Silk protein composite hard material as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biological materials, discloses a silk fibroin composite hard material as well as a preparation method and application thereof, and aims to solve the problems that a pure silk fibroin hard material is insufficient in toughness and easy to break. The preparation method comprises the following steps: preparing degummed silk; recovering sericin protein; dissolving degummed silk to obtain a silk fibroin solution; adding the recycled sericin protein into the silk fibroin solution to form a composite solution; and carrying out dialysis, solvent replacement and drying on the composite solution to finally obtain the composite material. The preparation method is characterized in that homologous sericin and silk fibroin are compounded, and the microphase structure of the sericin and the silk fibroin is remodeled. The structural remodeling optimizes the internal stress distribution of the material, so that the fracture strain is remarkably improved while the obtained composite material keeps high modulus, and good balance between rigidity and toughness is realized. The material can be further compounded with hydroxyapatite to enhance osseointegration activity, and is suitable for manufacturing artificial bone cartilage restoration and other medical implants and tissue engineering scaffolds.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, specifically to a silk fibroin composite rigid material and its preparation method. Background Technology

[0002] Silk fibroin is a structural protein extracted from natural silkworm silk. Its molecular chain is composed of 18 amino acids, including glycine, alanine, serine, and tyrosine, which are essential for the human body. This unique silk fibroin originates from the silkworm's silk gland and is a natural high-molecular-weight fibrous protein. By controlling processing conditions, its molecular chain can construct a multi-level structure ranging from random coils to highly ordered β-sheets. It is this tunability of the microstructure that allows regenerated silk fibroin materials to cover a wide range of mechanical properties, from soft hydrogels to high-strength, high-modulus rigid materials. Especially in fields requiring high modulus, such as osteochondral repair and dental temporary supports, regenerated silk fibroin can induce a tight molecular chain arrangement and high crystallinity, forming a dense β-sheet network maintained by numerous hydrogen bonds, thereby achieving excellent rigidity close to that of natural cortical bone. Coupled with its inherent good biocompatibility, controllable degradation, and mechanical strength, silk fibroin is considered a highly promising biomaterial for hard tissue engineering.

[0003] However, rigid materials made from pure regenerated silk fibroin using traditional processes often struggle to reconcile the contradiction between rigidity and toughness. Guo et al. reported in their paper "Thermoplastic molding of regenerated silk" (Nature Materials, 2020) that while the rigid material they prepared by hot-pressing regenerated silk fibroin powder achieved a modulus of approximately 5 GPa, its fracture strain was less than 8%, exhibiting typical brittle fracture characteristics. On the other hand, methods aimed at improving toughness often come at the cost of rigidity. For example, Chinese patent CN117045860A discloses a silk fibroin prosthesis material prepared using solvents such as hexafluoroisopropanol, which exhibits a compressive fracture strain exceeding 80%, demonstrating good flexibility, but its compressive modulus is less than 10 MPa. Introducing other flexible polymers such as polyvinyl alcohol, gelatin, or cellulose to improve toughness is a common approach, but poor compatibility between different components can easily lead to phase separation, significantly weakening the overall mechanical strength of the material.

[0004] Sericin is a naturally occurring hydrophilic colloidal protein that encapsulates the outer layer of silk fibroin fibers. It is homologous and symbiotic with silk fibroin and is known to possess excellent cell affinity and bioactivity. Theoretically, reintroducing sericin into the silk fibroin matrix could potentially enhance the toughness of composite materials through its flexible segments and interfacial interactions. However, most existing composite strategies have failed to fully realize this potential. In their subsequent study, "Protein composites from silkworm cocoons asversatile biomaterials" (Acta Biomaterialia, 2021), Guo et al. found that rigid materials made directly from intact, undegummed silk through hot pressing did not exhibit significantly different mechanical properties compared to pure silk fibroin materials, failing to demonstrate the expected synergistic toughening effect of sericin. This indicates that simple hot pressing cannot effectively reconstruct the stress transmission path of materials at the molecular and micrometer scales, thus hindering the overcoming of inherent performance bottlenecks. Furthermore, to apply these materials to bone repair, it is often necessary to introduce active components such as hydroxyapatite to impart osteoinductive properties, which places higher demands on the interfacial bonding and mechanical properties of the composite materials.

[0005] In summary, existing silk fibroin-based rigid materials are often characterized by high rigidity and low toughness or high toughness and low rigidity, lacking silk fibroin materials that possess both moderate rigidity and good toughness. This performance gap severely limits the application of such materials in physiological sites such as articular cartilage, ligaments, and menisci, which are subjected to dynamic or complex stress environments. Therefore, developing a silk fibroin-based rigid material that can effectively synergistically combine rigidity and toughness not only represents a clear technological innovation but also meets the urgent practical needs of high-end biomedical fields. Summary of the Invention

[0006] Addressing the technical bottlenecks mentioned in the background section—that existing pure silk fibroin rigid materials cannot simultaneously achieve high rigidity and high toughness, and that existing sericin composite methods cannot effectively control the microstructure and exert a synergistic toughening effect—this invention aims to provide a novel silk fibroin composite rigid material, its preparation method, and its applications. The primary objective of this invention is to prepare a silk fibroin composite material that achieves a good balance between rigidity and toughness through an innovative integrated recycling and composite process. A further objective of this invention is to provide an economical and environmentally friendly preparation method that allows for precise control of the material's mechanical properties, overcoming the problems of performance instability, biosafety risks, and high costs associated with the use of complex solvents or uncontrollable blending processes in existing technologies.

[0007] To achieve the above-mentioned objectives, the present invention adopts the following technical solution: On one hand, the present invention provides a method for preparing a silk fibroin composite rigid material, comprising the following steps: S1. Degumming raw silk is performed to simultaneously obtain degummed silk and a solution containing sericin. S2. Recover sericin from the sericin-containing solution obtained in step S1; S3. Dissolve the degummed silk obtained in step S1 to obtain a silk fibroin solution; S4. Add the sericin recovered in step S2 to the silk fibroin solution obtained in step S3, mix well, and form a composite protein solution; S5. Perform gradient dialysis on the composite protein solution obtained in step S4 to obtain gel blocks; S6. The gel block obtained in step S5 is solvent-displaced and then dried to finally obtain the silk protein composite rigid material.

[0008] Preferably, in step S3, the solvent used for dissolution is an aqueous solution of lithium bromide or a ternary solvent of calcium chloride / ethanol / water.

[0009] Preferably, in step S4, the amount of sericin added is 1% to 80% (w / w), based on the dry weight of the degummed silk. By controlling this amount, the mechanical properties of the final material can be precisely controlled.

[0010] Preferably, in step S5, the dialysate used in gradient dialysis is an aqueous solution of methanol or ethanol, wherein the volume fraction of alcohol varies from high to low, preferably within a gradient range of 50% to 100%. This process is crucial for regulating the crystallinity of the β-sheet of silk fibroin and the microstructure of the sericin-silk fibroin two-phase system.

[0011] Preferably, in step S6, solvent replacement is performed by immersing the gel block in water or a physiologically compatible buffer solution to completely remove the organic solvent.

[0012] In a second aspect, the present invention provides a silk fibroin composite rigid material prepared by the preparation method described in the first aspect.

[0013] Preferably, the silk protein composite rigid material according to the second aspect further comprises hydroxyapatite.

[0014] Preferably, when hydroxyapatite is included, the amount of hydroxyapatite added is 1% to 40% (w / w) based on the dry weight of degummed silk.

[0015] Preferably, the silk fibroin composite rigid material has the following mechanical properties: its compressive modulus is 10 MPa to 1000 MPa, and its fracture strain is not less than 10%. This performance window makes it particularly suitable for simulating and repairing tissues in the human body that need to withstand both load and deformation.

[0016] In another aspect, the present invention provides the use of the above-mentioned silk fibroin composite rigid material in the preparation of medical implants or tissue engineering scaffolds, particularly in the preparation of artificial bone and cartilage repairs, ligament or tendon reinforcing patches, meniscus substitutes, and craniofacial bone repair scaffolds.

[0017] Compared with the prior art, the silk fibroin composite rigid material and its preparation method provided by the present invention have the following significant advantages: (1) Achieving an excellent balance between material rigidity and toughness: This invention optimizes the stress transfer path at the molecular level by controllably recombining homologous sericin into the silk fibroin matrix and reshaping its microphase structure using a gradient dialysis process. This enables the final material to overcome the performance contradiction of traditional silk fibroin materials, which struggle to balance rigidity and toughness. It achieves a wide range of predictable synergistic control between high rigidity (compressive modulus 10-1000 MPa) and high toughness (fracture strain ≥10%), filling the gap in existing silk fibroin materials that combine rigidity and toughness.

[0018] (2) Predictable and controllable mechanical properties: Due to the adoption of the process route of separating and recombining sericin, the addition ratio of sericin becomes an independent and precise controllable variable. By systematically adjusting this ratio and combining it with gradient dialysis conditions, the rigidity and toughness of the final composite material can be controlled within a wide range, thereby allowing for customized design of material properties for different clinical application scenarios (such as ligaments, cartilage, and bone).

[0019] (3) Ensures component homogeneity and biosafety: Compared with methods such as direct processing without degumming or blending with purchased sericin, this invention separates and recovers sericin from the same batch of raw materials, ensuring the homology and optimal compatibility of sericin and silk fibroin, and avoiding product performance fluctuations due to batch differences or exogenous contamination. In addition, endotoxin residues in sericin are a significant safety hazard, and the independent sericin recovery step can further purify the material to reduce biosafety risks.

[0020] (4) Integrated, economical, and environmentally friendly process: The method of this invention forms a closed-loop process system with high raw material utilization. It transforms traditionally discarded sericin into a high-value toughening component, realizing the high-value utilization of all components of silk raw materials. Compared with existing processes that use regenerated silk fibroin to prepare rigid materials, this invention directly uses degummed silk for dissolution and compounding, eliminating the intermediate steps of preparing regenerated silk fibroin, significantly reducing raw material costs and processing energy consumption, while shortening the production cycle, demonstrating a high degree of process integration advantages. In addition, the entire process avoids the use of expensive and toxic solvents such as hexafluoroisopropanol, further reducing production costs, conforming to the concept of green manufacturing, and possessing the potential for large-scale industrialization. Attached Figure Description

[0021] Figure 1 Photographs of the silk fibroin composite rigid material prepared in this invention (from left to right: Examples 1 to 5, Comparative Examples 1 to 3 and Comparative Example 5).

[0022] Figure 2 The stress-strain curves of the compression test of the silk fibroin composite rigid materials prepared in Examples 1 to 3 and Comparative Examples 1 to 3 of this invention are shown.

[0023] Figure 3 The stress-strain curves of the silk fibroin composite rigid materials prepared in Examples 4 to 5 and Comparative Example 5 of this invention are shown in the compression test.

[0024] Figure 4 This refers to the compressive modulus of the silk fibroin composite rigid material prepared in this invention.

[0025] Figure 5 The fracture strain is the silk protein composite rigid material prepared in this invention. Detailed Implementation

[0026] In this invention, the rigidity of a material is quantitatively characterized by its compressive modulus; the higher the value, the stronger the rigidity. Toughness primarily characterizes its ability to absorb energy and withstand plastic deformation before fracture, and is quantitatively characterized by compressive fracture strain; the higher the value, the greater the deformation the material can withstand before fracture, indicating better toughness and stronger resistance to brittle fracture. To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments and comparative examples. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions in the art or as recommended by the manufacturer.

[0027] General materials and instruments: The silk was commercially available mulberry silk; anhydrous calcium chloride, anhydrous ethanol, lithium bromide, and sodium carbonate were all analytical grade and purchased from Sinopharm Reagent. Gelatin and nano-hydroxyapatite were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Example

[0028] (1) Cut the raw silk into pieces, place it in a 0.5% (w / v) sodium carbonate aqueous solution, boil it at 95-100℃ for 30 minutes to degumme, repeat three times to completely remove the sericin.

[0029] (2) Collect all the degumming liquids and combine them to obtain a solution containing sericin. After the sericin solution is evaporated and concentrated, the insoluble matter is removed by centrifugation. The endotoxin is removed by hydrophobic interaction chromatography. The collected liquid is injected into a dialysis bag with a molecular weight cutoff of 8000-14000 Da and dialyzed until the solution pH < 8. The dialysate is freeze-dried to obtain sericin powder. (3) The degummed silk obtained from the above degumming treatment is washed with deionized water, dried, and then dissolved in a ternary solvent of calcium chloride / ethanol / water at a material-to-liquid ratio of 1:5 g / mL, wherein the molar ratio of calcium chloride:ethanol:water is 1:2:8. The dissolution process is carried out by continuous stirring in a water bath at 60-90℃ until the solution is clear and transparent; (4) Slowly add sericin powder (1% of the dry weight of degummed silk) to the silk fibroin solution prepared in step 2 and stir continuously for 1 hour to form a uniform composite protein solution. (5) Inject the composite protein solution into the dialysis bag and dialyze it in 50%, 75% and 99% ethanol aqueous solution in sequence. Dialyze for 48 hours at each concentration to form a dense gel block. (6) Immerse the obtained gel block in deionized water for 48 hours to replace the solvent, and then place the gel block in a clean environment at 25°C and 40% relative humidity to air dry for 7 days to finally obtain a hard block material. Example

[0030] Compared with Example 1, the dissolving solvent in step 3 was changed to a 9.3M lithium bromide aqueous solution, and the amount of sericin added in step 4 was changed to 40% of the dry weight of degummed silk. The remaining steps and parameters were the same as in Example 1. Example

[0031] Compared with Example 1, the amount of sericin added in step 4 was changed to 80% of the dry weight of degummed silk, and the gradient dialysis external solution in step 5 was changed to an aqueous methanol solution with the same volume fraction. The remaining steps and parameters were the same as in Example 1. Example

[0032] Compared with Example 1, the amount of sericin added in step 4 was changed to 40% of the dry weight of degummed silk, and nano hydroxyapatite was added (the amount added was 1% of the dry weight of degummed silk). The remaining steps and parameters were the same as in Example 1. Example

[0033] Compared with Example 1, in step 4, the amount of sericin added was changed to 10% of the dry weight of degummed silk, and nano hydroxyapatite was added (the amount added was 40% of the dry weight of degummed silk). In step 6, the final replacement medium was changed from deionized water to phosphate buffer (PBS) with pH=7.4. The remaining steps and parameters were the same as in Example 1.

[0034] Comparative Example 1 Compared with Example 1, no sericin powder was added in step 4, and the remaining steps and parameters were the same as in Example 1.

[0035] Comparative Example 2 Compared with Example 1, the dissolving solvent in step 3 was replaced with a 9.3M lithium bromide aqueous solution, and the sericin in step 4 was replaced with gelatin, with the amount added being 40% of the dry weight of the degummed silk. The remaining steps and parameters were the same as in Example 1.

[0036] Comparative Example 3 Raw silkworm silk was cut into small pieces, washed with ultrapure water, and dried. It was then dissolved in a ternary solvent of calcium chloride / ethanol / water at a ratio of 1:5 g / mL, where the molar ratio of calcium chloride:ethanol:water was 1:2:8. The dissolution process was carried out with continuous stirring in a water bath at 60-90°C until the solution became clear and transparent. Subsequent steps were the same as steps 5 and 6 in Example 1.

[0037] Comparative Example 4 Compared with Example 1, the mass of sericin in step 4 was changed to 90% of the dry weight of degummed silk, while the remaining steps and parameters were the same as in Example 1.

[0038] Comparative Example 5 Compared with Example 1, in step 4, sericin was replaced with nano-hydroxyapatite, and the amount added was 1% of the dry weight of degummed silk. The remaining steps and parameters were the same as in Example 1.

[0039] Performance Testing and Result Analysis The materials obtained from all the above embodiments and comparative examples ( Figure 1 Mechanical property tests were conducted. The compressive properties were tested using a universal testing machine, following the standard GB / T 1041-2008. The results are shown in Table 1 and... Figures 2 to 5 As shown.

[0040] The bacterial endotoxin content of the silk fibroin composite rigid materials prepared in Example 3 and Comparative Example 3 was tested according to the provisions of the General Chapter 1143 of the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part IV, Bacterial Endotoxin Test Method. The results are shown in Table 2.

[0041] Table 1. Mechanical property test results of materials prepared in the examples and comparative examples. serial number Sericin addition amount Hydroxyapatite addition amount Dissolved solvent Dialysate Replacement medium Compression modulus (MPa) Fracture strain (%) Example 1 1% 0% Calcium chloride / ethanol / water ethanol water 336.22 10.99 Example 2 40% 0% Lithium bromide aqueous solution ethanol water 93.06 35.82 Example 3 80% 0% Calcium chloride / ethanol / water methanol water 18.96 50.44 Example 4 40% 1% Calcium chloride / ethanol / water ethanol water 102.16 33.08 Example 5 10% 40% Calcium chloride / ethanol / water ethanol PBS 902.18 12.58 Comparative Example 1 0% 0% Calcium chloride / ethanol / water ethanol water 413.89 8.51 Comparative Example 2 0% 0% Lithium bromide aqueous solution ethanol water 75.39 18.55 Comparative Example 3 0% 0% Calcium chloride / ethanol / water ethanol water 106.21 25.62 Comparative Example 4 90% 0% Calcium chloride / ethanol / water ethanol water —— —— Comparative Example 5 0% 1% Calcium chloride / ethanol / water ethanol water 471.73 7.34 Comparing the data from Comparative Example 1 (pure silk fibroin) and Examples 1-3 (with added homologous sericin), it can be seen that the introduction of sericin can systematically regulate the rigidity-toughness trade-off of the material. For example, when the sericin addition is 1% (Example 1), the material maintains a high modulus (336.22 MPa), while its fracture strain (10.99%) is significantly better than that of pure silk fibroin (Comparative Example 1, 8.51%), overcoming its inherent brittleness. As the sericin addition increases to 80% (Example 3), the material modulus decreases to 18.96 MPa, while the fracture strain increases significantly to 50.44%, exhibiting excellent ductility. This trend demonstrates that the process described in this invention can achieve predictable control of material properties, thereby precisely matching the differentiated mechanical performance requirements of different soft tissue repair scenarios.

[0042] Comparing Example 2 (40% sericin) with Comparative Example 2 (40% gelatin), it was found that, with similar toughening component contents, the mechanical properties of the homologous sericin composite material (modulus 93.06 MPa, strain 35.82%) were comprehensively superior to those of the gelatin composite material (modulus 75.39 MPa, strain 18.55%). This indicates that homologous sericin and the silk fibroin matrix have better interfacial compatibility and synergistic effects, and can more effectively transfer and dissipate stress.

[0043] Table 2 Results of bacterial endotoxin content detection serial number Bacterial endotoxin content (EU / mg) Example 3 ≤0.05 Comparative Example 3 ＞0.05 Further comparison of Example 3 and Comparative Example 3 (direct processing of undegummed silk) shows that the present invention, through the separation and purification of sericin, reduces the bacterial endotoxin content of the final material to ≤0.05 EU / mg, meeting the biological safety requirements for implantable medical devices, while Comparative Example 3 exceeds the standard. Regarding toughness, the strain of Comparative Example 3 is only 25.62%, while the strain can be increased to 50.44% by adding 80% purified sericin (Example 3) using the process of the present invention. This demonstrates that the preparation process of the present invention not only solves the biosafety problem but also fully releases the toughening potential of sericin through precise and controllable compounding of components, an effect that cannot be achieved by simple blending processes.

[0044] Comparative Example 5 shows that while combining hydroxyapatite with pure silk fibroin increases the modulus (471.73 MPa), it also leads to increased brittleness (fracture strain decreases to 7.34%). Examples 4 and 5 demonstrate that when hydroxyapatite is combined with sericin, the material achieves superior overall performance. In particular, Example 5 (10% sericin, 40% hydroxyapatite), with moderate toughening from sericin, shows a significant increase in modulus to 902.18 MPa due to the high hydroxyapatite content, while maintaining a fracture strain of 12.58%, successfully achieving a simultaneous improvement in both high rigidity and good toughness. Hydroxyapatite not only enhances the strength of the silk fibroin / sericin matrix, but its unique biological activity further expands the application range of silk fibroin composite rigid materials.

[0045] In Comparative Example 4, when the amount of sericin added was too high (90%), the material may experience severe phase separation due to excessive sericin phase, disrupting the uniform and continuous stress transmission network within the material. This makes the material prone to breakage after drying, failing to meet the compression test requirements. This result demonstrates, conversely, that only by controlling the amount of sericin added within an appropriate range (e.g., 1%-80%) can harmful phase separation be avoided, ensuring that sericin is effectively dispersed within the silk fibroin network, thereby achieving a synergistic improvement in rigidity and toughness. This clarifies the critical significance and preference of the addition range described in the claims of this invention.

[0046] The above embodiments are merely illustrative examples and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for preparing a silk fibroin composite rigid material, characterized in that, Includes the following steps: S1. Degumming raw silk to obtain degummed silk and a solution containing sericin; S2. The solution containing sericin obtained in step S1 is concentrated, centrifuged and dried to obtain sericin; S3. Dissolve the degummed silk obtained in step S1 to obtain a silk fibroin solution; S4. Add the sericin obtained in step S2 to the silk fibroin solution obtained in step S3, mix, and form a composite protein solution; S5. Perform gradient dialysis on the composite protein solution obtained in step S4 to obtain gel blocks; S6. The gel block obtained in step S5 is solvent-displaced and then dried to obtain a silk protein composite rigid material.

2. The preparation method according to claim 1, characterized in that, In step S3, the solvent used to dissolve the degummed silk is an aqueous solution of lithium bromide or a ternary solvent of calcium chloride / ethanol / water.

3. The preparation method according to claim 1, characterized in that, In step S4, the amount of sericin added is 1% to 80% based on the dry weight of the degummed silk.

4. The preparation method according to claim 1, characterized in that, In step S5, the dialysate used in the gradient dialysis is an aqueous solution of methanol or ethanol, wherein the volume fraction of methanol or ethanol is 50% to 100%.

5. The preparation method according to claim 1, characterized in that, In step S6, the solvent replacement is performed by immersing the gel block in water or a physiologically compatible buffer solution.

6. A rigid material composed of silk protein, characterized in that, It is prepared by any one of claims 1 to 5.

7. The silk fibroin composite rigid material according to claim 6, characterized in that, The material also contains hydroxyapatite.

8. The silk fibroin composite rigid material according to claim 7, characterized in that, Based on the dry weight of the degummed silk, the amount of hydroxyapatite added is 1% to 40%.

9. The rigid material composited with silk fibroin according to any one of claims 6 to 8, characterized in that, The material has a compressive modulus of 10-1000 MPa and a fracture strain of not less than 10%.

10. The use of the silk fibroin composite rigid material according to any one of claims 6 to 9 in the preparation of medical implants or tissue engineering scaffolds.

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