A composite bioactive hydrogel scaffold and its preparation method and application

Abstract

The application relates to the technical field of scaffolds, and particularly discloses a composite bioactive hydrogel scaffold as well as a preparation method and application thereof. The composite bioactive hydrogel provided by the application simultaneously comprises methacrylated hyaluronic acid and methacrylated silk fibroin, and HAMA and SFMA have significant complementarity in performance, that is, the high-viscosity characteristic of HAMA can be used as a thickening agent to improve the rheological performance of SFMA, and the strong and tough mechanical performance of SFMA can make up for the brittleness defect of HAMA, and the synergistic effect can enable the mixed application of the two to form a composite hydrogel which is more superior in performance and more extensive in application range. Furthermore, platelet-rich plasma is loaded on the basis, rich cytokines are provided, and the composite bioactive hydrogel is endowed with bioactivity.

Description

Technical Field

[0001] This application relates to the field of scaffold technology, and in particular to a composite bioactive hydrogel scaffold, its preparation method, and its application. Background Technology

[0002] Currently, breast cancer treatment is a comprehensive approach based on surgery, combined with radiotherapy or other drug therapies. However, surgical removal of the tumor inevitably causes physical and psychological trauma to patients. With advancements in breast cancer treatment, the survival time of breast cancer patients has significantly increased, and patients' demands for postoperative physical restoration and quality of life are constantly rising, leading to a continuous increase in the clinical demand for breast reconstruction.

[0003] Currently, breast reconstruction methods mainly include implant placement, autologous fat grafting, autologous tissue transplantation, and tissue-engineered scaffold implantation. Implant placement offers advantages such as good results, minimal trauma, and short operation time, making it suitable for patients with small skin defects, sufficient subcutaneous tissue thickness, and relatively small breast size. However, this method may lead to serious complications such as implant rupture, displacement, deformation, and implant-associated anaplastic large cell lymphoma. Autologous fat grafting offers advantages such as convenient fat harvesting and minimal trauma, but its practical application is often limited by issues such as fat necrosis and absorption, low volume maintenance, and the need for repeated grafting. Autologous tissue transplantation is an important method for breast reconstruction, offering long-lasting cosmetic results, good sensory effects, and avoiding the complications of implant placement, especially those related to adjuvant radiotherapy. Currently, autologous tissue flaps such as latissimus dorsi flap, single-pedicle or double-pedicle rectus abdominis myocutaneous flap, free deep abdominal wall artery perforator flap, and superficial abdominal wall artery flap are commonly used for breast reconstruction. However, this method is difficult to perform and carries the risks of flap necrosis, wound infection, and significant secondary damage to the donor site.

[0004] In recent years, with the rapid development of tissue engineering technology and biomaterials, the research and application of emerging tissue engineering scaffolds in breast reconstruction have attracted increasing attention from researchers. Zhang Julang et al. (Zhang Julang, Yao Qing, Huang Meiling, et al. Computer-aided 3D printing technology for primary breast reconstruction after breast-conserving surgery [J]. Chinese Journal of Breast Disease (Electronic Edition), 2018, 12(01):12-6.) conducted a study on the application of tissue engineering scaffolds in breast repair after breast-conserving surgery in China. Based on the preoperative breast MRI images of patients, they designed and 3D printed personalized polycaprolactone (PCL) scaffolds and implanted them into the local defect sites after breast-conserving surgery. The results of the two-year follow-up showed that the breast shape of the patients was well maintained, and the implanted scaffolds gradually degraded and were replaced by fibrogranulation tissue, which suggests that simply providing physical support does not achieve ideal results in fat tissue regeneration. In addition, some scholars have proposed combining tissue engineering scaffolds with autologous fat grafting for breast reconstruction. However, research results indicate that while tissue-engineered scaffolds can provide sufficient physical support for transplanted adipose tissue, thus avoiding some tension absorption, large-volume adipose tissue transplants still face the risk of tissue necrosis and absorption due to slow early angiogenesis. To address this issue, Chhaya et al. (CHHAYA MP, BALMAYOR ER, HUTMACHER DW, et al. Transformation of Breast Reconstruction via Additive Biomanufacturing [J]. Sci Rep, 2016, 6: 28030.) proposed a delayed fat injection strategy. This involves implanting the tissue-engineered scaffold into the animal for 2 weeks, allowing some neovascularization to form within the scaffold, before injecting the adipose tissue into the scaffold. The results of this study confirmed the feasibility of delayed fat injection, but histological results showed that nearly half of the tissue within the scaffold was still fibrous.

[0005] The key factor determining the quality of tissue regeneration is the structural and functional similarity between the tissue-engineered scaffold and natural tissue. Therefore, selecting appropriate scaffold preparation components and methods is crucial. Hydrogels are three-dimensional polymer materials formed by the cross-linking of hydrophilic polymers, possessing properties similar to the natural extracellular matrix. Numerous studies have shown that the three-dimensional network structure formed between hydrogel molecules can provide delivery channels for the transport of oxygen, nutrients, and cellular metabolites, and can achieve efficient encapsulation and long-term sustained release of cytokines. Furthermore, their processing conditions are mild and do not affect the loaded bioactive components. Therefore, hydrogels are considered highly promising scaffold materials in tissue engineering. In previous studies, Falguni et al. (PATI F, HA DH, JANG J, et al. Biomimetic 3D tissue printing for soft tissue regeneration [J]. Biomaterials, 2015, 62: 164-75.) mixed adipose-derived matrix hydrogel with human adipose-derived stem cells (hADSCs) to make bio-ink, and then printed hemispherical breast scaffolds by alternating the mixture with PCL. Animal experiments showed that this strategy could promote the survival and adipogenic differentiation of hADSCs on both the periphery and inside of the scaffold. Furthermore, Zhu et al. (ZHU D, BAO W, WEI B, et al. Innovative regenerative strategy for reconstructing breast defect: Gas-foamedgelatin methacryloyl scaffolds combined with human adipose-derived stem cell pheroids [J]. Applied Materials Today, 2023, 31: 101772.) prepared an injectable photosensitive gelatin hydrogel with a porous structure and encapsulated hADSCs cell spheres within it. Finally, the cells were photoformed under the support of a hemispherical PCL scaffold. The results showed that this method could promote adipogenic differentiation and angiogenesis in cells both in vivo and in vitro. It is worth noting that although hydrogel materials are significantly more effective than polymeric materials in adipose tissue regeneration, their mechanical properties are generally poor, often requiring the use of high-strength polymeric materials such as PCL for physical support. However, the mechanical properties of polymeric materials are not compatible with the surrounding tissue, and their slow degradation often has a significant impact on soft tissue repair.Furthermore, although loading ADSCs into hydrogels can effectively promote fat regeneration, the cell-loaded scaffold strategy still faces many challenges in practical applications. These include: long in vitro cell expansion cycles; potential changes in cell characteristics during culture; low cell survival rates after transplantation; risks of infection, immune rejection, or allergies; and uncertainties regarding the differentiation behavior and long-term safety of stem cells in vivo. To address these challenges, researchers have proposed cell-free scaffold strategies. For example, Zhu et al. (ZHU Y, HIDEYOSHI S, JIANG H, et al. Injectable, porous, biohybrid hydrogels incorporating decellularized tissue components for soft tissue applications[J]. Acta Biomater, 2018, 73: 112-26.) prepared a porous, injectable cell-free hydrogel based on decellularized bladder matrix. They found that injecting this hydrogel in a rabbit model of localized fat defects resulted in good fat regeneration, demonstrating the potential of cell-free hydrogels for the regeneration and repair of adipose soft tissue.

[0006] Furthermore, traditional filler scaffolds formed through injection molding or hydrogel casting generally suffer from insufficient biomimetic design and poor mechanical properties. In recent years, the development of bio-3D printing technology has provided a new strategy for constructing tissue engineering scaffolds. Compared to traditional hydrogel scaffold fabrication techniques, 3D printing technology uses digital control to stack bio-inks layer by layer, enabling precise distribution and positioning of biomaterials and bioactive components. It can rapidly and accurately prepare hydrogel structures with complex biological structures, thus better promoting tissue regeneration and repair in complex injury environments. In addition, the rapid development of research on photosensitive biomaterials in recent years has provided a convenient way to prepare functional hydrogels with complex and precise structures. Simply put, photosensitive biomaterials are materials that have undergone photosensitive modification, allowing them to undergo rapid cross-linking and curing through a free radical polymerization reaction initiated by light in the presence of a photoinitiator. Their advantages are mainly reflected in two aspects: first, the photocuring characteristics make them particularly suitable for bio-3D printing; second, the physicochemical properties of the hydrogel can be precisely adjusted by controlling the preparation and application parameters, thereby meeting differentiated tissue engineering needs.

[0007] Currently, commonly used hydrogel materials for bioprinting include gelatin, hyaluronic acid (HA), silk fibroin (SF), and sodium alginate. Among these, HA, as a core component of the natural extracellular matrix, is widely involved in regulating important cellular processes such as cell adhesion, proliferation, and differentiation. Studies have shown that HA possesses anti-inflammatory and immunosuppressive functions, stimulating the production of heat shock proteins, thereby exhibiting properties such as promoting angiogenesis, anti-apoptosis, and immunostimulation. Simultaneously, HA is widely used in local cosmetic and plastic surgery due to its high biocompatibility, ability to penetrate the epidermis, and ability to improve local tissue hydration and promote wound healing, making it the most widely used biodegradable material in clinical applications. Therefore, in the field of bioprinting, the photosensitive modification product of HA, hyaluronic acidmethacrylate (HAMA), has received widespread attention. Studies have found that HAMA has a strong ability to adsorb water molecules and can make the solution viscous by establishing intramolecular and intermolecular hydrogen bonds, thus exhibiting good rheological properties and printability. However, its brittleness and poor mechanical properties limit its application effectiveness. In contrast, SF, a natural protein material certified by the US Food and Drug Administration (FDA) for clinical use, has several medical-based products that have received FDA approval for clinical use due to its tunable degradation rate, excellent mechanical properties, and outstanding oxygen / water permeability. However, its photosensitive modified product (Silk fibroinmethacrylate, SFMA), while possessing strong mechanical properties, suffers from difficulties in maintaining the fiber structure during extrusion 3D printing due to the low viscosity of the precursor solution. To address this issue, researchers have attempted to increase the viscosity of the SF precursor solution by inducing gelation to adapt it to extrusion 3D printing. However, this strategy requires high precision and simultaneous adaptation of parameters such as crosslinking conditions and extrusion rate, making the operation complex.

[0008] Ideal tissue-engineered scaffolds, besides possessing precise biomimetic structures and providing physical support, also need to exhibit excellent biological functions. Therefore, the functionalization of tissue-engineered scaffolds has received increasing attention in recent years, involving the incorporation of active biomolecules such as drugs, growth factors, chemokines, peptides, and aptamers into the scaffolds through physical incorporation, chemical covalent coupling, and encapsulation. Among numerous candidate active substances, platelet-rich plasma (PRP), a bioactive plasma product extracted from blood, contains platelets that, upon activation, undergo degranulation and release large concentrations of cytokines, including vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), and transforming growth factor-β (TGF-β). Among them, VEGF is an important cytokine for angiogenesis, which can promote the proliferation and differentiation of vascular endothelial cells, thereby inducing the formation of new blood vessels; bFGF has functions such as promoting cell proliferation and migration, promoting angiogenesis and wound healing, and plays an important role in the proliferation and differentiation of human adipose stem cells. Therefore, PRP may have multiple functions such as promoting adipogenic differentiation of adipose stem cells and stimulating angiogenesis. In clinical application, Liu Xiaofei et al. (Liu Xiaofei, Tang Yusen. Study on the effect of PRP injection combined with autologous fat transplantation and its influence on fat survival rate[J]. Chinese Journal of Aesthetic Medicine, 2020, 29(07): 67-70.) explored the effect of PRP injection combined with autologous fat transplantation on the survival rate and transplantation effect of transplanted fat. They found that PRP injection combined with autologous fat transplantation can significantly improve the survival rate of transplanted fat, and the transplantation effect is better. The facial contour of patients after surgery is significantly improved. Han Mi et al. (Han Mi. Clinical observation on the efficacy of platelet-rich fibrin-assisted autologous fat breast augmentation [D], 2021.) investigated the effect of platelet-rich fibrin-assisted autologous fat breast augmentation on fat survival rate. The results showed that platelet-rich fibrin-assisted autologous fat breast augmentation can improve the survival rate of fat transplantation, with a lower incidence of postoperative complications and relatively higher patient satisfaction. Therefore, PRP, as an important bioactive component, has great research and application potential in adipose tissue engineering.

[0009] Currently, there are no existing studies on HAMA / SFMA composite bioactive hydrogel scaffolds loaded with platelet-rich plasma. Summary of the Invention

[0010] The purpose of this application is to overcome the shortcomings of the prior art and provide a composite bioactive hydrogel scaffold, its preparation method, and its application. This application utilizes bio-3D printing technology to prepare a personalized composite bioactive hydrogel scaffold from a bio-ink composed of methacrylamide hyaluronic acid, methacrylamide silk fibroin, and platelet-rich plasma, for application in the repair of breast tissue defects.

[0011] To achieve the above objectives, the technical solution adopted in this application is as follows: This application provides a composite bioactive hydrogel comprising methacrylamide hyaluronic acid, methacrylamide silk fibroin, and platelet-rich plasma; The mass concentration of methacrylamide hyaluronic acid in the composite bioactive hydrogel is 1-3%; The mass concentration of methacrylamide silk fibroin in the composite bioactive hydrogel is 1-5%.

[0012] Compared to pure HAMA and SFMA hydrogels, pure HAMA precursor hydrogels, while possessing high viscosity and shear-thinning properties, making them suitable for extrusion 3D printing, exhibit poor mechanical properties and high brittleness in the hydrogels formed after photopolymerization, limiting their applications. On the other hand, while pure SFMA hydrogels exhibit excellent mechanical properties after photopolymerization, their precursor solution has low viscosity, making them unsuitable for extrusion 3D printing and limiting their application in complex scenarios.

[0013] In the technical solution of this application, the composite bioactive hydrogel provided by this application simultaneously comprises methacrylamide hyaluronic acid (HAMA) and methacrylamide fibroin (SFMA). HAMA and SFMA have significant complementary properties; that is, the high viscosity of HAMA can act as a thickener to improve the rheological properties of SFMA, while the strong mechanical properties of SFMA can compensate for the brittleness of HAMA. This synergistic effect allows the combined application of the two to form a composite hydrogel with superior performance and a wider range of applications. Furthermore, by loading platelet-rich plasma, abundant cytokines are provided, endowing the composite bioactive hydrogel with biological activity.

[0014] Furthermore, by using a specific mass concentration range for both methacrylamide hyaluronic acid and methacrylamide silk fibroin in the composite bioactive hydrogel, the resulting hydrogel exhibits superior mechanical properties, including higher compressive strength, better rheological properties, higher viscosity, and shear-thinning characteristics, resulting in a better overall performance and suitability for extrusion 3D printing. In some specific embodiments, the mass concentration of methacrylamide hyaluronic acid and the mass concentration of methacrylamide silk fibroin in the composite bioactive hydrogel are both 2%.

[0015] The composite bioactive hydrogel of this application uses the above-mentioned specific mass concentrations of methacrylamide hyaluronic acid and methacrylamide silk fibroin, which can achieve a better overall effect.

[0016] The technical solution of this application prepares a composite bioactive hydrogel with good rheological properties (shear thinning properties) by mixing methacrylamide hyaluronic acid and methacrylamide silk fibroin. Compared with the simple methacrylamide silk fibroin hydrogel, it has higher viscosity, can be adapted to extrusion 3D printing, and shows good molding effect in extrusion 3D printing test.

[0017] In a preferred embodiment of the composite bioactive hydrogel described in this application, the mass concentration of methacrylamide silk fibroin in the composite bioactive hydrogel is 2%.

[0018] The composite bioactive hydrogel also includes platelet-rich plasma, and the mass concentration of platelet-rich plasma in the composite bioactive hydrogel is 25-75%, preferably 50%.

[0019] The composite bioactive hydrogel of this application also includes platelet-rich plasma (PRP). PRP is rich in fibrin and various cytokines, endowing the composite bioactive hydrogel with bioactivity. The composite bioactive hydrogel possesses superior cell adhesion properties and promotes angiogenesis and adipogenesis. Furthermore, compared to traditional PRP gels, the composite bioactive hydrogel effectively retains cytokines in PRP, preventing explosive release of cytokines and achieving a long-term sustained release effect.

[0020] The composite bioactive hydrogel provided in this application has a suitable viscosity, making it suitable for extrusion 3D printing. Secondly, methacrylamide silk fibroin (SFMA) endows the composite bioactive hydrogel with strong mechanical properties and compressive strength. Finally, platelet-rich plasma (PRP) endows the composite bioactive hydrogel with bioactivity, which can effectively achieve the regeneration and repair of adipose tissue.

[0021] This application also provides a composite bioactive hydrogel scaffold, which is obtained by 3D printing the composite bioactive hydrogel.

[0022] Using extrusion 3D printing technology, the configured composite bioactive hydrogel is 3D printed into a composite bioactive hydrogel scaffold with a specific shape and a multi-level porous structure.

[0023] This application utilizes a mixture of methacrylamide hyaluronic acid and methacrylamide silk fibroin to create a composite bioactive hydrogel scaffold that combines the excellent rheological properties of hyaluronic acid, making it suitable for extrusion 3D printing, with the superior mechanical properties of silk fibroin. The composite bioactive hydrogel is then used in bio-3D printing technology to fabricate personalized tissue engineering scaffolds (composite bioactive hydrogel scaffolds). Its excellent function in promoting adipose tissue regeneration is demonstrated in animal adipose tissue defect models, providing a theoretical basis for its clinical application in breast tissue defect repair.

[0024] The extrusion-based 3D printing system provided in this application can print at room temperature. A major advantage of extrusion-based 3D printing over projection-based 3D printing is its faster printing speed, making it suitable for rapid, large-scale printing and production. Secondly, the composite bioactive hydrogel scaffold provided in this application is used for adipose tissue repair. Adipose tissue regeneration involves the proliferation and differentiation of tissue cells, requiring high levels of cytokines, which is why PRP is added in this application. Furthermore, animal experimental results from this application show that the addition of PRP significantly promotes adipose tissue regeneration, while HAMA / SFMA alone is not as effective. This application also provides a method for preparing the above-mentioned composite bioactive hydrogel scaffold, characterized by the following steps: S1. Add the photoinitiator to the buffer solution to form a photoinitiator solution; S2. Add the lyophilized sponge of methacrylamide hyaluronic acid to the photoinitiator solution obtained in step S1 to form a methacrylamide hyaluronic acid precursor solution; S3. Add the lyophilized sponge of methacrylamide silk fibroin to the methacrylamide hyaluronic acid precursor solution obtained in step S2, stir and mix well to form HAMA / SFMA composite hydrogel solution. S4. After activating platelet-rich plasma with calcium gluconate solution, add it to the HAMA / SFMA composite hydrogel solution obtained in step S3 to form a composite bioactive hydrogel. S5. The composite bioactive hydrogel obtained in step S4 is used to obtain a composite bioactive hydrogel scaffold through 3D printing, UV curing and freeze drying.

[0025] To address the current challenges in breast reconstruction tissue engineering, and considering the excellent physicochemical properties of SF and HA, as well as the promising biological functions of PRP, this application employs HAMA and SFMA. The performance of HAMA / SFMA composite hydrogels with different component ratios was characterized through tests on mechanical properties, pore structure, degradation rate, swelling properties, rheological properties, and 3D printing molding effects. Then, PRP was added to a specific concentration ratio of HAMA / SFMA composite hydrogel exhibiting superior overall performance to prepare a composite bioactive hydrogel. Its cellular properties were then assessed through in vitro experiments. Finally, a bioactive hydrogel scaffold was fabricated using 3D printing. The efficacy and function of the prepared composite bioactive hydrogel scaffold in repairing localized adipose tissue defects and reconstructing large volumes of adipose tissue were further investigated in animals, providing a research foundation and theoretical basis for future clinical translation and application.

[0026] In the technical solution of this application, SFMA (methacrylamide silk fibroin) has high solubility, but due to its inherent characteristics, it is sensitive to physical factors (stirring / ultrasound) and easily self-crosslinks and solidifies. Therefore, this application first dissolves HAMA, and then adds SFMA to the HAMA precursor solution (methacrylamide hyaluronic acid precursor solution) to overcome the above-mentioned problems of SFMA. As a preferred embodiment of the preparation method of the composite bioactive hydrogel scaffold described in this application, in step S1, the photoinitiator includes LAP photoinitiator; And / or, the mass concentration of the photoinitiator is 0.25~0.5 w / v, preferably 0.25 w / v.

[0027] This application incorporates a photoinitiator, resulting in a composite bioactive hydrogel that exhibits superior mechanical properties and higher compressive strength after curing under ultraviolet light.

[0028] In a preferred embodiment of the preparation method of the composite bioactive hydrogel scaffold described in this application, the buffer solution in step S1 includes PBS buffer.

[0029] In a preferred embodiment of the preparation method of the composite bioactive hydrogel scaffold described in this application, in step S4, the mass concentration of calcium gluconate in the calcium gluconate solution is 8-12%, preferably 10%.

[0030] In a preferred embodiment of the preparation method of the composite bioactive hydrogel scaffold described in this application, the printing parameters for step S4, 3D printing, include: The filling pattern is a grid, the fill rate is 30%, the layer height is 0.2mm, the print head moving speed is 10mm / s, and the syringe extrusion speed is 0.001mm / s.

[0031] In a preferred embodiment of the method for preparing the composite bioactive hydrogel scaffold described in this application, step S4, 3D printing, includes the following steps: Inject the HAMA / SFMA / PRP precursor hydrogel solution into the printhead slot of the 3D printer. Install and connect the 27G printing needle at the bottom of the syringe slot. Adjust the height of the printing platform. Set the slicing parameters and 3D printing parameters of the model in the Slic3r software. Convert the STL format file into a G-Code program that the 3D printer can recognize. Turn on the compressor, connect the 3D printer and the operating software, select the printing channel, and start printing.

[0032] This application also provides the application of the above-mentioned composite bioactive hydrogel and composite bioactive hydrogel scaffold in the preparation of breast tissue defect repair products.

[0033] Compared to single HAMA or SFMA, the composite bioactive hydrogel provided in this application has superior mechanical properties, stronger compressive strength, better rheological properties, higher viscosity, and exhibits shear-thinning properties, making it suitable for extrusion 3D printing.

[0034] Compared to HAMA hydrogel, the composite bioactive hydrogel of this application has better molding effect in extrusion 3D printing. In the radial printing test, the extruded ink radial lines are finer and more uniform. In the pore structure printing test, the printed pore structure is uniform and regular with clear structure edges (SFMA cannot be tested for extrusion 3D printing effect due to its low viscosity).

[0035] Furthermore, compared to traditional PRP gels, the composite bioactive hydrogel of this application effectively retains cytokines in PRP, preventing explosive release of cytokines and achieving a long-term sustained-release effect. This allows the hydrogel to have a long-term repair effect on local tissues and cells.

[0036] In vitro cell experiments and animal experiments have verified that this biological scaffold can effectively promote adipogenic differentiation and angiogenesis of adipose stem cells, thereby promoting the regeneration and repair of adipose tissue.

[0037] Compared with the prior art, this application has the following beneficial effects: This application provides a composite bioactive hydrogel scaffold, its preparation method, and its application. This application combines two materials to prepare a HAMA / SFMA composite hydrogel. The precursor solution possesses excellent rheological properties and good shear-thinning properties, making it suitable for extrusion 3D printing. Furthermore, the hydrogel formed after photocuring exhibits excellent mechanical properties. This application also imparts bioactivity to the HAMA / SFMA composite hydrogel by adding PRP. Finally, using bio-3D printing technology, the prepared hydrogel material is 3D printed to fabricate a tissue engineering repair scaffold with a multi-level porous structure, enabling it to adapt to various complex application environments and achieve personalized applications. Animal experiments have verified the function of this invention in adipose tissue regeneration and repair, providing new ideas for future clinical application in breast tissue defect repair. Attached Figure Description

[0038] Figure 1 This is a flowchart illustrating the fabrication process of a composite bioactive hydrogel scaffold. Figure 2 Macroscopic and scanning electron microscope images of the HAMA / SFMA / PRP scaffold prepared in Example 1; Figure 3 Characterization diagram of the micropore structure of HAMA / SFMA composite hydrogels with different component ratios; Figure 4 The mechanical properties of the HAMA / SFMA / PRP hydrogel scaffold and the HAMA hydrogel scaffold (Comparative Example 1) prepared in Example 1 are shown in the figure. Figure 5 Figure 1 shows the characterization results of the compressive mechanical properties of HAMA / SFMA composite hydrogels with different component ratios. Figure 6 The graph shows the degradation test results of the HAMA / SFMA / PRP hydrogel scaffold prepared in Example 1. Figure 7 The results of viscosity tests for HAMA / SFMA composite hydrogels with different proportions are shown in the figure. Figure 8 The image shows the test results of 3D extrusion printing of HAMA / SFMA composite hydrogels with different ratios. Figure 9 The figure shows the cell adhesion test results of the HAMA / SFMA / PRP hydrogel scaffold and the HAMA hydrogel scaffold prepared in Example 1. Figure 10 The results of cell proliferation and biocompatibility tests on the HAMA / SFMA / PRP hydrogel scaffold and the HAMA hydrogel scaffold prepared in Example 1 are shown in the figure. Figure 11Figure 1 shows the in vitro angiogenesis results of the HAMA / SFMA / PRP hydrogel scaffold and the HAMA hydrogel scaffold prepared in Example 1. Figure 12 The in vitro adipogenic differentiation experiment results of the HAMA / SFMA / PRP hydrogel scaffold and the HAMA hydrogel scaffold prepared in Example 1 are shown in the figure. Figure 13 Figure I shows the in vivo efficacy evaluation results of the HAMA / SFMA / PRP hydrogel scaffold prepared in Example 1; Figure 14 Figure II shows the in vivo efficacy evaluation results of the HAMA / SFMA / PRP hydrogel scaffold prepared in Example 1. Detailed Implementation

[0039] To better illustrate the purpose, technical solution, and advantages of this application, the following description will be provided in conjunction with the accompanying drawings and specific embodiments.

[0040] In the following examples and comparative examples, unless otherwise specified, the experimental methods used are conventional methods, and the materials and reagents used are commercially available unless otherwise specified. Furthermore, the raw materials used in each parallel experiment are the same.

[0041] The following will be referred to as: HAMA (Hyaluronic acid methacrylate): methacrylated hyaluronic acid; SFMA (Silk fibroin methacrylate): Methacrylated silk fibroin; PRP (Platelet-rich plasma): Platelet-rich plasma; HV: Ultraviolet light; 3D printing: 3D printing; Freezing drying: Freeze-drying; Platelet: blood cell count; Cytokine: cytokine; Animal model: animal experiment; Scaffold; ADSCs (Adipose-derived stem cells): adipose-derived mesenchymal stem cells Adipose-differentiated ADSCs: adipose-derived mesenchymal stem cells; Adipocyte: fat cell; ECM (extra cellular matrix): extracellular matrix; Blood vessel: blood vessel.

[0042] This embodiment provides a composite bioactive hydrogel scaffold and its preparation method, the specific process of which is as follows: Figure 1 As shown; Figure 1 The dashed box section: Methacrylamide hyaluronic acid (HAMA), methacrylamide silk fibroin (SFMA), and platelet-rich plasma (PRP) were mixed to prepare a composite bioactive hydrogel, which was then 3D printed and cured under ultraviolet light using a bio-3D printer. Finally, it was freeze-dried to prepare a composite bioactive hydrogel scaffold with a multi-level porous structure.

[0043] In the following experiments, the mechanical properties, degradation properties, and viscosity properties were obtained from Xia H, Zhao D, Zhu H, Hua Y, Xiao K, Xu Y, Liu Y, Chen W, Liu Y, Zhang W, Liu W, Tang S, Cao Y, Wang X, Chen HH, Zhou G. Lyophilized Scaffolds Fabricated from 3D-Printed Photocurable Natural Hydrogel for Cartilage Regeneration. ACS Appl MaterInterfaces. 2018 Sep 19;10(37):31704-31715. doi: 10.1021 / acsami.8b10926. Epub2018 Sep 10. PMID: 30157627. Example 1: A composite bioactive hydrogel scaffold and its preparation method This embodiment provides a composite bioactive hydrogel scaffold and its preparation method, the preparation method including the following steps: S1. Weigh 12.5 mg of photoinitiator LAP powder and add it to 4 mL of PBS buffer to form a 0.25% (w / v) LAP solution. Store in the dark for later use. S2. Add 100 mg of lyophilized sponge containing methacrylamide hyaluronic acid (HAMA) to 5 mL of the photoinitiator solution obtained in step S1, and stir thoroughly on a magnetic stirrer to dissolve it, forming a methacrylamide hyaluronic acid precursor solution (the mass concentration of methacrylamide hyaluronic acid in the methacrylamide hyaluronic acid precursor solution is 2% (w / v)). S3. Add 100 mg of lyophilized methacrylamide silk fibroin (SFMA) sponge to the methacrylamide hyaluronic acid precursor solution obtained in step S2, and stir to mix it into a uniformly dispersed solution to form a HAMA / SFMA composite hydrogel solution (abbreviated as 2H2S), wherein the mass concentration of methacrylamide silk fibroin in the HAMA / SFMA composite hydrogel solution is 2% (w / v). S4. After activating 5 mL of platelet-rich plasma with 0.5 mL of 10% calcium gluconate solution, immediately add 5 mL of LPRP activation solution to the HAMA / SFMA composite hydrogel solution obtained in step S3 to form a composite bioactive hydrogel. S5. Composite bioactive hydrogel scaffolds (also known as HAMA / SFMA / PRP hydrogel scaffolds) were obtained by 3D printing, UV curing (405nm UV light irradiation for 30s) and freeze drying.

[0044] The specific steps involved in 3D printing include: Aspirate the composite bioactive hydrogel into a 10mL syringe. Slowly draw the hydrogel into the syringe using the 10mL syringe, avoiding air bubbles during the process. Place the syringe in the 3D printer's print head slot, attach the 27G printing needle to the bottom of the syringe slot, and adjust the printing platform height to an appropriate level. In the Slic3r software, set the model's slicing parameters and 3D printing parameters as follows: grid pattern, infill rate 30%, layer height 0.2mm, print head movement speed 10mm / s, and syringe extrusion speed 0.001mm / s. Import a cube model (10mm long, 10mm wide, 3mm high) into the printing software and convert the STL file into a G-Code program recognizable by the 3D printer. Turn on the compressor, connect the Lepu Bio 3D printer and the operating software, and select the printing channel to begin printing. After printing, freeze the 3D printing stand at -80℃ for 12 hours, then freeze-dry it in a freeze dryer. Collect the freeze-dried supports and sterilize them with a high-energy electron beam (15-20KGy). After sterilization, store the supports in a -80℃ refrigerator for later use.

[0045] Example 2: A composite bioactive hydrogel scaffold and its preparation method Compared with Example 1, the difference in Example 2 is that the weight of the freeze-dried sponge of methacrylamide silk fibroin (SFMA) is 50 mg, and the mass concentration of methacrylamide silk fibroin in the obtained HAMA / SFMA composite hydrogel solution is 1% (w / v). The rest of the preparation method is the same as in Example 1.

[0046] Among them, the HAMA / SFMA composite hydrogel solution (abbreviated as 2H1S) formed in step S3.

[0047] Example 3: A composite bioactive hydrogel scaffold and its preparation method Compared with Example 1, the difference in Example 2 is that the weight of the freeze-dried sponge of methacrylamide silk fibroin (SFMA) is 250 mg, the mass concentration of methacrylamide silk fibroin in the HAMA / SFMA composite hydrogel solution is 5% (w / v), and the rest of the preparation method is the same as in Example 1.

[0048] Among them, the HAMA / SFMA composite hydrogel solution (abbreviated as 2H5S) formed in step S3.

[0049] Comparative Example 1 Weigh 12.5 mg of photoinitiator LAP powder and add it to 4 mL of PBS solution to prepare a 0.25% (w / v) LAP solution. Store in the dark. Weigh 100 mg of HAMA lyophilized sponge and add it to 5 mL of the 0.25% (w / v) photoinitiator LAP solution. Stir thoroughly on a magnetic stirrer to dissolve the sponge and prepare a 2% (w / v) HAMA solution (abbreviated as 2H).

[0050] Comparative Example 2 Weigh 12.5 mg of photoinitiator LAP powder and add it to 5 mL of PBS solution to prepare a 0.25% (w / v) LAP solution. Store in the dark. Weigh 100 mg of SFMA lyophilized sponge and add it to 5 mL of the 0.25% (w / v) photoinitiator LAP solution. Stir thoroughly on a magnetic stirrer to dissolve the dissolved SFMA solution to prepare a 2% (w / v) SFMA solution (abbreviated as 2S).

[0051] Comparative Example 3 Compared with Example 1, the difference in Comparative Example 3 is that cellulose nanofibers (CNF) were used instead of HAMA to obtain a composite hydrogel (CNF / 2S), while the rest of the preparation method was the same as in Example 1.

[0052] Comparative Example 4 Compared with Example 1, the difference in Comparative Example 4 is that methacrylamide chitosan (CSMA) was used instead of SFMA to obtain a composite hydrogel (2H / CSMA). The rest of the preparation method is the same as in Example 1.

[0053] Comparative Example 5 Compared with Example 1, the difference in Comparative Example 5 is that the weight of the lyophilized sponge of methacrylamide silk fibroin (SFMA) is 25 mg, and the mass concentration of methacrylamide silk fibroin in the obtained HAMA / SFMA composite hydrogel solution (abbreviated as 2H0.5S) is 0.5% (w / v). The rest of the preparation method is the same as that in Example 1.

[0054] Comparative Example 6 Compared with Example 1, the difference in Comparative Example 6 is that the weight of the lyophilized sponge of methacrylamide silk fibroin (SFMA) is 500 mg, and the mass concentration of methacrylamide silk fibroin in the obtained HAMA / SFMA composite hydrogel solution (abbreviated as 2H10S) is 10% (w / v). The rest of the preparation method is the same as that in Example 1.

[0055] Experimental Example 1: Testing the performance of composite bioactive hydrogel scaffolds 1. Basic characterization of HAMA / SFMA / PRP hydrogel scaffolds: Figure 2 Macroscopic and scanning electron microscopic characterization of the HAMA / SFMA / PRP scaffold prepared in Example 1. Among them, as shown... Figure 2 As shown in Figure B, the HAMA / SFMA / PRP hydrogel scaffold prepared according to the method of this application is generally off-white, and a grid-like macroporous structure can be observed in the middle part of the scaffold. Its microstructure was observed using a scanning electron microscope, as shown... Figure 2 As shown in Figure C, the HAMA / SFMA / PRP scaffold prepared according to the method of this application exhibits an interconnected microporous structure, indicating that the 3D printed HAMA / SFMA / PRP hydrogel scaffold possesses a multi-level porous structure.

[0056] like Figure 3 This section showcases the microporous structure characterization of HAMA / SFMA composite hydrogels with different component ratios. The microporous structure of hydrogels significantly influences the transport of oxygen, nutrients, and cellular metabolic waste within the gel, as well as cell-matrix interactions and cell growth and implantation. High porosity and appropriately sized pores provide a suitable growth environment for cell survival and growth. The microporous structure characteristics of hydrogels can be observed using SEM. Figure 3 As shown in Figure A, all groups of hydrogel samples formed interconnected pore structures inside. The pore structure inside the hydrogel in group 2H was the most porous. The pore size inside the composite hydrogel gradually decreased with increasing SFMA concentration, forming a denser pore structure. Figure 3As shown in Figure B, the pore diameter distributions of the hydrogels in each group are 2H (510.64±44.43 μm), 2H1S (345.09±31.06 μm), 2H2S (247.18±31.57 μm), and 2H5S (74.83±13.72 μm). This suggests that adding excessively high concentrations of SFMA can significantly affect the pore structure of the hydrogel, potentially hindering tissue regeneration.

[0057] 2. Mechanical properties of HAMA / SFMA / PRP hydrogel scaffolds: The biomechanical microenvironment plays a crucial role in cell behavior and function during tissue repair. In tissue engineering research and applications, the mechanical properties of repair materials are typically adjusted according to the specific application scenario to meet the requirements of the application. Although breast tissue is mostly composed of glandular and adipose tissue, in the repair of breast tissue defects, larger tissue defects still require sufficient support from the repair material to maintain the overall aesthetic appearance of the breast. Therefore, certain requirements are placed on the mechanical properties of the repair material.

[0058] Figure 4 The HAMA / SFMA / PRP hydrogel scaffold and the HAMA hydrogel scaffold (Comparative Example 1) prepared in Example 1 were subjected to compression mechanics tests using a universal testing machine. Compared to the HAMA hydrogel scaffold, the HAMA / SFMA / PRP hydrogel scaffold of Example 1 exhibited higher compressive strength.

[0059] like Figure 5 Characterization of the compressive mechanical properties of HAMA / SFMA composite hydrogels with different component ratios. From Figure 5 As can be seen from the stress-strain curves of the composite hydrogel, the simple HAMA hydrogel has poor mechanical strength and toughness, and structural fracture occurs after only about 25% compression, while the strain at the time of fracture of the composite hydrogel increases to over 40%. Figure 5 As shown in Figure C, the compressive strengths of the composite hydrogels in the 2H, 2H1S, 2H2S, and 2H5S groups are 2.34±0.63 N, 4.07±1.55 N, 7.61±0.68 N, and 10.53±0.86 N, respectively. It can be seen that the compressive strength of the composite hydrogel gradually increases with the increase of SFMA concentration. This indicates that the addition of SFMA significantly improves the stress resistance of the composite hydrogel, and the composite hydrogel exhibits superior strength and toughness compared to HAMA. Figure 5 As shown in Figure D, the compressive modulus of the composite hydrogel is lower than that of the HAMA hydrogel, indicating that the composite hydrogel is softer than the HAMA hydrogel. However, there is no significant difference in compressive modulus among the various composite hydrogel groups.

[0060] The results show that the compressive strength of 2% HAMA is low, while 2% SFMA cannot cure due to its low concentration. Adding CNF to SFMA (Comparative Example 3) can increase mechanical properties to some extent, but the compressive strength is still very weak. Adding CSMA to HAMA (Comparative Example 4) can increase mechanical properties to some extent, but the compressive strength of this hydrogel is still lower than that of the SFMA-added group. Adding SFMA to HAMA can significantly increase the compressive strength of the composite hydrogel, especially the 2H2S group with 2% SFMA. The 2H10S group with a higher SFMA concentration (Comparative Example 6) does not show a significant increase in compressive strength compared to the 2H2S group.

[0061] 3. Degradability test of HAMA / SFMA / PRP hydrogel scaffold: Figure 6 The HAMA / SFMA / PRP hydrogel scaffold prepared in Example 1 was evaluated for in vitro degradation characteristics using an enzymatic method, with the HAMA scaffold serving as a control. After lyophilization, each group of hydrogel scaffolds underwent irradiation sterilization and was weighed, denoted as M0. Each group of samples was treated with 2 U / mL protease XIV solution. Hydrogels from the corresponding groups were collected at 1, 2, 3, 7, 14, and 21 days, rinsed with deionized water, and then lyophilized for 48 hours. After lyophilization, the samples were weighed and denoted as M1. The degradation rate DR of the hydrogel was calculated using the formula DR = (M0 - M1) / M0 × 100%. The results indicate that, compared to the HAMA scaffold, the HAMA / SFMA / PRP hydrogel scaffold degrades more slowly and exhibits more suitable degradation performance.

[0062] 4. Bio-inks suitable for extrusion 3D printing need to possess a certain viscosity and good shear-thinning properties. This application first tests the viscosity of HAMA / SFMA composite hydrogels with different component ratios, from... Figure 7 The viscosity-shear rate curves show that the hydrogel (2S) in Comparative Example 2 has extremely low viscosity, and no obvious shear-thinning phenomenon was observed. The viscosity of the 2H group and all composite hydrogel groups is much higher than that of the 5S group, and the viscosity of the 2H group and all composite hydrogel groups gradually decreases with increasing shear rate, exhibiting good shear-thinning properties. The viscosity of CNF / 2S is significantly lower than that of the 2H group and other composite hydrogel groups. In the HAMA / SFMA composite hydrogel group, although the viscosity shows a certain upward trend with increasing SFMA concentration, there is no significant difference, indicating that the viscosity characteristics of the composite hydrogel are mainly influenced by the HAMA component.

[0063] Experiment Example 2: Test on the effect of extrusion 3D printing of HAMA / SFMA composite hydrogels with different ratios As mentioned earlier, the hydrogel in Comparative Example 2 has a low viscosity, making it difficult to directly apply to extrusion-based bio-3D printing due to its rheological properties. In contrast, the hydrogel in Comparative Example 1 possesses higher viscosity and better shear-thinning properties, which can be used to increase the viscosity of SFMA solutions and impart good shear-thinning properties to the composite precursor hydrogel, thus enabling its application in extrusion-based 3D printing.

[0064] This experiment demonstrates the extrusion 3D printing of composite hydrogels with different component ratios to test their extrusion printing effect. Figure 8 Image A shows the printing results of hydrogels with different component ratios for linear and pore size printing. The printing results of different groups are analyzed. Figure 8 The diameter of the printed lines in groups B is visible. Compared to group 2H (735.1±40μm), groups 2H1S (562.9±5.6μm) and 2H2S (563.7±17.1μm) have finer and more uniform ink lines. However, when the SFMA concentration increases to 5%, the diameter of the printed lines in group 2H5S is 558.1±76.9μm, showing a large difference in the size of the printed lines. This may be due to the higher ink concentration and viscosity, leading to uneven material output and a poorer printing effect. In the pore size printing test, group 2H, due to its coarser ink lines, produced the smallest and most irregularly shaped pore structures. Groups 2H1S and 2H2S showed better forming results than group 2H, producing uniform and regular pore structures with clear edges. Group 2H2S was able to print regular square pore structures. Figure 8 The pore area of ​​each group printed in group C is shown to be the largest (592619.7±26941.3μm). 2 The 2H2S group produced the best 3D printing results, with the pore size being closest to the preset size and exhibiting uniformity. In contrast, the 2H5S group suffered from uneven ink distribution, resulting in significant variations in the pore structure and poorer printing quality. Therefore, the 2H2S group demonstrated the best extrusion-based 3D printing performance.

[0065] The performance tests above demonstrate that the composite hydrogel prepared by mixing HAMA and SFMA effectively combines the excellent properties of both. The addition of SFMA effectively improves the mechanical properties of HAMA, resulting in superior compressive strength in the composite hydrogel. HAMA, in turn, compensates for the rheological deficiencies of SFMA, giving the composite hydrogel certain viscosity and shear-thinning properties. However, excessively high concentrations of SFMA can further reduce the micropore structure of the hydrogel, making it more compact and potentially affecting its cell biological properties, hindering cell growth and implantation. Furthermore, high concentrations of SFMA can lead to uneven dispensing of the hydrogel precursor solution during printing, resulting in poor printing quality. Therefore, considering the results of all performance tests, the composite hydrogel with a medium SFMA concentration (the composite hydrogel of Example 1) exhibits the best overall performance.

[0066] Experimental Example 3: Cell Adhesion and Cell Proliferation Tests of HAMA / SFMA / PRP Hydrogel Scaffolds Four hours after seeding human adipose-derived mesenchymal stem cells with the HAMA / SFMA / PRP hydrogel scaffold and HAMA hydrogel scaffold prepared in Example 1, it was confirmed that... Figure 9 As shown, the HAMA / SFMA / PRP hydrogel scaffold exhibits superior cell adhesion compared to the HAMA hydrogel scaffold.

[0067] After seeding human adipose-derived mesenchymal stem cells with the HAMA / SFMA / PRP hydrogel scaffold and HAMA hydrogel scaffold prepared in Example 1, in vitro biocompatibility analysis was performed using the MTT assay and cell live / dead staining. The results confirmed that, Figure 10 As shown, compared to the HAMA hydrogel scaffold, the HAMA / SFMA / PRP hydrogel scaffold has higher biocompatibility and better cell proliferation effect.

[0068] Experimental Example 4: In vitro angiogenesis and adipogenic differentiation experiments of HAMA / SFMA / PRP hydrogel scaffolds Figure 11 In vitro angiogenesis experiments were conducted on the HAMA / SFMA / PRP hydrogel scaffold and the HAMA hydrogel scaffold prepared in Example 1. After co-culturing human umbilical vein endothelial cells seeded in a matrix gel with the hydrogel scaffold, it was confirmed that the HAMA / SFMA / PRP hydrogel scaffold exhibited superior angiogenesis-promoting properties compared to the HAMA hydrogel scaffold.

[0069] Figure 12In vitro adipogenic differentiation experiments were conducted on the HAMA / SFMA / PRP hydrogel scaffold and the HAMA hydrogel scaffold prepared in Example 1. After co-culturing the hydrogel scaffold with human adipose-derived mesenchymal stem cells and inducing adipogenic differentiation for 14 days using adipogenic induction medium, it was confirmed that the HAMA / SFMA / PRP hydrogel scaffold synergistically promoted adipogenic differentiation of adipose-derived stem cells more effectively than the HAMA hydrogel scaffold.

[0070] Case Study 5: In vivo efficacy evaluation of HAMA / SFMA / PRP hydrogel scaffold A rabbit animal experiment was conducted to construct a model for repairing adipose tissue defects, simulating tissue defects caused by clinical breast-conserving surgery.

[0071] The process of constructing a fat tissue defect repair model includes the following steps: Rabbits were sedated by intramuscular injection of xylazine (3 mg / kg) and ketamine (35 mg / kg). The surgical area was fully exposed, and the rabbits were moved to a warming blanket on the operating table. Anesthesia was maintained by continuous inhalation of 2% isoflurane via a ventilator mask. The surgical area was disinfected with povidone-iodine and draped with surgical towels. A 3-4 cm incision was made along the groin direction, and the tissue was bluntly dissected with surgical scissors to fully expose the deep adipose tissue at the incision site. A 10×10×1 mm adipose tissue was completely removed from the fat pad to simulate the local tissue defect caused by breast-conserving surgery in humans. After creating the local adipose tissue defect model, a sterilized scaffold was carefully placed at the defect site. The scaffold was fixed to the surrounding tissue at the four corners with non-absorbable sutures to prevent displacement within the body. The surgical incision was sutured, and the incision site was disinfected again with povidone-iodine after suturing. Postoperatively, each rabbit received an intramuscular injection of 400,000 units of sodium penicillin daily for one week. The rabbits' condition was observed daily. Eight weeks post-surgery, the animals were euthanized to fully expose the inguinal fat pad. The modeling site was determined by markings made with non-absorbable sutures, photographed, and then the tissue was harvested.

[0072] Figure 13 In Example A, the HAMA / SFMA / PRP hydrogel scaffold prepared in Example 1 was implanted into a local defect (1.0cm × 1.0cm) in the inguinal fat pad of a New Zealand rabbit. Eight weeks later, tissue from the surgical area was harvested for HE staining. Figure 13 Figure A was compared with that of physiological saline treatment. The number of blood vessels in the newly formed tissue was also quantitatively analyzed. Figure 13 (Figure C).

[0073] Figure 13 In Example B, the HAMA / SFMA / PRP hydrogel scaffold prepared in Example 1 was implanted into a local defect (1.0cm × 1.0cm) of the inguinal fat pad in New Zealand rabbits. Eight weeks later, tissue samples from the surgical area were taken for MASSON staining, treated with physiological saline for comparison, and the fibrous tissue area was quantitatively and statistically analyzed. Figure 13 (D diagram).

[0074] Figure 14 In Example A, the HAMA / SFMA / PRP hydrogel scaffold prepared in Example 1 was implanted into a local defect (1.0cm × 1.0cm) of the inguinal fat pad in New Zealand rabbits. Eight weeks later, tissue samples from the surgical area were taken for Oil Red O staining, treated with physiological saline for comparison, and the adipose tissue area was quantified and statistically analyzed. Figure 14 (B)

[0075] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application and are not intended to limit the scope of protection of this application. Although this application has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of this application without departing from the substance and scope of the technical solutions of this application.

Claims

1. A composite bioactive hydrogel, characterized in that, Including methacrylamide hyaluronic acid, methacrylamide silk fibroin, and platelet-rich plasma; The mass concentration of methacrylamide hyaluronic acid in the composite bioactive hydrogel is 1-3%; The mass concentration of methacrylamide silk fibroin in the composite bioactive hydrogel is 1-5%.

2. The composite bioactive hydrogel as described in claim 1, characterized in that, The composite bioactive hydrogel also includes platelet-rich plasma, with a mass concentration of 25-75% in the composite bioactive hydrogel.

3. A composite bioactive hydrogel scaffold, characterized in that, The composite bioactive hydrogel scaffold is obtained by 3D printing the composite bioactive hydrogel as described in claim 1.

4. A method for preparing the composite bioactive hydrogel scaffold as described in claim 3, characterized in that, Includes the following steps: S1. Add the photoinitiator to the buffer solution to form a photoinitiator solution; S2. Add the lyophilized sponge of methacrylamide hyaluronic acid to the photoinitiator solution obtained in step S1 to form a methacrylamide hyaluronic acid precursor solution; S3. Add the lyophilized sponge of methacrylamide silk fibroin to the methacrylamide hyaluronic acid precursor solution obtained in step S2, stir and mix well to form HAMA / SFMA composite hydrogel solution. S4. After activating platelet-rich plasma with calcium gluconate solution, add it to the HAMA / SFMA composite hydrogel solution obtained in step S3 to form a composite bioactive hydrogel. S5. The composite bioactive hydrogel obtained in step S4 is used to obtain a composite bioactive hydrogel scaffold through 3D printing, UV curing and freeze drying.

5. The method for preparing the composite bioactive hydrogel scaffold as described in claim 4, characterized in that, In step S1, the photoinitiator includes LAP photoinitiator; And / or, the mass concentration of the photoinitiator is 0.25~0.5 w / v.

6. The method for preparing the composite bioactive hydrogel scaffold as described in claim 4, characterized in that, In step S1, the buffer solution includes PBS buffer.

7. The method for preparing the composite bioactive hydrogel scaffold as described in claim 4, characterized in that, In step S4, the mass concentration of calcium gluconate in the calcium gluconate solution is 8-12%.

8. The method for preparing the composite bioactive hydrogel scaffold as described in claim 4, characterized in that, In step S5, the 3D printing parameters include: The filling pattern is a grid, the fill rate is 30%, the layer height is 0.2mm, the print head moving speed is 10mm / s, and the syringe extrusion speed is 0.001mm / s.

9. The method for preparing the composite bioactive hydrogel scaffold as described in claim 4, characterized in that, Step S5, 3D printing includes the following steps: Inject the HAMA / SFMA / PRP precursor hydrogel solution into the printhead slot of the 3D printer. Install and connect the 27G printing needle at the bottom of the syringe slot. Adjust the height of the printing platform. Set the slicing parameters and 3D printing parameters of the model in the Slic3r software. Convert the STL format file into a G-Code program that the 3D printer can recognize. Turn on the compressor, connect the 3D printer and the operating software, select the printing channel, and start printing.

10. The application of the composite bioactive hydrogel as described in claim 1 or 2, or the composite bioactive hydrogel scaffold as described in claim 3, in the preparation of breast tissue defect repair products.

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