Preparation method of high-concentration collagen gel and application thereof in promoting bone regeneration

Abstract

The application discloses a preparation method of high-concentration collagen gel and application of the high-concentration collagen gel in promoting bone regeneration. Collagen is dissolved in an acetic acid solution to prepare a collagen solution, the collagen solution is loaded into a dialysis bag, and the dialysis bag is sealed and placed in a PEG solution; the collagen solution is concentrated through reverse dialysis; the PEG solution outside the dialysis bag is poured out; the concentrated collagen solution in the dialysis bag is taken out; the concentrated collagen solution is mixed with PBS; the neutralized collagen solution is transferred into a mold; the mold is placed in a 37 DEG C constant-temperature box for incubation; and a stable high-concentration collagen gel is formed. The application uses a low-concentration collagen acid solution as raw material, and the low-concentration collagen acid solution is physically concentrated to high concentration through reverse dialysis and drying technology; no chemical cross-linking agent is added in the whole preparation process, and no foreign material is introduced; and the obtained gel is pure collagen component. The gel is implanted into a bone defect site, mechanical support and suitable degradation performance provided by the high-density collagen network of the gel promote host cell ingrowth and new bone formation.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, specifically relating to a method for preparing a high-concentration collagen gel and its application in promoting bone regeneration. Background Technology

[0002] In existing technologies, collagen gels are typically prepared using low-concentration collagen solutions (generally 1-20 mg / mL) through self-assembly or chemical cross-linking. Preparation methods include dissolving collagen in acidic solutions such as acetic acid, adjusting the pH to neutral, and incubating at 37°C to form a gel. Some studies have improved the mechanical strength of collagen gels by adding chemical cross-linking agents (such as glutaraldehyde, carbodiimide EDC, etc.). The disadvantages of this approach are: insufficient mechanical strength: conventional low-concentration collagen gels have poor mechanical properties, making it difficult to provide sufficient space support at bone defect sites, and they are prone to collapse or shrinkage; rapid degradation: low-concentration collagen gels degrade rapidly in vivo (usually within several days to two weeks), which does not match the long cycle of bone regeneration and cannot provide a continuous scaffold for new bone ingrowth. Limited bone regeneration capacity: Low-concentration collagen gels have weak bone induction capacity when used alone, and usually require compound growth factors (such as BMP), stem cells or inorganic materials (such as hydroxyapatite) to achieve better bone repair effects; Toxicity risks of chemical cross-linking agents: Chemical cross-linking agents (such as glutaraldehyde and EDC) added to improve mechanical properties may have residual cytotoxicity, posing a biosafety hazard.

[0003] In recent years, researchers have attempted to prepare high-concentration collagen gels. For example, the patent CN105641745B published by Sichuan University (a method for preparing type I collagen lyotropic liquid crystal three-dimensional gel) uses a tangential flow system for pre-concentration combined with PEG dialysis dehydration: first, an acidic dilute collagen solution is directionally sheared and concentrated to 1-3 times its original concentration in a tangential flow system, and then placed in a PEG solution with a mass / volume concentration of 20%-50% for dialysis dehydration, further increasing the concentration by 10-40 times, ultimately obtaining a high-viscosity liquid crystal collagen fluid with a concentration of 200-300 mg / mL or even higher; subsequently, it is placed in an alkaline gas generator for neutralization and incubated at 4℃-50℃, and the self-assembly of microfibers and fiber branching cross-linking are regulated by the acid-base environment to obtain a collagen three-dimensional gel. Furthermore, Guangdong Marubi Biotechnology's patent CN121550493A (Collagen Hydrogel for Injection and its Preparation Method) dissolves recombinant collagen in citric acid solution, achieving a collagen concentration of 100-200 mg / mL. Heating at 80-100℃ causes esterification and cross-linking of the citric acid and collagen, forming a three-dimensional network hydrogel. The disadvantages of this method are: high equipment requirements: CN105641745B requires a tangential flow system for pre-concentration, which is costly and hinders technology promotion and large-scale production; complex process and cumbersome operation: the multi-step process of tangential flow concentration + PEG dialysis dehydration + alkaline gas neutralization is cumbersome and difficult to control batch-to-batch consistency; limited concentration efficiency: when concentrating collagen solely through PEG dialysis, the dialysis efficiency drops significantly after reaching approximately 100 mg / mL, making further concentration to 300 mg / mL difficult. mg / ml and higher concentrations; introduction of chemical cross-linking or harsh treatment conditions: CN121550493A uses high-temperature esterification cross-linking (80-100℃), which may damage the triple helix structure and bioactivity of collagen; CN105641745B uses alkaline gas neutralization, and the alkaline environment may affect the collagen structure; impure composition: CN121550493A introduces citric acid as a cross-linking agent, resulting in an esterification reaction, and the resulting gel is not a pure collagen system; non-bone regeneration application: none of the above patents involve bone defect repair. CN121550493A is explicitly used for medical aesthetic filling (such as wrinkle repair and soft tissue filling), while CN105641745B does not explicitly specify its use for bone regeneration, and the applicability of its high-concentration collagen in the field of bone regeneration has not been verified; lack of experimental evidence for bone regeneration effect: none of the above patents provide any animal experimental data on in vivo bone defect repair, and whether they have the ability to promote bone regeneration is still unknown.

[0004] To improve the mechanical properties and bone regeneration capacity of collagen raw materials, existing technologies combine collagen with other materials. For example, collagen can be combined with nano-hydroxyapatite to mimic the organic-inorganic composition of natural bone; collagen can be combined with silk fibroin, with degradation time controlled by adjusting the ratio; or collagen can be combined with synthetic polymers (such as PEG) or bioactive factors. The disadvantages of this approach are: complex composition and difficult control: the composite system involves multiple components (inorganic particles, cross-linking agents, active factors, etc.), and the proportion, distribution, and interactions of each component are difficult to control precisely, resulting in poor batch-to-batch consistency; cumbersome preparation process: multi-component composites require multiple reaction and purification processes, resulting in a long preparation cycle, which is not conducive to large-scale production and clinical translation; safety issues of exogenous additives: added growth factors (such as BMP) are costly, have short half-lives, and high-dose use may lead to side effects such as ectopic ossification; the use of exogenous cells involves immune rejection and ethical issues; and the problem of inorganic particle aggregation: inorganic particles such as nano-hydroxyapatite are prone to aggregation in the collagen matrix, affecting material uniformity and mechanical properties.

[0005] Based on the above-mentioned existing technical solutions, the following technical challenges still exist in this field: Complex preparation methods or stringent conditions: To obtain high-concentration collagen, existing technologies either rely on specialized equipment such as tangential flow systems or require the introduction of chemical cross-linking agents or high temperatures. This is not only cumbersome, inefficient, and costly, but may also damage the natural biological activity of collagen; Impure composition: Existing high-concentration collagen gels either introduce chemical cross-linking agents or are compounded with other polymer materials, resulting in a composition that is not a pure collagen system, posing potential toxicity risks or difficulties in control; Limited application areas: Currently disclosed high-concentration collagen gel technologies are mainly applied to non-bone repair fields such as cosmetic fillings. Research on using high-concentration pure collagen gels to promote bone regeneration has not been reported domestically or internationally; Unclear bone repair mechanism: Existing technologies have not conducted in vivo animal experiments on bone defect repair. Whether they have the ability to promote bone regeneration and through what mechanism they promote bone regeneration remains unknown. Summary of the Invention

[0006] The purpose of this invention is to provide a method for preparing high-concentration collagen gel and its application in promoting bone regeneration.

[0007] A method for preparing a high-concentration collagen gel, comprising the following steps: (1) Dissolve collagen in 0.3-0.7 M acetic acid solution and stir at 0-8℃ for 2-4 hours until completely dissolved to prepare collagen solution; (2) Take 20-40 mL of the collagen solution prepared in step (1) and put it into a dialysis bag. After sealing, place it in 1-3 L of 90-130 g / L PEG solution and concentrate it by reverse dialysis at 0-8℃ for 20-28 hours. During this period, replace the PEG solution 1-3 times. (3) After concentration, pour off the PEG solution on the outside of the dialysis bag and take out the concentrated collagen solution inside the dialysis bag; (4) Add 0.05-0.15M PBS to the concentrated collagen solution at a volume ratio of 10:(0.5-2) and mix gently. (5) Transfer the neutralized collagen solution to the mold and incubate it in a 37°C constant temperature box for 0.5-1.5 hours to form a stable high-concentration collagen gel.

[0008] The concentration of the collagen solution prepared in step (1) is 3-7 mg / mL.

[0009] The dialysis bag described in step (2) retains protein molecules with a molecular weight of 50 kDa.

[0010] The PEG in step (2) has a molecular weight of 6000-20000 Da.

[0011] The pH of the PBS in step (4) is 7-8.

[0012] The concentration of the high-concentration collagen gel is 100-350 mg / mL.

[0013] The application of the high-concentration collagen gel in the preparation of drugs that promote bone regeneration.

[0014] The high-concentration collagen gel can induce bone marrow mesenchymal stem cells to align in a specific direction.

[0015] The core idea of ​​this invention is as follows: using a low-concentration acidic collagen solution as raw material, it is physically concentrated to a high concentration (above 100 mg / mL) through reverse dialysis and other drying techniques. After neutralization, it is incubated at 37°C, utilizing the self-assembly properties of collagen molecules under physiological temperature and neutral pH conditions to form a gel. The entire preparation process does not add any chemical cross-linking agents or introduce any exogenous materials, resulting in a gel composed entirely of collagen. When this gel is implanted into bone defect sites, its high-density collagen network provides mechanical support and suitable degradation properties, promoting host cell ingrowth and new bone formation.

[0016] Technical Principle: Collagen molecules disperse as single molecules or small aggregates under acidic conditions (pH≈2-3). After dehydration, concentration, or drying, the intermolecular distance decreases and the collision frequency increases. Upon neutralization, collagen molecules self-assemble through electrostatic and hydrophobic interactions to form fibers with a natural D-periodic structure. During incubation at 37°C, the fibers further cross-link to form a three-dimensional network, encapsulating a large amount of water to form a gel. High concentrations (above 100 mg / mL) endow the gel with a high-density fiber network, giving it high mechanical strength and resistance to degradation, providing continuous spatial support and a matrix environment to guide bone regeneration at bone defect sites.

[0017] The beneficial effects of this invention are as follows: (1) The preparation method is simple and efficient: only conventional laboratory equipment (dialysis bag, PEG, constant temperature and humidity chamber) is required, and a high concentration of collagen gel of 100-350 mg / mL can be obtained through three steps of "concentration-neutralization-37℃ incubation". Compared with the prior art, the equipment requirements of this invention are low, the operation is simple, the energy consumption is low, and it is easy to scale up production. (2) The components are pure and the biosafety is high: no chemical cross-linking agent, cross-linking enzyme or other exogenous materials are added in the whole process, and the resulting gel components contain only collagen and water. Compared with the technical solutions of introducing cross-linking agents or compounding other materials in the prior art, this invention avoids the toxicity risk of exogenous substances and batch-to-batch differences, and preserves the natural biological activity of collagen to the greatest extent. (3) It can guide the directional arrangement of cells: this invention is the first to discover that bone marrow mesenchymal stem cells (BMSCs) show obvious directional arrangement on the surface of pure collagen gel at a concentration of 100 mg / mL, while cells on the surface of low concentration collagen gel are randomly arranged. This directional arrangement is beneficial to the orderly regeneration of bone tissue. (4) First successful application in bone regeneration: Existing high-concentration collagen patents do not involve bone regeneration applications, nor do they provide any experimental data on promoting bone regeneration. This invention is the first to use high-concentration pure collagen gel for bone defect repair, and its effectiveness has been verified through a rat cranial critical defect model, filling this technological gap. (5) Significant bone regeneration effect: Micro-CT quantitative analysis showed that 6 weeks after surgery, the BV / TV parameters of the high-concentration collagen gel group of this invention were higher than those of the blank control group and the low-concentration group. HE, Masson, and Goldner staining all confirmed that the high-concentration collagen gel of this invention can effectively promote the formation of new bone tissue, collagen deposition, bone matrix mineralization, and osteoblast activity. (6) Good storage stability: The high-concentration collagen gel of this invention remains stable when stored at 0-4℃, which is convenient for transportation and use, and has good prospects for clinical translation. Attached Figure Description

[0018] Figure 1 A comparison of the morphology of bone marrow mesenchymal stem cells cultured on surfaces of low-concentration and high-concentration collagen gels.

[0019] Figure 2 Results of hematoxylin-eosin (HE) staining.

[0020] Figure 3 The results of Masson's trichrome staining.

[0021] Figure 4 The results of Goldner trichrome staining.

[0022] Figure 5 To verify the bone regeneration effect of 100 mg / mL collagen gel. Detailed Implementation

[0023] To facilitate understanding of the present invention, a more comprehensive description will be given below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention. Example 1

[0024] A method for preparing a high-concentration collagen gel, comprising the following steps: (1) Dissolve collagen (type I collagen from bovine Achilles tendon) in 0.5 M acetic acid solution and stir at 4°C for 3 hours until completely dissolved to prepare a 5 mg / mL collagen solution; (2) Take 30 mL of the collagen solution prepared in step (1) and put it into a dialysis bag (retaining protein molecules with a molecular weight of 50 kDa). After sealing, place it in 2 L of 110 g / L PEG (molecular weight of 10000 Da) solution and concentrate it by reverse dialysis at 4℃ for 24 hours, during which the PEG solution is replaced twice. (3) After concentration, pour off the PEG solution on the outside of the dialysis bag and take out the concentrated collagen solution inside the dialysis bag; (4) Add 0.01M PBS (pH 7.4) to the concentrated collagen solution at a volume ratio of 10:1 and mix gently. (5) Transfer the neutralized collagen solution to the mold and incubate it in a 37°C constant temperature box for 1 hour to form a stable high-concentration collagen gel with a concentration of 100 mg / mL. Example 2

[0025] A method for preparing a high-concentration collagen gel, comprising the following steps: (1) Dissolve collagen (type I collagen from bovine Achilles tendon) in 0.4 M acetic acid solution and stir at 4°C for 2 hours until completely dissolved to prepare a 4 mg / mL collagen solution; (2) Take 25 mL of the collagen solution prepared in step (1) and put it into a dialysis bag (retaining protein molecules with a molecular weight of 50 kDa). After sealing, place it in 1 L of 90 g / L PEG (molecular weight of 8000 Da) solution and concentrate it by reverse dialysis at 4℃ for 22 hours, changing the PEG solution once during the period. (3) After concentration, pour off the PEG solution on the outside of the dialysis bag and take out the concentrated collagen solution inside the dialysis bag; (4) Add 0.08M PBS (pH 7.2) to the concentrated collagen solution at a volume ratio of 10:0.8 and mix gently. (5) Transfer the neutralized collagen solution to the mold and incubate it in a 37°C constant temperature box for 0.8 hours to form a stable high-concentration collagen gel with a concentration of 120 mg / mL. Example 3

[0026] A method for preparing a high-concentration collagen gel, comprising the following steps: (1) Dissolve collagen (type I collagen from bovine Achilles tendon) in 0.6 M acetic acid solution and stir at 4°C for 4 hours until completely dissolved to prepare a 7 mg / mL collagen solution; (2) Take 40 mL of the collagen solution prepared in step (1) and put it into a dialysis bag (retaining protein molecules with a molecular weight of 50 kDa). After sealing, place it in 3 L of 130 g / L PEG (molecular weight of 14000 Da) solution and concentrate it by reverse dialysis at 4℃ for 28 hours, during which the PEG solution is replaced 3 times. (3) After concentration, pour off the PEG solution on the outside of the dialysis bag and take out the concentrated collagen solution inside the dialysis bag; (4) Add 0.15M PBS (pH 7.8) to the concentrated collagen solution at a volume ratio of 10:1.5 and mix gently. (5) Transfer the neutralized collagen solution to the mold and incubate it in a constant temperature oven at 37°C for 1.5 hours. After gradient evaporation and concentration, a stable high-concentration collagen gel with a concentration of 350 mg / mL is formed.

[0027] Experimental example: 1. Collagen Concentration Determination: Take a high-concentration collagen gel (V, unit mL), freeze-dry it in a freeze dryer until constant weight, and weigh it (W, unit mg). The collagen concentration is calculated using the following formula: Collagen concentration (mg / mL) = W / V Results: The high-concentration collagen gel prepared by the method of Example 1 of this invention has a collagen concentration of 100 mg / mL, the collagen prepared by Example 2 has a collagen concentration of 120 mg / mL, and the collagen prepared by Example 3 has a collagen concentration of 350 mg / mL.

[0028] 2. Observation of cell compatibility and cell behavior: The growth and arrangement of human bone marrow mesenchymal stem cells (HBMSCs) on the surface of the high-concentration collagen gel of this invention were observed. Experimental materials: 20 mg / mL and 100 mg / mL collagen gels. Staining: Phalloidin was used to label the cytoskeleton (F-actin), and DAPI was used to label the cell nucleus. Observation: Cell morphology and arrangement were observed using confocal microscopy.

[0029] like Figure 1As shown, on the surface of a collagen gel with a concentration of 100 mg / mL, BMSCs exhibit a distinct directional arrangement, with the cytoskeleton extending in a specific direction. In contrast, cells on the surface of low-concentration collagen gels (e.g., 20 mg / mL) are randomly arranged. These results indicate that the high-concentration collagen gel of this invention can guide the directional arrangement of BMSCs, which is beneficial for the orderly regeneration of bone tissue.

[0030] 3. Storage stability: The high-concentration collagen gels prepared in Examples 1-3 were sealed and stored at 4°C, and the changes in gel morphology were observed periodically.

[0031] Experimental results: Under storage conditions of 4°C, the gel maintained its intact shape without shrinkage or collapse, indicating that the high-concentration collagen gel of the present invention has good storage stability.

[0032] 4. Verification of bone regeneration promotion effect 4.1 Animal Model Laboratory animals: SPF-grade male SD rats, 8 weeks old, weighing 250-300 g. All animal experimental procedures were approved by the animal ethics committee.

[0033] Bone defect model: A rat model of critical skull defect. Rats were anesthetized by isoflurane inhalation, and the top of the head was prepared and disinfected. The skin and periosteum were incised along the midline to expose the skull. A full-thickness bone defect with a diameter of 5 mm was prepared on each side of the midline of the skull. This size is the critical size at which rat skull defects cannot heal spontaneously.

[0034] 4.2 Experimental Grouping

[0035] Implantation procedure: The collagen gel is filled into the bone defect area, level with the bone surface, and the periosteum and skin are sutured.

[0036] Sampling time: Rats were sacrificed 6 weeks after surgery, and skull specimens were collected for testing.

[0037] 4.3 Micro-CT Detection Detection method: Skull specimens were fixed in 4% paraformaldehyde. Micro-CT scanners were used for scanning, and the reconstructed bone defect areas were selected as regions of interest (ROIs) to analyze parameters such as bone volume fraction (BV / TV, %).

[0038] Test results:

[0039] Conclusion: Six weeks after surgery, the BV / TV and other parameters of the high-concentration collagen gel group in Example 1 were significantly higher than those of the blank control group and the low-concentration group, indicating that the high-concentration collagen gel of the present invention can effectively promote the regeneration and repair of skull defects in rats.

[0040] 4.4 Histological staining Detection method: After Micro-CT scanning, the bone defect specimen was decalcified, graded dehydrated, and embedded in paraffin. Sagittal sections were prepared along the center of the defect and stained as follows: (1) Hematoxylin-eosin (HE) staining Objective: To observe the overall tissue morphology, newly formed bone tissue, residual collagen gel, and inflammatory response. Results: In Example 1, the high-concentration group showed abundant new bone tissue in the defect area, good continuity of bone trabeculae, partial degradation of the collagen raw material, and no obvious inflammatory response. Figure 2 ).

[0041] (2) Masson's trichrome staining Objective: To differentiate between collagen fibers (blue) and bone tissue (red) Results: In Example 1, the high-concentration group showed a large area of ​​blue staining in the defect area, indicating a large amount of mature collagen deposition and good integration with the newly formed bone tissue. Figure 3 ).

[0042] (3) Goldner trichrome staining Objective: To differentiate between mineralized bone (green) and osteoid (red / orange) Results: In Example 1, the high-concentration group showed green-stained mineralized bone and a small amount of red / orange osteoid in the defect area, indicating that bone formation and mineralization were underway. Figure 4 ).

[0043] 4.5 Verification of the bone regeneration effect of 100 mg / mL collagen gel Animal experiments: Following the method described in Section 4, 100 mg / mL collagen gel was implanted into a skull defect (5 mm in diameter) in rats, and samples were collected for testing 6 weeks post-surgery.

[0044] Results: Micro-CT showed that the defect area of ​​the high-concentration collagen gel group in Example 1 was filled with a large amount of new bone tissue, demonstrating a good bone regeneration effect. Figure 5 ).

[0045] 4.6 Effects of high-concentration collagen gel on the cytoskeleton of HBMSCs Cellular experiments: HBMSCs were seeded on the surface of a high-concentration collagen gel (100 mg / mL), with a low-concentration collagen gel (20 mg / mL) as a control. After 2 days of culture, the cytoskeleton (F-actin) was stained with phalloidin (FITC / TRITC labeled), and the nuclei were labeled with DAPI. Cell morphology and cytoskeleton alignment were observed using a confocal microscope.

[0046] Results: On the surface of a high-concentration collagen gel (100 mg / mL), bone MSCs exhibited a distinct directional arrangement, with the cytoskeleton extending along a specific direction, displaying a regular orientation. However, on the surface of a low-concentration collagen gel (20 mg / mL), BMSCs were randomly arranged without obvious directionality. These results indicate that the high-concentration collagen gel of this invention can induce directional alignment of the BMSC cytoskeleton. This directional alignment is beneficial for orderly cell migration and differentiation, and may provide a favorable cellular behavioral basis for the orderly regeneration of bone tissue.

[0047] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A method for preparing a high-concentration collagen gel, characterized in that, Follow these steps: (1) Dissolve collagen in 0.3-0.7 M acetic acid solution and stir at 0-8℃ for 2-4 hours until completely dissolved to prepare a collagen solution; (2) Take 20-40 mL of the collagen solution prepared in step (1) and put it into a dialysis bag. After sealing, place it in 1-3 L of 90-130 g / L PEG solution and concentrate it by reverse dialysis at 0-8℃ for 20-28 hours. During this period, replace the PEG solution 1-3 times. (3) After concentration, pour off the PEG solution on the outside of the dialysis bag and take out the concentrated collagen solution inside the dialysis bag; (4) Add 0.05-0.15M PBS to the concentrated collagen solution at a volume ratio of 10:(0.5-2) and mix gently. (5) Transfer the neutralized collagen solution to the mold and incubate it in a 37°C constant temperature box for 0.5-1.5 hours to form a stable high-concentration collagen gel.

2. The method for preparing high-concentration collagen gel according to claim 1, characterized in that, The concentration of the collagen solution prepared in step (1) is 3-7 mg / mL.

3. The method for preparing high-concentration collagen gel according to claim 1, characterized in that, The dialysis bag described in step (2) retains protein molecules with a molecular weight of 50 kDa.

4. The method for preparing high-concentration collagen gel according to claim 1, characterized in that, The PEG molecular weight in step (2) is 6000-20000 Da.

5. The method for preparing high-concentration collagen gel according to claim 1, characterized in that, The pH of the PBS in step (4) is 7-8.

6. The method for preparing high-concentration collagen gel according to claim 1, characterized in that, The concentration of the high-concentration collagen gel is 100-350 mg / mL.

7. The use of the high-concentration collagen gel prepared according to claim 1 in the preparation of a drug that promotes bone regeneration.

8. The application of the high-concentration collagen gel prepared according to claim 1 in the preparation of a drug promoting bone regeneration, characterized in that, The high-concentration collagen gel can induce bone marrow mesenchymal stem cells to align in a specific direction.

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