≤1%, and fat content ≤1%.
3. The method for preparing the collagen implant according to claim 1, characterized in that, The method for homogenization is as follows: Bovine Achilles tendon collagen is added to a solution with a pH of 4.0 to 7.0 to obtain a collagen slurry with a concentration of 0.5 to 1 wt%, and homogenized at 6 to 20°C and 8000 to 12000 rpm for 1 to 3 hours; then filtered to obtain a homogenized slurry.
4. The method for preparing the collagen implant according to claim 1, characterized in that, The freeze-drying parameters are as follows: the homogenized slurry is transferred to a freeze-drying mold with a thickness of 5-6 cm, and cooled to -4-4℃ and held for 20-60 min; then cooled to below -20℃ and held for 1.5-3 h; the temperature is raised to -4-0℃ and held for 15-40 h under a pressure ≤-0.3 mbar; then the temperature is raised to 20-35℃ at a heating rate of 0.5-3℃ / min and held for 0.5-4 h.
5. The method for preparing the collagen implant according to claim 4, characterized in that, The process parameters for freeze-drying are as follows: the homogenized slurry is transferred to a freeze-drying mold with a thickness of 5-6 cm, cooled to 0-2℃ and held for 20-60 min; cooled to below -20℃ and held for 1.5-3 h; heated to -1-0℃ and held for 20-30 h under a pressure ≤ -0.3 mbar; then heated to 20-32℃ at a heating rate of 1-2℃ / min and held for 0.5-2 h.
6. The method for preparing the collagen implant according to claim 1, characterized in that, The secondary homogenization method is as follows: add sterile collagen raw material to 0.1~0.2M phosphate buffer solution with pH 7.0~7.4 to obtain collagen slurry with a concentration of 3~5wt%, and homogenize at 15~25℃ and 8000-12000rpm for 3~8h.
7. The method for preparing the collagen implant according to claim 1, characterized in that, The crosslinking method is as follows: 0.001~0.05wt% of crosslinking agent is added to the slurry, and homogenous crosslinking is carried out at 15~25℃ and 8000-15000rpm for 8~36h; then aseptic filling is performed to obtain the collagen implant; the crosslinking agent is selected from glutaraldehyde or EDC / NHS.
8. The method for preparing the collagen implant according to claim 7, characterized in that, The crosslinking agent is a glutaraldehyde solution with a concentration of 0.1~5wt%.
9. A collagen implant, characterized in that, It is prepared using the preparation method according to any one of claims 1-8.
10. The use of the collagen implant as described in claim 9 in the preparation of medical materials.