Preparation method of GelMA composite photosensitive hydrogel containing aquatic biomass calcium powder

Abstract

This invention discloses a method for preparing a GelMA composite photosensitive hydrogel containing aquatic biomass calcium powder. The method includes: obtaining methacrylamide gelatin; mixing the methacrylamide gelatin with a photoinitiator solution at a mass-to-volume ratio of (0.05–0.35) g:1 mL to obtain a photosensitive methacrylamide gelatin prepolymer; mixing the aquatic biomass calcium powder with the photosensitive methacrylamide gelatin prepolymer at a mass-to-volume ratio of (0.05–0.15) g:1 mL to obtain a mixed solution; placing the mixed solution in a mold and subjecting it to photo-irradiation curing to obtain the GelMA composite photosensitive hydrogel containing aquatic biomass calcium powder. The composite photosensitive hydrogel prepared by this invention exhibits high osteogenic activity and good biocompatibility, providing a new repair material option for filling bone defects.

Description

Technical Field

[0001] This invention belongs to the field of bone repair gel material technology, specifically relating to a method for preparing a GelMA composite photosensitive hydrogel containing aquatic biomass calcium powder. Background Technology

[0002] Clinically, the treatment of bone defects urgently requires tissue-engineered bone repair materials to fill the defect sites and achieve structural and functional reconstruction. Therefore, developing a bone repair substitute material that combines good biocompatibility with biomimetic natural bone structure and function is of great significance for repairing bone defects, reducing complications, and improving patient rehabilitation. Bone tissue engineering offers a new approach to solving this problem, with bio-scaffold materials being key to achieving bone repair and reconstruction.

[0003] Methacrylamide gelatin (GelMA) can be used to construct a photosensitive three-dimensional scaffold material that can mimic the extracellular matrix environment and has good biocompatibility, and has been widely used in tissue engineering fields such as cartilage and bone. However, the osteogenic induction ability of pure GelMA hydrogel is limited, and it usually needs to be compounded or loaded with osteogenic active substances to enhance its osteogenic capacity.

[0004] It should be noted that the information disclosed in the background section above is only used to enhance the understanding of the background of the present invention, and therefore may include information that does not constitute prior art known to those skilled in the art. Summary of the Invention

[0005] To address the aforementioned problems in the prior art, this invention provides a method for preparing a GelMA composite photosensitive hydrogel containing aquatic biomass calcium powder. The technical problem to be solved by this invention is achieved through the following technical solution: This invention provides a method for preparing a GelMA composite photosensitive hydrogel containing aquatic biomass calcium powder, comprising the following steps: S1. Obtain methacrylamide gelatin; S2. The methacrylamide gelatin is added to a photoinitiator solution to obtain a photosensitive methacrylamide gelatin prepolymer solution; wherein the mass-to-volume ratio of the methacrylamide gelatin to the photoinitiator solution is (0.05-0.35) g: 1 mL. S3. Add aquatic biomass calcium powder to the photosensitive methacrylamide gelatin prepolymer solution, mix evenly, and obtain a mixed solution; wherein, the mass-volume ratio of the aquatic biomass calcium powder to the photosensitive methacrylamide gelatin prepolymer solution is (0.05~0.15) g: 1 mL. S4. Place the mixed solution in a mold and cure it by ultraviolet light to obtain a GelMA composite photosensitive hydrogel containing aquatic biomass calcium powder.

[0006] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. The present invention provides a method for preparing a GelMA (methacrylamide gelatin) composite photosensitive hydrogel containing aquatic biomass calcium powder, wherein aquatic biomass calcium powder is added as a filler to the GelMA hydrogel system. The main components of aquatic biomass calcium powder are calcium carbonate, calcium silicate, or hydroxyapatite, and it also contains various trace elements and amino acids. The addition of aquatic biomass calcium powder results in the formation of a calcium salt particle reinforcing phase inside the hydrogel. The calcium salt particles physically infuse and fill the GelMA polymer network, constructing a denser three-dimensional hydrogel network structure, thereby improving the mechanical properties of the hydrogel. Thus, based on bioactive substances derived from natural bone, the osteogenic activity of methacrylamide gelatin (GelMA) is improved, providing a new repair material option for bone defect filling.

[0007] 2. The addition of aquatic biomass calcium powder in this invention provides osteogenic active sites within the hydrogel. Calcium salts can degrade in body fluids, releasing calcium ions and carbonate, silicate, or phosphate ions, which participate in bone mineralization and promote the adhesion, proliferation, and osteogenic differentiation of bone marrow mesenchymal stem cells. Simultaneously, the trace elements and amino acids contained in the aquatic biomass calcium powder can further promote the expression of osteogenic-related genes, enhancing the osteogenic induction capacity of the hydrogel.

[0008] 3. This invention uses photo-irradiation curing to prepare hydrogel blocks. By controlling the wavelength and irradiation time of ultraviolet light, the methacryloyl groups in the GelMA molecules undergo free radical polymerization to form a three-dimensional network structure. The photo-irradiation curing conditions are mild and the curing speed is fast, enabling the hydrogel to be formed in a short time while maintaining the uniform dispersion of aquatic biomass calcium powder in the system.

[0009] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0010] Figure 1 This is a flowchart illustrating a method for preparing a GelMA composite photosensitive hydrogel containing aquatic biomass calcium powder, as provided in an embodiment of the present invention. Figure 2 The images show SEM characterization images of the GelMA photosensitive hydrogel and the GelMA composite photosensitive hydrogel containing aquatic biomass calcium powder prepared in Examples 1 and 2 of this invention. Figure 3 EDS characterization images of the GelMA photosensitive hydrogel and the GelMA composite photosensitive hydrogel containing aquatic biomass calcium powder prepared in Examples 1 and 2 of this invention. Figure 4The XRD characterization diagrams are of the GelMA photosensitive hydrogel and the GelMA composite photosensitive hydrogel containing aquatic biomass calcium powder prepared in Examples 1 and 2 of this invention. Figure 5 The images provided in this embodiment of the invention show cell viability and mortality staining (A) and a statistical chart of the number of dead cells (B) after co-culturing the extract with MC-3T3 cells for 24 hours (scale bar 200 μm). Figure 6 The images shown are ALP staining (A) and relative quantitative statistics of alkaline phosphatase (B) after 7 days of co-culturing the extract with MC-3T3 cells provided in this embodiment of the invention (scale bar 200 μm). Detailed Implementation

[0011] To further illustrate the technical means and effects adopted by the present invention to achieve the intended purpose, the following describes in detail, with reference to the accompanying drawings and specific embodiments, a method for preparing a GelMA composite photosensitive hydrogel containing aquatic biomass calcium powder according to the present invention.

[0012] The foregoing and other technical contents, features, and effects of the present invention will be clearly presented in the following detailed description of specific embodiments in conjunction with the accompanying drawings. Through the description of the specific embodiments, a more in-depth and concrete understanding can be gained of the technical means and effects adopted by the present invention to achieve its intended purpose. However, the accompanying drawings are for reference and illustration only and are not intended to limit the technical solutions of the present invention.

[0013] It should be noted that, in this document, the terms “comprising,” “including,” or any other variations are intended to cover non-exclusive inclusion, such that an article or device that comprises a list of elements includes not only those elements but also other elements not expressly listed.

[0014] Oyster shells (rich in calcium and trace elements) derived from the exoskeleton and tortoise shells (rich in hydroxyapatite and organic components, commonly used in traditional Chinese medicine) show promise as potential bone repair materials for treating clinical bone defects. Based on this, this invention provides a method for preparing a GelMA composite photosensitive hydrogel containing aquatic biomass calcium powder, see [link to relevant documentation]. Figure 1 This includes the following steps: S1. Obtain methacrylamide gelatin (GelMA).

[0015] S2. Methacrylated gelatin is added to a photoinitiator solution to obtain a photosensitive methacrylated gelatin prepolymer. The mass-to-volume ratio of methacrylated gelatin to the photoinitiator solution is (0.05–0.35) g:1 mL. This ratio determines the viscosity of the photosensitive methacrylated gelatin prepolymer, thus affecting the moldability and mechanical properties of the subsequent composite photosensitive hydrogel. When the mass-to-volume ratio of GelMA to the photoinitiator solution is close to the lower limit (0.05 g:1 mL), the prepared hydrogel is softer and has relatively lower mechanical strength, but possesses good elasticity and flexibility, making it suitable for repairing non-load-bearing bone defects. When the mass-to-volume ratio of GelMA to the photoinitiator solution is close to the upper limit (0.35 g:1 mL), the prepared hydrogel is harder and has higher mechanical strength, but its brittleness increases, making it suitable for load-bearing bone defects. In practical applications, an appropriate ratio can be selected based on the specific conditions of the bone defect. It is worth noting that exceeding the above ratio range, for example, too low a GelMA ratio (less than 0.05g:1mL) will result in insufficient mechanical strength of the hydrogel; too high a GelMA ratio (greater than 0.35g:1mL) will result in excessive viscosity of the prepolymer, making it difficult to handle and mold.

[0016] Step S2 is performed in a light-protected environment. Specifically, methacrylamide gelatin is added to a container containing a photoinitiator solution and placed in a water bath at 35°C–40°C. The mixture is then shaken for 20–30 minutes to ensure the photoinitiator solution completely wets the methacrylamide gelatin, resulting in a photosensitive methacrylamide gelatin prepolymer. Shaking is used to ensure thorough and uniform mixing of the photoinitiator solution and the methacrylamide gelatin solution, facilitating uniform photocuring of the prepolymer. Further warming promotes complete dissolution, and shaking is used to ensure uniform mixing and avoid excessive air bubbles.

[0017] In one example, the photoinitiator is lithium phenyl-2,4,6-trimethylbenzoylphosphinic acid, and the mass-to-volume ratio of lithium phenyl-2,4,6-trimethylbenzoylphosphinic acid to PBS solution (phosphate buffer) in the photoinitiator solution is (1–5) mg:1 mL. The concentration of the photoinitiator has a significant impact on the rate and extent of the photocuring reaction. Too low a concentration (less than 1 mg:1 mL) will result in incomplete photocuring, while too high a concentration (greater than 5 mg:1 mL) will cause excessively rapid curing, uneven gelation, and affect the microstructure of the hydrogel. During preparation, the photoinitiator powder can be dissolved by sonication in a small amount of PBS solution first, and then the remaining PBS solution can be added to reach the target volume. After preparation, the photoinitiator solution needs to be stored away from light to prevent decomposition and inactivation due to light exposure.

[0018] S3. Add aquatic biomass calcium powder to the photosensitive methacrylamide gelatin prepolymer solution and mix thoroughly to obtain a mixed solution. The mass-to-volume ratio of aquatic biomass calcium powder to the photosensitive methacrylamide gelatin prepolymer solution is (0.05–0.15) g:1 mL. This ratio determines the content of calcium salt particles in the hydrogel, thus affecting its mechanical properties and osteogenic induction ability. When the mass-to-volume ratio of aquatic biomass calcium powder to the photosensitive methacrylamide gelatin prepolymer solution is close to the lower limit (0.05 g:1 mL), the improvement effect on the mechanical properties of the hydrogel is relatively weak, but the number of osteogenic active sites is moderate, which can meet the needs of general bone repair. When the mass-to-volume ratio of aquatic biomass calcium powder to photosensitive methacrylamide gelatin prepolymer solution is close to the upper limit (0.15 g: 1 mL), the mechanical properties of the hydrogel are significantly improved, and the number of osteogenic active sites increases. However, excessively high calcium powder content (e.g., a mass-to-volume ratio of aquatic biomass calcium powder to photosensitive methacrylamide gelatin prepolymer solution greater than 0.15 g: 1 mL) may lead to particle aggregation within the hydrogel, affecting the uniformity of the microstructure. In practical applications, an appropriate ratio can be selected based on the size of the bone defect and the repair requirements.

[0019] This step is performed in a light-protected environment. Specifically, aquatic biomass calcium powder is added to the photosensitive methacrylamide gelatin prepolymer solution, and then the solution is mixed by shaking.

[0020] In one example, the aquatic biomass calcium powder is one of oyster shell powder (exoskeleton source) or tortoise shell powder (endoskeleton source). Preferably, the particle size of the oyster shell powder and tortoise shell powder is in the range of 10 μm to 50 μm.

[0021] S4. Place the mixed solution in a mold and cure it by light irradiation to obtain a GelMA composite photosensitive hydrogel containing aquatic biomass calcium powder with a three-dimensional network structure.

[0022] In one example, photocuring uses ultraviolet light with a wavelength of 405 nm and an irradiation time of 30–60 s. The 405 nm wavelength effectively excites the phenyl-2,4,6-trimethylbenzoyl lithium phosphinoate photoinitiator, generating free radicals that subsequently cause the methacryloyl groups to polymerize. Too short an irradiation time leads to incomplete polymerization, resulting in weak hydrogel formability and insufficient strength; too long an irradiation time may cause over-curing, affecting the hydrogel's flexibility and biocompatibility. For example, a photocuring UV lamp with a wavelength of 405 nm, an irradiation power of 3 W, and an irradiation time of 30 s is used.

[0023] In one embodiment of the present invention, obtaining methacrylamide gelatin includes: S11. Add gelatin to a volume of V mL of PBS solution and stir at 50℃~80℃ until the gelatin dissolves to obtain a gelatin solution. The mass-to-volume ratio of gelatin to PBS solution is (0.05~0.20) g:1 mL. If the gelatin solution concentration is too low, the final hydrogel will have weak formability and insufficient mechanical strength; if the gelatin solution concentration is too high, the reaction system will be too viscous and have poor flowability, affecting the uniformity of the reaction.

[0024] S12. Maintaining the temperature within the range of 40℃ to 60℃, add methacrylic anhydride to the gelatin solution and continue stirring for 2 to 4 hours. Terminate the reaction by adding at least (5×V) mL of PBS solution to obtain a crude methacrylamide gelatin solution; wherein the mass-to-volume ratio of gelatin to methacrylic anhydride is (1.5–2.5) g:1 mL. In this step, the proportion of methacrylic anhydride added directly affects the degree of substitution of the methacrylamide gelatin. A higher degree of substitution results in faster photocuring of the hydrogel, but excessively high substitution may affect the biocompatibility of the gelatin. During the reaction, methacrylic anhydride undergoes an amidation reaction with the amino groups on the gelatin molecular chain, introducing methacryloyl groups onto the gelatin molecule.

[0025] S13. The crude methacrylamide gelatin solution was purified by dialysis and dried to obtain methacrylamide gelatin.

[0026] In one example, the dialysis purification of crude methacrylamide gelatin solution includes: placing the crude methacrylamide gelatin solution in a dialysis bag with a molecular weight cutoff of 8 kDa to 15 kDa, and dialyzing it for 3 to 10 days at a temperature range of 30°C to 60°C to remove unreacted methacrylic anhydride and reaction byproducts.

[0027] Specifically, the dialysis bag containing the crude solution is placed in a large beaker containing deionized water, and dialysis is performed within a temperature range of 30℃ to 60℃. During dialysis, the deionized water needs to be changed 2-3 times daily to maintain a clean dialysis environment. The dialysis time is controlled between 3 and 10 days, with the specific time determined based on the clarity of the solution and laboratory conditions. After dialysis, the solution in the dialysis bag is removed, yielding the purified methacrylamide gelatin solution.

[0028] In one example, in step S13, the drying is performed by freeze-drying. Specifically, the purified methacrylamide gelatin solution can be pre-frozen at -20°C for 4 hours, then transferred to the sample tray of a freeze dryer and freeze-dried at -40°C for 24–48 hours until the sample is completely dry. After freeze-drying, a white or light yellow foamy methacrylamide gelatin solid is obtained.

[0029] This invention uses GelMA hydrogel as a matrix and combines it with oyster shell powder (exoskeleton source) or tortoise shell powder (endoskeleton source) to construct a three-dimensional network structure of composite photosensitive hydrogel of GelMA / oyster shell powder and GelMA / tortoise shell powder.

[0030] The performance of the GelMA composite photosensitive hydrogel containing aquatic biomass calcium powder prepared by the method provided in this invention will be further explained below with reference to specific embodiments.

[0031] Materials preparation: Preparation of PBS solution: Take 8.0g of sodium chloride, 0.2g of potassium chloride, 0.24g of potassium dihydrogen phosphate, and 1.44g of disodium hydrogen phosphate, add deionized water to make up to 1000mL, and adjust the pH to 7.4.

[0032] The gelatin used was Grade A gelatin derived from pigskin, and the methacrylic anhydride used was an analytical grade reagent.

[0033] Comparative Example 1 Step 1: Take 10g of gelatin and add it to 100mL of PBS solution. Stir at 60℃ until completely dissolved to obtain a gelatin solution. Maintain the temperature at 50℃ and slowly add 5mL of methacrylic anhydride dropwise to the gelatin solution, stirring continuously during the addition. After the addition is complete, continue stirring at 50℃ for 3 hours. After the reaction is complete, add 1000mL of PBS solution to terminate the reaction, obtaining a crude methacrylamide gelatin solution. Place the crude methacrylamide gelatin solution into a dialysis bag with a molecular weight cutoff of 10kDa and dialyze at 40℃ for 7 days, changing the water 3 times a day. After dialysis, collect the purified solution and freeze-dry to obtain solid methacrylamide gelatin. Figures 2 to 4 The diagram shows the SEM (scanning electron microscope) characterization, EDS (energy dispersive spectroscopy) characterization, and XRD (X-ray diffraction) characterization of GelMA.

[0034] Step 2: Take 0.04 g of phenyl-2,4,6-trimethylbenzoyl lithium phosphine and add it to 20 mL of PBS solution to prepare a 2 mg / mL photoinitiator solution. Take 0.10 g of methacrylamide gelatin and add it to 1 mL of the photoinitiator solution. Shake and dissolve at 37 °C for 25 min to obtain a photosensitive methacrylamide gelatin prepolymer solution. The mass-to-volume ratio of methacrylamide gelatin to photoinitiator solution is 0.10 g: 1 mL.

[0035] Step 3: Place the photosensitive methacrylamide gelatin prepolymer in a silicone mold and directly photocur and irradiate (UV irradiation wavelength 405nm, irradiation power 30W, irradiation time 45s) to obtain GelMA photosensitive hydrogel (e.g. Figure 2 , Figure 3 , Figure 5 and Figure 6 (GelMA as illustrated in the image).

[0036] Example 1 Step 1: Take 10g of gelatin and add it to 100mL of PBS solution. Stir at 60℃ until completely dissolved to obtain a gelatin solution. Maintain the temperature at 50℃ and slowly add 5mL of methacrylic anhydride dropwise to the gelatin solution, stirring continuously during the addition. After the addition is complete, continue stirring at 50℃ for 3 hours. After the reaction is complete, add 1000mL of PBS solution to terminate the reaction, obtaining a crude methacrylamide gelatin solution. Place the crude methacrylamide gelatin solution into a dialysis bag with a molecular weight cutoff of 10kDa and dialyze at 40℃ for 7 days, changing the water 3 times a day. After dialysis, collect the purified solution and freeze-dry to obtain solid methacrylamide gelatin. Figures 2 to 4 The diagram shows the SEM (scanning electron microscope) characterization, EDS (energy dispersive spectroscopy) characterization, and XRD (X-ray diffraction) characterization of GelMA.

[0037] Step 2: Take 0.04 g of phenyl-2,4,6-trimethylbenzoyl lithium phosphine and add it to 20 mL of PBS solution to prepare a 2 mg / mL photoinitiator solution. Take 0.10 g of methacrylamide gelatin and add it to 1 mL of the photoinitiator solution. Shake and dissolve at 37 °C for 25 min to obtain a photosensitive methacrylamide gelatin prepolymer solution. The mass-to-volume ratio of methacrylamide gelatin to photoinitiator solution is 0.10 g: 1 mL.

[0038] Step 3: Take 0.10 g of oyster shell powder and add it to 1 mL of the above photosensitive methacrylamide gelatin prepolymer solution. Shake and mix for 15 min to obtain a mixed solution. The mass-volume ratio of oyster shell powder to photosensitive methacrylamide gelatin prepolymer solution is 0.10 g: 1 mL. Pour the mixed solution into a silicone mold and irradiate it with ultraviolet light at a wavelength of 405 nm and a power of 30 W for 45 s to complete the photo-irradiation curing. Remove the cured hydrogel from the mold to obtain the GelMA composite photosensitive hydrogel containing aquatic biomass calcium powder. Define the GelMA composite photosensitive hydrogel containing aquatic biomass calcium powder prepared in Example 1 as composite hydrogel 1.

[0039] like Figure 2 The image shown is a SEM characterization image of hydrogel 1 (GelMA+ML shown in the figure) prepared in Example 1. The GelMA composite photosensitive hydrogel containing oyster shell powder exhibits a three-dimensional network structure, in which oyster shell powder is diffusely distributed.

[0040] like Figure 3The image shows the EDS characterization of hydrogel 1 (GelMA+ML as indicated in the figure) prepared in Example 1, illustrating the types of elements contained in this composite hydrogel. This is compared to the GelMA photosensitive hydrogel (prepared in Comparative Example 1) which does not contain aquatic biomass calcium powder. Figure 3 The elements in GelMA (illustrated in the diagram), and the newly added elements in GelMA+ML, demonstrate the successful inclusion of oyster shell powder in the hydrogel network.

[0041] like Figure 4 The XRD characterization diagram of hydrogel 1 (GelMA+ML shown in the figure) prepared in Example 1 is shown. Compared with the characteristic peaks of oyster shell powder (ML shown in the figure), the GelMA+ML spectrum shows corresponding characteristic peaks (such as calcium carbonate and calcium silicate) at the corresponding positions.

[0042] Example 2 The difference between Example 2 and Example 1 is that tortoise shell powder is used in step 3.

[0043] The remaining steps are the same as those in Example 1. The GelMA composite photosensitive hydrogel containing aquatic biomass calcium powder prepared in Example 2 is defined as hydrogel 2.

[0044] like Figure 2 The image shown is a SEM characterization image of hydrogel 2 (GelMA+GB shown in the figure) prepared in Example 2. The GelMA composite photosensitive hydrogel containing tortoise shell powder exhibits a three-dimensional network structure, in which the tortoise shell powder is dispersed.

[0045] like Figure 3 The image shows the EDS characterization of hydrogel 2 (GelMA+GB as indicated in the figure) prepared in Example 2, illustrating the types of elements contained in this composite hydrogel. This is compared to the GelMA photosensitive hydrogel (prepared in Comparative Example 1) which does not contain water-soluble aquatic biomass calcium powder. Figure 3 The elements in GelMA (illustrated in the diagram), and the newly added elements in GelMA+ML, indicate the successful inclusion of tortoise shell powder in the hydrogel network.

[0046] like Figure 4 The XRD characterization diagram of hydrogel 2 prepared in Example 2 is shown. Compared with the characteristic peaks of tortoise shell powder (GB shown in the figure), the GelMA+GB spectrum shows the corresponding hydroxyapatite characteristic peaks at the corresponding positions.

[0047] Example 3 The difference between Example 3 and Example 1 is that in step 2, the mass-to-volume ratio of methacrylamide gelatin to photoinitiator solution is 0.05 g: 1 mL; and in step 3, the mass-to-volume ratio of oyster shell powder to photosensitive methacrylamide gelatin prepolymer solution is 0.10 g: 1 mL.

[0048] The remaining steps are the same as those in Example 1. The GelMA composite photosensitive hydrogel containing aquatic biomass calcium powder prepared in Example 3 is defined as hydrogel 3. The SEM, EDS, and XRD characterization images of hydrogel 3 are respectively compared with... Figures 2 to 4 The representation diagrams shown in the figure are similar to those corresponding to GelMA+ML.

[0049] Example 4 The difference between Example 4 and Example 1 is that in step 2, the mass-to-volume ratio of methacrylamide gelatin to photoinitiator solution is 0.35 g: 1 mL; and in step 3, the mass-to-volume ratio of oyster shell powder to photosensitive methacrylamide gelatin prepolymer solution is 0.15 g: 1 mL.

[0050] The remaining steps are the same as those in Example 1. The GelMA composite photosensitive hydrogel containing aquatic biomass calcium powder prepared in Example 4 is defined as hydrogel 4. The SEM, EDS, and XRD characterization images of hydrogel 4 are respectively compared with... Figures 2 to 4 The representation diagrams shown in the figure are similar to those corresponding to GelMA+ML.

[0051] Example 5 The difference between Example 5 and Example 1 is that in step 2, the mass-to-volume ratio of methacrylamide gelatin to photoinitiator solution is 0.10 g: 1 mL; and in step 3, the mass-to-volume ratio of oyster shell powder to photosensitive methacrylamide gelatin prepolymer solution is 0.05 g: 1 mL.

[0052] The remaining steps are the same as those in Example 1. The GelMA composite photosensitive hydrogel containing aquatic biomass calcium powder prepared in Example 5 is defined as hydrogel 5. The SEM, EDS, and XRD characterization images of hydrogel 5 are respectively compared with... Figures 2 to 4 The representation diagrams shown in the figure are similar to those corresponding to GelMA+ML.

[0053] Example 6 The difference between Example 6 and Example 1 is that in step 2, the mass-to-volume ratio of methacrylamide gelatin to photoinitiator solution is 0.20 g: 1 mL; and in step 3, the mass-to-volume ratio of oyster shell powder to photosensitive methacrylamide gelatin prepolymer solution is 0.10 g: 1 mL.

[0054] The remaining steps are the same as those in Example 1. The GelMA composite photosensitive hydrogel containing aquatic biomass calcium powder prepared in Example 6 is defined as hydrogel 6. The SEM, EDS, and XRD characterization images of hydrogel 6 are respectively compared with... Figures 2 to 4 The representation diagrams shown in the figure are similar to those corresponding to GelMA+ML.

[0055] To further illustrate the biocompatibility and osteogenic capacity of the GelMA composite photosensitive hydrogel containing aquatic biomass calcium powder prepared in this invention, in vitro verification experiments were conducted using hydrogel 1 and hydrogel 2 prepared in Examples 1 and 2, and MC-3T3 cells.

[0056] Preparation of extract: Based on α-MEM medium, add 1% penicillin-streptomycin antibiotics, 10% fetal bovine serum, 50 μg / mL vitamin C, 10 nM dexamethasone and 10 mM β-glycerophosphate sodium, mix thoroughly and filter to sterilize to obtain mineralization induction solution.

[0057] Take the GelMA composite photosensitive hydrogel containing oyster shell powder prepared in Example 1, add it to the above mineralization induction solution at a solid-liquid ratio of 0.1g:1mL, and soak for 48h to obtain the extract of the GelMA composite photosensitive hydrogel containing oyster shell powder.

[0058] Take the GelMA composite photosensitive hydrogel containing tortoise shell powder prepared in Example 2, add it to the above mineralization induction solution at a solid-liquid ratio of 0.1g:1mL, and soak for 48h to obtain the extract of the GelMA composite photosensitive hydrogel containing tortoise shell powder.

[0059] Take the GelMA photosensitive hydrogel prepared in Comparative Example 1, add it to the above mineralization induction solution at a solid-liquid ratio of 0.1g:1mL, and soak for 48h to obtain the extract of GelMA.

[0060] Example 7 Collect MC-3T3 cells in good growth condition, count them, and then divide them into groups of 5 × 10⁶ cells per well. 4 100 individuals were seeded at a density of 100 individuals in a 24-well plate.

[0061] After overnight incubation, the complete culture medium in the wells was aspirated, and 1 mL of extraction solution (extraction solution of GelMA composite photosensitive hydrogel containing oyster shell powder, or extraction solution of GelMA composite photosensitive hydrogel containing tortoise shell powder, or extraction solution of GelMA) was added to each well according to the experimental group, and the wells were incubated for another 24 h at 37℃ and 5% CO2.

[0062] Staining was performed using the Calcein-AM / PI live / dead cell double staining kit, specifically following the steps of: discarding the culture medium, gently washing with PBS, adding the prepared Calcein-AM / PI detection working solution, and incubating at room temperature in the dark for 1 hour. After incubation, the cell staining was observed and recorded under a fluorescence microscope.

[0063] The results are as follows Figure 5As shown, Calcein-AM is calcein acetoxymethyl ester, a live cell-specific dye used to label live cells; PI is propidium iodide, a dead cell-specific dye used to label dead cells with damaged cell membranes. Figure 5 (A) is a staining diagram, and (B) is a statistical diagram of dead cells. The results show that both composite hydrogels have good cell compatibility, with a large number of live cells and uniform distribution, and very few dead cells. This indicates that the composite hydrogel system constructed in this invention has no obvious toxicity to MC-3T3 cells and meets the biosafety requirements of bone tissue engineering scaffold materials.

[0064] Example 8 Select MC-3T3 cells in good growth condition, count the cells, and then distribute them at a ratio of 5 × 10⁶ cells per well. 4 The cells were seeded at a density of 100 cells per well in a 12-well plate. 1 mL of complete culture medium was added to each well. The plates were then gently shaken and incubated in a 37°C, 5% CO2 incubator.

[0065] Cell growth was observed every other day. When the cell density reached approximately 80% (the monolayer of cells covered 80% of the bottom of the culture well), the culture medium in the well was completely aspirated. According to the experimental group settings, 1 mL of extraction solution (extraction solution of GelMA composite photosensitive hydrogel containing oyster shell powder, or extraction solution of GelMA composite photosensitive hydrogel containing tortoise shell powder, or extraction solution of GelMA) was added to each well. The cells were then cultured at 37℃ and 5% CO2 for 7 days.

[0066] Staining was performed using the BCIP / NBT alkaline phosphatase colorimetric kit (Shanghai Beyotime Biotechnology Co., Ltd.). The specific steps included: removing the induced cell culture medium, discarding the medium, and washing three times with PBS. Adding 4% paraformaldehyde to each well and fixing for 30 min. Preparing the BCIP / NBT staining working solution according to the kit instructions, removing the fixative, and adding approximately 1 mL of staining working solution to each well to ensure complete cell coverage. Incubating at room temperature in the dark for 2 h, observing the staining progress during this time. Once the desired staining effect was achieved, removing the staining working solution, adding 1 mL of distilled water to each well to stop the staining process, and then photographing and saving the images.

[0067] See results Figure 6 (A) is a schematic diagram after cell staining, and (B) is a schematic diagram comparing the relative quantitative analysis results of alkaline phosphatase. This example compares the osteogenic activity of hydrogels prepared using oyster shell powder (derived from exoskeleton) or tortoise shell powder (derived from endoskeleton) combined with methacrylamide gelatin. Figure 6As shown in (A) and (B), ALP staining results revealed that both composite hydrogel systems exhibited superior osteogenic capacity compared to single methacrylamide hydrogel (GelMA). Comparing alkaline phosphatase levels, the oyster shell powder group derived from the exoskeleton showed a deeper blue-purple color, indicating higher alkaline phosphatase activity and stronger osteogenic differentiation induction capacity. Based on these differences in osteogenic activity, potential filling and repair materials can be screened for specific areas such as bone or cartilage defects.

[0068] The above description, in conjunction with specific preferred embodiments, provides a further detailed explanation of the present invention. It should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, various simple deductions or substitutions can be made without departing from the concept of the present invention, and all such modifications and substitutions should be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a GelMA composite photoreactive hydrogel containing an aqueous biomass calcium powder, characterized in that, Includes the following steps: S1. Obtain methacrylamide gelatin; S2. The methacrylamide gelatin is added to a photoinitiator solution to obtain a photosensitive methacrylamide gelatin prepolymer solution; wherein the mass-to-volume ratio of the methacrylamide gelatin to the photoinitiator solution is (0.05-0.35) g: 1 mL. S3. Add aquatic biomass calcium powder to the photosensitive methacrylamide gelatin prepolymer solution, mix evenly, and obtain a mixed solution; wherein, the mass-volume ratio of the aquatic biomass calcium powder to the photosensitive methacrylamide gelatin prepolymer solution is (0.05~0.15) g: 1 mL. S4. Place the mixed solution in a mold and cure it by ultraviolet light to obtain a GelMA composite photosensitive hydrogel containing aquatic biomass calcium powder.

2. The method for preparing an aqueous photogel of GelMA composite with aquatic biomass calcium powder according to claim 1, characterized in that, The photoinitiator is lithium phenyl-2,4,6-trimethylbenzoylphosphine, and the mass-to-volume ratio of lithium phenyl-2,4,6-trimethylbenzoylphosphine to phosphate buffer in the photoinitiator solution is (1-5) mg:1 mL.

3. The method of claim 2, wherein the method further comprises the step of adding the calcium powder to the aqueous biomass solution prior to the step of adding the GelMA solution to the aqueous biomass solution. The aquatic biomass calcium powder is one of oyster shell powder and tortoise shell powder.

4. The method of claim 1, wherein the method of preparing the aqueous photogel of living biomass calcium powder-embedded GelMA composite photogel is characterized by, The process of obtaining methacrylamide gelatin includes: S11. Add gelatin to a phosphate buffer solution of volume V mL and stir at 50℃~80℃ until the gelatin dissolves to obtain a gelatin solution; wherein the mass-volume ratio of the gelatin to the phosphate buffer solution is (0.05~0.20) g:1 mL; S12. Maintain the temperature within the range of 40℃ to 60℃, add methacrylic anhydride to the gelatin solution and continue stirring for 2 to 4 hours, then add at least (5×V) mL of phosphate buffer to terminate the reaction, and obtain a crude methacrylamide gelatin solution; wherein the mass-volume ratio of the gelatin to the methacrylic anhydride is (1.5 to 2.5) g: 1 mL. S13. The crude methacrylamide gelatin solution is purified by dialysis and dried to obtain the methacrylamide gelatin.

5. The method of claim 4, wherein the method further comprises the step of adding the aqueous biomass calcium powder to the GelMA solution. 5 The crude methacrylamide gelatin solution is purified by dialysis by placing the crude methacrylamide gelatin solution in a dialysis bag and dialyzing it for 3 to 10 days at a temperature range of 30°C to 60°C; the molecular weight cutoff of the dialysis bag is 8 kDa to 15 kDa.

6. The method for preparing the GelMA composite photosensitive hydrogel containing aquatic biomass calcium powder according to claim 5, characterized in that, The drying process employs freeze drying.

7. The method for preparing the GelMA composite photosensitive hydrogel containing aquatic biomass calcium powder according to claim 1, characterized in that, Step S2 includes: in a light-protected environment, adding the methacrylamide gelatin to a container containing a photoinitiator solution, and shaking and dissolving it in a water bath at 35°C to 40°C for 20 to 30 minutes to completely impregnate the methacrylamide gelatin with the photoinitiator solution, thereby obtaining a photosensitive methacrylamide gelatin prepolymer solution.

8. The method for preparing the GelMA composite photosensitive hydrogel containing aquatic biomass calcium powder according to claim 1, characterized in that, Step S3 includes: adding aquatic biomass calcium powder to the photosensitive methacrylamide gelatin prepolymer solution, and mixing by shaking to obtain a mixed solution.

9. The method for preparing the GelMA composite photosensitive hydrogel containing aquatic biomass calcium powder according to claim 1, characterized in that, In step S4, the wavelength of the ultraviolet lamp used for light irradiation curing is 405 nm, and the irradiation time is 30–60 s.

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