A composite hydrogel with bone defect repair function of marine origin and a preparation method and application thereof

Abstract

This invention discloses a marine-derived composite hydrogel with bone defect repair function, its preparation method, and its applications, belonging to the field of biomedical materials technology. The invention provides a composite hydrogel with bone defect repair function, which is prepared by combining marine-derived collagen with hydroxyapatite to form a biomimetic matrix, and innovatively doping it with fucoidan, which has multiple biological functions. The composite hydrogel achieves a synergistic effect between material structure and function, breaking through the limitations of organic phase biomimetic microenvironment construction capabilities, inorganic phase osteoconductive properties, and fucoidan anti-inflammatory functions to form a three-dimensional network composite material. This invention also provides a simple and easy-to-implement method for preparing the composite hydrogel, with low preparation cost and significant potential for widespread application. The composite hydrogel provided by this invention not only possesses excellent biocompatibility but also significantly improves the coordination of cellular response, matrix remodeling, and functional reconstruction during bone defect repair, bringing a breakthrough solution to the regeneration and repair of complex bone defects, with broad application prospects.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, specifically relating to a marine-derived composite hydrogel with bone defect repair function, its preparation method, and its application. Background Technology

[0002] Bone defect repair is a major challenge in clinical medicine and regenerative engineering, with a large number of patients suffering from bone defects each year due to congenital defects, diseases, or trauma. Traditional repair materials, such as autologous bone grafting, suffer from problems such as donor site damage and limited availability, while allogeneic bone or synthetic materials (such as metals and ceramics) face limitations such as immune rejection, insufficient mechanical compatibility, or potential toxicity of degradation products. In recent years, marine collagen raw materials have emerged as a promising candidate due to their abundant natural sources, high biocompatibility, and molecular structure similar to human collagen. Studies have shown that marine collagen can promote osteoblast adhesion and proliferation by mimicking the extracellular matrix microenvironment, accelerating the mineralization and remodeling of new bone tissue at the defect site. By combining porous scaffolds constructed using nano-hydroxyapatite or photocrosslinking technology, this material not only possesses excellent osteoconductivity but also enables minimally invasive and precise filling, reducing the risk of surgical trauma. This type of novel material, which combines biomimetic characteristics, customizable functions, and clinical applicability, opens up innovative pathways for efficient and safe bone repair strategies.

[0003] Hydroxyapatite (HA) is a major inorganic component of human bones and teeth, possessing a crystal structure highly similar to natural bone tissue, thus exhibiting excellent biocompatibility, osteoconductivity, and osteointegration. As a key material for bone defect repair, nHA can directly form chemical bonds with host bone through surface ion exchange, promoting osteoblast adhesion, proliferation, and bone matrix deposition. Simultaneously, its slow degradation rate matches the new bone formation process, preventing structural collapse due to premature material absorption. However, single nHA materials have limited osteoinductive activity and are brittle with insufficient compressive strength, making it difficult to meet the mechanical support requirements of large bone defects. In recent years, researchers have constructed multifunctional scaffolds by combining bioactive glass (BAG), collagen, or growth factors. This utilizes the stable mineralization properties of nHA while introducing the rapid ion release activity of BAG, synergistically regulating the local microenvironment to promote vascularization and bone regeneration. Furthermore, the combination of nano-sized HA with collagen enables precise control of material porosity, degradation rate, and mechanical properties, providing a new direction for personalized repair of complex bone defects.

[0004] Marine-derived collagen, as a natural polymer material, is widely and sustainably sourced from abundant marine biological resources, and its extraction process is relatively mature. It possesses unique biocompatibility and biodegradability, making it suitable for hydrogel construction, and showing particular potential in the field of bone tissue defect repair. However, marine-derived collagen also has some drawbacks, such as lower gel strength, making it difficult to meet the high mechanical strength requirements of bone repair scenarios; and poor toughness, making it prone to breakage or deformation, affecting long-term effectiveness and potentially leading to unsatisfactory repair results.

[0005] Fucoidan, a natural polysaccharide derived from the ocean, is structurally rich in fucose units and often modified with functional groups such as sulfate groups, endowing it with excellent biological activity and functional diversity. However, there are currently no reports on the use of hydrogels prepared from marine-derived collagen combined with organic fucoidan and the inorganic component hydroxyapatite for bone defect repair. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a marine-derived composite hydrogel with bone defect repair function, its preparation method, and its applications. This composite hydrogel is specifically designed for bone defect repair, cleverly combining the inorganic component hydroxyapatite with the organic component fucoidan. It not only integrates the excellent properties of both but also possesses the unique advantage of significantly promoting new bone formation. The composite hydrogel provided by this invention exhibits bone tissue filling capacity and highly efficient bone defect repair efficacy, offering a promising solution in the field of bone tissue engineering technology.

[0007] To achieve the above objectives, the present invention is implemented through the following solution: This invention provides a method for preparing a marine-derived composite hydrogel with bone defect repair function, the method specifically comprising the following steps: (1) The fish skin is repeatedly washed and soaked to obtain swollen fish skin; (2) The swollen fish skin was enzymatically hydrolyzed with pepsin, and after complete enzymatic hydrolysis, it was centrifuged and the supernatant was collected. (3) Salt out and filter the supernatant, and dissolve the precipitate obtained after filtration in acetic acid to obtain a fish skin collagen solution; (4) Add NaH2PO4·2H2O to the fish skin collagen solution and stir to adjust the solution to neutral to obtain pure collagen precursor solution; (5) Add the pure collagen precursor solution to fucoidan to obtain a mixed solution, add NaH2PO4 solution and CaCl2 solution in sequence and stir at a constant speed, adjust the pH of the solution to 8~10, and obtain a composite collagen precursor solution. (6) The composite collagen precursor solution is left to stand for 20-30 hours to induce the self-assembly and mineralization of collagen. Genipin is added during the mineralization process for cross-linking to obtain a composite hydrogel.

[0008] Furthermore, the pepsin has an enzyme activity of 3000~10000 U / g and an enzymatic hydrolysis time of 20~55 h.

[0009] Furthermore, the mass ratio of fucoidan to collagen in the mixture is 5% to 20%.

[0010] Furthermore, the molar concentration of the phosphate solution is 0.3~3M, and the molar concentration of the CaCl2 solution is 1~5M.

[0011] Furthermore, the Ca / P ratio in the composite collagen precursor solution is 1.0~2.0.

[0012] Furthermore, the temperature is controlled at 3~10 ℃ during the preparation of the composite hydrogel.

[0013] Furthermore, the mineralization time is 24-72 h, and the mass amount of genipin is 0.05-0.2% of the mass of the composite collagen precursor solution.

[0014] The present invention also provides a composite hydrogel prepared by the aforementioned preparation method.

[0015] Furthermore, the composite hydrogel has a homogeneous and porous structure, and its fiber structure shows visible nano-sized hydroxyapatite, with mineralization both inside and outside the fibers.

[0016] The present invention also provides the application of the aforementioned composite hydrogel in the preparation of formulations with bone defect repair function.

[0017] Furthermore, the bone defect includes inflammatory microbone defects, and the composite hydrogel can promote bone defect repair and healing under inflammatory conditions. The composite hydrogel can increase the expression of osteogenic genes Runx-2 and ALP, and upregulate the expression of TGF-β1.

[0018] Compared with the prior art, the present invention has the following advantages: 1. This invention addresses the shortcomings of current bone defect repair materials in terms of bioactivity, mechanical properties, and ease of use. It innovatively utilizes marine-derived natural collagen and fucoidan through covalent cross-linking to prepare a mineralized hydrogel suitable for bone tissue defect repair. Nano-hydroxyapatite (nHA), a classic bioactive inorganic component, is specifically introduced into this hydrogel system to enhance its mineralization capacity and further improve the material's osteoinductive properties. Marine-derived collagen not only endows the material with excellent biocompatibility and cell affinity but also significantly enhances the hydrogel's stability through synergistic effects with fucoidan. The addition of fucoidan further provides multiple bioactivities, including antioxidant and anti-inflammatory properties, which help promote the healing process of bone defects. This invention achieves a perfect combination of bioactivity, mechanical strength, and ease of use, providing an innovative and efficient solution for bone tissue defect repair.

[0019] 2. In the marine collagen of this invention, hydroxyapatite (Ca) is formed through calcium ions and phosphate ions. 10 (PO4)6(OH)2), with its crystal composition and micro / nano structure highly similar to the inorganic phase of natural bone, can directly form chemical bonds with host bone, promoting bone integration. Simultaneously, the surface of nHA can provide active sites for osteoblast adhesion and facilitate ion release (such as Ca2+). 2+ PO4 3- It regulates the cellular microenvironment, guides the directional migration and differentiation of osteocytes, and accelerates the growth of new bone along its surface. nHA formed in the marine collagen network, through organic-inorganic interface bonding (such as phosphorylation modification), retains the flexible three-dimensional scaffold function of collagen while endowing the composite material with a gradient modulus close to that of natural bone, avoiding stress shielding effects. Furthermore, HA can be slowly degraded in vivo through a cell-mediated dissolution-redeposition process, and its rate can be regulated by crystal size / crystallinity to ensure dynamic synchronization with new bone formation and maintain the structural stability of the repair zone.

[0020] 3. The raw materials in this invention are marine collagen and fucoidan. Marine collagen is a natural protein extracted from marine organisms. Its structure is highly similar to human collagen, exhibiting good biodegradability and tissue compatibility, and effectively promoting cell proliferation and tissue repair. Fucoidan is a natural polysaccharide extracted from brown algae, possessing excellent bioactivity, including anti-inflammatory, antioxidant, and wound-healing properties. These two materials are not only naturally derived, safe, and non-toxic, but have also undergone extensive experimental verification, demonstrating good compatibility with human tissues and not triggering significant immune rejection or toxic reactions. Therefore, they have broad application prospects in the biomedical field.

[0021] 4. The preparation method of this invention has significant advantages such as inexpensive and readily available raw materials, low cost, and simple synthesis steps. The raw materials used in this invention are all derived from marine organisms, and are characterized by being natural, renewable, and abundant. Marine-derived materials are not only inexpensive, but also avoid the occurrence of zoonotic diseases. Moreover, their unique bioactivity and structural characteristics give them wide application value in the biomedical field. Attached Figure Description

[0022] Figure 1 SEM images of the hydrogels prepared in Examples 1-5 of this invention.

[0023] Figure 2 These are TEM images of the hydrogels prepared in Examples 1-5 of this invention, where A represents COL, B represents COL-nHA, C represents 5% Fc-COL-nHA, D represents 10% Fc-COL-nHA, and E represents 20% Fc-COL-nHA.

[0024] Figure 3 The images show the CCK-8 diagrams of the hydrogels prepared in Examples 1-5 of this invention.

[0025] Figure 4 The PCR images are of the hydrogels prepared in Examples 1-5 of this invention, n=3, *p < 0.05, **p < 0.01, ***p < 0.001.

[0026] Figure 5 This is a schematic diagram illustrating the principle of composite hydrogel in the repair of inflammatory microbone defects. Detailed Implementation

[0027] The technical solutions and beneficial effects of the present invention will be described in detail below with reference to embodiments, so as to fully understand the purpose and features of the present invention. It should be noted that the embodiments described here are merely illustrative and are not intended to limit the entirety of the present invention. Other embodiments obtained by those skilled in the art based on the content of the present invention without creative effort are all within the protection scope of the present invention.

[0028] Example 1 This embodiment provides the preparation of pure collagen precursor solution (COL), which is carried out entirely at 4 °C, and specifically includes the following: 1. Extraction of collagen from fish skin (1) Clean the monkfish skin, repeatedly wash with deionized water, weigh, and cut into small pieces; pre-cool with 5% sodium bicarbonate solution for 4 h and then wash the skin to remove the fishy smell. Pre-cool with 10% n-butanol (v / v: 1 / 20) for 4 h, soak the skin for 24 h to remove fat tissue (stir for 10 min every 6 h), pre-cool with anhydrous ethanol, 90% ethanol, 80% ethanol, 70% ethanol, 50% ethanol, 30% ethanol, and deionized water in sequence to replace the n-butanol, wash with deionized water multiple times, and weigh the wet weight; after washing, soak the skin in pre-cooled PBS solution for 24 h to remove impurities and proteins, wash with deionized water, collect the skin, drain the water and weigh. Pre-cool with 0.5 MHAc acetic acid and soak the collected skin for 24 h to make it swell, stir mechanically and observe the swelling effect.

[0029] (2) Pepsin digestion: Pepsin (dissolved in 0.01M HCl and added in multiple batches with stirring) was added at a mass ratio of enzyme:substrate (swollen fish skin) = 1:300. The enzyme activity of the pepsin was 3000 U / g. The mixture was stirred for 48 h. The mixture was centrifuged at 9000 rpm for 15 min at low temperature, and the supernatant was collected. The supernatant was stirred at low temperature, and the pH of the system was adjusted to 7.0 with 2M NaOH and maintained for 30 min to inactivate the pepsin. The supernatant after enzyme inactivation was added to pre-cooled 2M HAc solution to adjust the pH to 3, and then NaCl was used for salting out until white flocculent matter was produced. The precipitate was filtered and collected. The precipitate was soaked in 0.5M HAc for 24 h to re-dissolve and the swelling effect was observed.

[0030] Salting out again with sodium chloride solution until a white flocculent substance is formed, filter, collect the precipitate, and dialyze with deionized water until the conductivity of the dialysate is <30 μS / cm. -1 Change the water every 8 hours. Concentrate the dialysis product to 10 mg / ml using 0.5M HAc. -1 (The solution after concentration is fish skin collagen solution, which mainly contains type I collagen) for later use.

[0031] 2. Preparation of pure collagen precursor solution (COL) 10 mg mL -1 The anglerfish skin collagen solution was adjusted to pH 7.0 with 3M NaH2PO4·2H2O and 2M NaOH under uniform stirring at low temperature (4℃), and then adjusted to a final collagen concentration of 5 mg / mL with deionized water. -1 Pure collagen precursor solution (COL) was obtained.

[0032] Example 2 This embodiment provides the preparation of a composite collagen precursor solution, specifically including the following: At 4 °C, the pure collagen precursor solution prepared in Example 1 was added to 1.2 M NaH2PO4 solution and stirred for 1 h. Then, 2 M CaCl2 solution was added to make the Ca / P ratio in the system about 1.67. The pH was adjusted and kept at 9.0±0.2. The mixture was stirred at a constant speed for 2 h to obtain the composite collagen precursor solution, denoted as COL / nHA.

[0033] Example 3 This embodiment provides the preparation of a composite collagen precursor solution, specifically including the following: At 4 °C, the pure collagen precursor solution prepared in Example 1 was added to a fucoidan solution (20 mg / mL). -1 The mixture was prepared and adjusted to a mass ratio of fucose to collagen of 5% with deionized water. Then, 1.2 M NaH2PO4 solution was added and stirred for 1 h. 2 M CaCl2 solution was added to make the Ca / P ratio of the system about 1.67. The pH was adjusted and kept at 9.0±0.2. The mixture was stirred at a constant speed for 2 h to obtain the composite collagen precursor solution, denoted as 5% Fc-COL-nHA.

[0034] Example 4 This embodiment provides the preparation of a composite collagen precursor solution, specifically including the following: At 4 °C, the pure collagen precursor solution prepared in Example 1 was added to a fucoidan solution (20 mg / mL). -1 The mixture was prepared and adjusted with deionized water to make the mass ratio of fucoidan to collagen 10%. Then, 1.2 M NaH2PO4 solution was added and stirred for 1 h. Then, 2 M CaCl2 solution was added to make the Ca / P ratio of the system about 1.67. The pH was adjusted and kept at 9.0±0.2. The mixture was stirred at a constant speed for 2 h to obtain the composite collagen precursor solution, denoted as 10% Fc-COL-nHA.

[0035] Example 5 This embodiment provides the preparation of a composite collagen precursor solution, specifically including the following: At 4 °C, the pure collagen precursor solution prepared in Example 1 was added to a fucoidan solution (20 mg / mL). -1 The mixture was prepared by adding 1.2 M NaH2PO4 solution and stirring for 1 h. Then, 2 M CaCl2 solution was added to make the Ca / P ratio of the system about 1.67. The pH was adjusted and kept at 9.0±0.2. The mixture was stirred at a constant speed for 2 h to obtain the composite collagen precursor solution, denoted as 20% Fc-COL-nHA.

[0036] Example 6 The pure collagen precursor solution prepared in Example 1 and the composite collagen precursor solutions prepared in Examples 2-5 were respectively added dropwise to 24-well plates and placed at 4 °C for 24 h to initiate collagen self-assembly and mineralization. After gelation, impurities on the surface of the materials were rinsed with ultrapure water and then freeze-dried. The morphology of each group of mineralized samples was observed using a scanning electron microscope. The freeze-dried samples were fixed on a special stage with conductive adhesive, sputter-coated with gold under vacuum for 120 s, and the fibrous porous structure and micromorphology of each group of materials were observed using SEM.

[0037] Experimental results are as follows Figure 1 As shown, the microstructure of different samples was characterized by SEM. In the self-assembled system of pure fish skin collagen, fiber bundles were clearly visible, with a relatively smooth surface and visible transverse striations characteristic of collagen. The fiber bundles intertwined to form a porous three-dimensional network structure, with a relatively uniform fiber diameter distribution. After the introduction of calcium phosphate, nanoparticles were deposited on the surface or inside of the collagen fibers, forming mineralization outside or inside the fibers, resulting in thicker fiber diameters and rougher surfaces. The pore size also changed significantly, with the originally dense and ordered structure becoming loose and disordered. At fiber intersections or along the fiber direction, denser nanoparticle deposition occurred, forming obvious mineralized nodules, and the gaps between collagen fiber bundles became irregular or blocked by mineralized particles. At the same time, with the introduction of Fc (fucoidan), the fiber morphology and pore characteristics changed further, with some fibers becoming thicker and wider, and the corresponding pores also showing diversification. Low concentrations of Fc made the distribution of calcium phosphate particles in collagen slightly more uniform, and reduced the number of aggregated nodules. Higher concentrations of Fc may affect the self-assembly process of collagen fibers, forming thicker fibers or a more uniform composite network. Mineralization may be distributed within the formed gel network rather than concentrated on the surface of the coarse fibers, and large mineralized nodules are significantly reduced or disappear. The addition of Fc makes the gel material structure more homogeneous and porous, or forms smaller, more uniform micropores composed of a fine fiber network.

[0038] Example 7 The effects of different concentrations of fucoidan (precursor solutions prepared in Examples 1-5) on the microstructure of mineralized materials were observed using TEM. Each composite material was thoroughly dispersed in deionized water using an ultrasonic instrument. Then, a drop of each well-dispersed suspension was placed on a copper grid with a carbon support film. After thorough air drying, the suspension was observed using a transmission electron microscope.

[0039] Experimental results are as follows Figure 2 As shown, TEM results revealed the structural relationship between collagen fibers and apatite minerals. Under the microscope, the self-assembled collagen fibers all exhibited alternating light and dark stripe structures, which are manifestations of the periodic arrangement of their molecules, known as D-periodic bands, consistent with the structure of natural collagen fibrils. Figure 2As shown in Figure A, pure COL fibers exhibit a typical, regular morphology, with relatively orderly arrangement between fibers and no obvious mineralization structure. The darker color is a result of fiber overlap. Combined with... Figure 2 As shown in Figure B, the basic morphology of collagen fibers was preserved after the addition of nHA, while obvious mineralization traces appeared on the fibers, with particles beginning to deposit around and inside the fibers. Figure 2 As shown in C, D and E, with the introduction of Fc, the nano-dispersion characteristics of nHA can be seen in the fiber structure. When the Fc content reaches 20%, the fiber mineralization is obvious, and mineralization inside and outside the fiber exists simultaneously.

[0040] Example 8 This embodiment conducts cell experiments on the collagen precursor solutions prepared in Examples 1-5 to further explore their biological activity.

[0041] The COL, COL-nHA, 5% Fc-COL-nHA, 10% Fc-COL-nHA, and 20% Fc-COL-nHA composite collagen precursor solutions synthesized in Examples 1-5 were respectively added dropwise to 48-well plates and incubated at 4 °C for 24 h to initiate collagen self-assembly and mineralization. After gelation, 5 mM genipin was added for cross-linking. The samples were then sterilized by soaking in 75% ethanol (v / v) for 6 h, washed three times with sterile PBS, and subsequently incubated in serum-free α-MEM cell culture medium at 37 °C for 24 h in a 95% air and 5% CO2 atmosphere, with the serum-free α-MEM cell culture medium being replaced once during incubation. The incubated samples were then used for cell experiments.

[0042] BMSCs (Bone Marrow Mesenchymal Stem Cells) were placed in a cell culture incubator at 37 ℃ and 5% CO2 and cultured in α-MEM complete medium containing 10% fetal bovine serum and 1% penicillin / streptomycin. When the cell density was greater than 80% of the bottom area of ​​the culture flask, the cells were passaged. The old medium in the flask was discarded, and the cells were washed with 1×PBS. 0.25% trypsin was added, and the cells were gently shaken to cover them. The cells were then placed in a cell culture incubator for digestion for 1 min. α-MEM medium was added to stop the digestion. The cell suspension was collected by pipetting and centrifugation at 1000 rps for 5 min. The supernatant was discarded, and the cells were resuspended in fresh medium for passage. Cell counts were performed, and the cell concentration was adjusted to the required level for subsequent use.

[0043] BMSCs were divided into 1 × 10 4Cells were seeded at the specified concentrations per well onto the surfaces of the pre-treated materials and co-cultured. On days 1, 3, and 5, the old culture medium was discarded, and the cells were washed with 1×PBS. CCK-8 assay solution was prepared in the dark at a ratio of 1:10 to the culture medium, and 100 μL of the prepared assay solution was added to each well. The plates were incubated in the dark for 1 h. After 1 h, the solution from each well was aspirated into a 96-well plate, and the absorbance at 450 nm was measured and recorded using a microplate reader.

[0044] After adding the same proportion of n-HA and different proportions of Fc, the cell proliferation capacity of each group of materials was measured after co-culturing with cells for 1, 3, and 5 days. The CCK-8 results are as follows: Figure 3 As shown, the number of cells in each group increased over time. Compared with the blank control group, the cells in the pure COL group and each composite material group maintained good proliferative activity. After the introduction of Fc, the cell proliferation trend of each Fc-COL-nHA group was similar to that of the COL group, and there was no significant inhibitory or promoting effect on cell proliferation, indicating that the introduction of Fc and nHA did not affect the biocompatibility of the material and the overall safety was good.

[0045] Example 9 In this embodiment, the collagen precursor solutions prepared in Examples 1-5 were placed at 4°C for 24 h to induce collagen self-assembly and mineralization, respectively, to prepare corresponding gel materials. The bone-promoting and anti-inflammatory capabilities of the prepared gel materials were evaluated.

[0046] To further evaluate the osteogenic and anti-inflammatory capabilities of the material, this embodiment used real-time PCR to measure the expression of osteogenic-related genes (ALP, Runt-related transcription factor 2 (Runx-2)) and inflammation-related genes (transforming growth factor-β1, TGF-β1). After cells adhered to the surface of the gel material, LPS was added to a final concentration of 10 µg / mL. -1 After a suitable stimulation time, the supernatant from each group was collected. At days 7, 14, and 21 of BMSCs induction, cells were lysed using RNA Easy to extract RNA. The concentration and purity of each sample at 260 nm and 280 nm were determined using Nano Drop One. cDNA was reverse transcribed using total mRNA as a template, and PCR amplification was performed using the cDNA as a template. 2 -ΔΔCt The method calculates the gene expression level for each sample, and the method is repeated three times.

[0047] Experimental results are as follows Figure 4 As shown, the expression of osteogenic genes Runx-2, ALP, and inflammatory gene TGF-β1 in Fc-COL-nHA material cells from different groups was assessed on days 7 and 14, respectively. Figure 4 As shown in the results, on days 7 and 14, the osteogenic gene Runx-2 maintained high expression levels in the 10% Fc-COL-nHA group, while Runx-2 expression was significantly downregulated in the 20% Fc group, and each group showed a time-dependent increase. On day 14, the gene expression level in the 10% Fc-COL-nHA group was 5.51 times that of the control group and 1.61 times that of the COL-nHA group. On day 7, ALP expression was highest in the COL-nHA group, followed by the 10% Fc-COL-nHA group, both significantly higher than the control group; ALP expression was lowest in the 20% Fc-COL-nHA group, with no significant difference from the control group, suggesting that high concentrations of Fc did not promote early ALP activity. On day 14, except for the COL-nHA group, ALP expression in all other groups was significantly higher than on day 7. The 20% Fc-COL-nHA group showed the highest expression level, followed by the 10% Fc-COL-nHA group. On day 14, the gene expression level in the 10% Fc-COL-nHA group was 4.47 times that of the control group and 3.07 times that of the COL-nHA group; the gene expression level in the 20% Fc-COL-nHA group was 6.30 times that of the control group and 4.33 times that of the COL-nHA group. TGF-β1 expression levels are closely related to the regulation of the bone repair microenvironment. On day 7, TGF-β1 expression levels were similar across groups, with the COL-nHA, 10% Fc-COL, and 20% Fc-COL-nHA groups all slightly higher than the control group, suggesting that the early regulatory effect of each group on TGF-β1 was relatively weak. On day 14, TGF-β1 expression was significantly upregulated in all groups, exhibiting a clear concentration-dependent increase: the 20% Fc-COL-nHA group showed the highest expression level, followed by the 10% Fc-COL-nHA group, and then the COL-nHA group; all three groups were significantly higher than the control group. On day 14, the gene expression level in the 10% Fc-COL-nHA group was 3.04 times that of the control group and 1.83 times that of the COL-nHA group; the gene expression level in the 20% Fc-COL-nHA group was 4.24 times that of the control group and 2.55 times that of the COL-nHA group. Figure 5 This is a schematic diagram illustrating the principle of composite hydrogel in the repair of inflammatory microbone defects.

[0048] Example 10 Based on the experiments and analyses described above, this embodiment provides a preparation process for a marine-derived composite hydrogel with bone defect repair function, providing a theoretical basis for subsequent industrial production and promotion. The preparation process includes the following: 1. The fish skin is repeatedly washed and soaked to obtain swollen fish skin; the soaking time is 20-50 hours.

[0049] 2. The swollen fish skin is enzymatically hydrolyzed using pepsin, with an enzyme activity of 3000~10000 U / g and a hydrolysis time of 20~55 h; after hydrolysis, the fish skin is centrifuged and the supernatant is collected.

[0050] 3. The supernatant is salted out and filtered. The precipitate obtained after filtration is dissolved in acetic acid to obtain a fish skin collagen solution.

[0051] 4. Add 1-3M NaH2PO4·2H2O to the fish skin collagen solution and stir. Adjust the solution to neutral with NaOH to obtain pure collagen precursor solution.

[0052] 5. Add the pure collagen precursor solution to fucoidan to obtain a mixed solution, and adjust the mass ratio of fucoidan to collagen in the mixed solution to 5-20% with deionized water solution; add NaH2PO4 solution with a molar concentration of 1-3M and CaCl2 solution with a molar concentration of 1-5M successively and stir at a constant speed, and adjust the pH of the solution to 8-10 to obtain a composite collagen precursor solution; the Ca / P ratio in the composite collagen precursor solution is 1.0-2.0.

[0053] 6. The composite collagen precursor solution is allowed to stand for 20-30 h to initiate the self-assembly and mineralization of collagen. The mineralization time is 24-72 h. Genipin is added during the mineralization process for cross-linking. The mass amount of genipin is 0.05-0.2% of the mass of the composite collagen precursor solution to obtain a composite hydrogel with bone defect repair function.

[0054] The ambient temperature was controlled at 3~10℃ throughout the entire preparation process.

[0055] The core innovation of this invention lies in the construction of an organic-inorganic material synergistic system. By combining marine collagen with hydroxyapatite to form a biomimetic matrix and innovatively doping it with fucoidan, which has multiple biological functions, a synergistic effect between material structure and function is achieved. This breakthrough integrates the biomimetic microenvironment construction capability of the organic phase (marine collagen), the osteoconductive properties of the inorganic phase (hydroxyapatite), and the anti-inflammatory function of fucoidan to form a three-dimensional network composite material.

[0056] The marine collagen provided by this invention forms a three-dimensional network scaffold through self-assembly, mimicking the topology of the natural extracellular matrix and providing a biomimetic microenvironment for cell adhesion, proliferation, and migration. Hydroxyapatite is directionally deposited on the surface of collagen fibers to form a mineralized substrate with a crystal structure highly similar to the inorganic phase of bone, endowing the material with excellent osteoconductivity and guiding bone tissue regeneration along specific directions. Fucoidan binds to the collagen network through dynamic hydrogen bonds, exerting anti-inflammatory effects and improving the regenerative microenvironment by inhibiting NF-κB pathway-mediated macrophage polarization. Furthermore, it releases loaded osteogenic active factors (such as BMP-2) in a pH-responsive manner, achieving time-controllable pro-angiogenic and osteoinductive effects. Through organic / inorganic interfacial chemical bonding and biomolecular interactions, these three components construct a smart composite material system with gradient modulus, dynamic degradation, and bioactive delivery characteristics. This significantly improves the coordination of cellular response, matrix remodeling, and functional reconstruction during bone defect repair, providing a breakthrough solution for the regeneration and repair of complex bone defects.

[0057] This invention addresses the limitations of existing marine-derived bone tissue repair materials by innovatively utilizing biocompatible marine collagen and fucoidan to prepare a composite hydrogel suitable for repairing bone defects under inflammatory conditions through covalent cross-linking technology. This composite hydrogel not only possesses excellent biocompatibility but also achieves mineralization through loading hydroxyapatite, significantly enhancing its mechanical properties and bioactivity. When applied to bone defect sites, it can achieve efficient in vivo transformation, completing the bone defect repair and mineralization process under inflammatory conditions, providing an efficient, convenient, and widely applicable solution for bone tissue engineering.

[0058] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions claimed by the present invention.

Claims

1. A method for preparing a marine-derived composite hydrogel with bone defect repair function, characterized in that, The preparation method of the composite hydrogel specifically includes the following steps: (1) The fish skin is repeatedly washed and soaked to obtain swollen fish skin; (2) The swollen fish skin was enzymatically hydrolyzed with pepsin, and after complete enzymatic hydrolysis, it was centrifuged and the supernatant was collected. (3) Salt out and filter the supernatant, and dissolve the precipitate obtained after filtration in acetic acid to obtain a fish skin collagen solution; (4) Add phosphate to the fish skin collagen solution and stir to adjust the solution to neutral to obtain pure collagen precursor solution; (5) Add the pure collagen precursor solution to fucoidan to obtain a mixed solution, add phosphate solution and CaCl2 solution in sequence and stir at a constant speed, adjust the pH of the solution to 8~10, and obtain a composite collagen precursor solution. (6) The composite collagen precursor solution is left to stand for 20-30 hours to induce the self-assembly and mineralization of collagen. Genipin is added during the mineralization process for cross-linking to obtain a composite hydrogel.

2. The preparation method according to claim 1, characterized in that, The activity of the pepsin is 3000~10000 U / g.

3. The preparation method according to claim 1, characterized in that, The mass ratio of fucoidan to collagen in the mixture is 5% to 20%.

4. The preparation method according to claim 1, characterized in that, The phosphate solution has a molar concentration of 0.3~3M, and the CaCl2 solution has a molar concentration of 1~5M.

5. The preparation method according to claim 1, characterized in that, The Ca / P ratio in the composite collagen precursor solution is 1.0~2.

0.

6. The preparation method according to claim 1, characterized in that, The temperature is controlled at 3~10℃ during the preparation of the composite hydrogel.

7. The preparation method according to claim 1, characterized in that, The mineralization time is 24-72 h, and the mass of genipin used is 0.05-0.2% of the collagen mass in the composite collagen precursor solution.

8. The composite hydrogel prepared by any one of the preparation methods according to claims 1 to 7.

9. The application of the composite hydrogel according to claim 8 in the preparation of formulations with bone defect repair function.

10. The application according to claim 9, characterized in that, The bone defects include inflammatory microbone defects, and the composite hydrogel can increase the expression of osteogenic genes Runx-2 and ALP, and upregulate the expression of TGF-β1.

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