SS / CTGF composite biological material based on carboxyl chitosan as well as preparation method and application of SS / CTGF composite biological material

Abstract

The invention discloses an SS / CTGF composite biological material based on carboxyl chitosan as well as a preparation method and application of the SS / CTGF composite biological material. A substance containing active sericin (SS) and CTGF at the same time is extracted from genetic engineering silkworm cocoons over-expressing human connective tissue growth factors (CTGF) by utilizing an alkali extraction-acid protection method. Furthermore, the active substance, carboxyl chitosan and beta-glycerophosphate are used as raw materials, and the CH-SS / CTGF composite hydrogel biological material with the core-shell structure is prepared through thermal induction gelation. The material has the characteristics that the water content is adjustable, the mechanical property is controllable, and active protein can be quickly released. Compared with pure sericin, the active SS / CTGF substance and the composite hydrogel thereof can more remarkably promote cell proliferation and migration, and show good biocompatibility. The biological material prepared by the invention has wide application prospects in the fields of wound healing, drug delivery and regenerative medicine.

Description

Technical Field

[0001] This invention relates to the field of biomaterials, specifically to an SS / CTGF composite biomaterial based on carboxy-chitosan, and also to a method for preparing the composite biomaterial and its application. Background Technology

[0002] Functional biomaterials hold immense potential for future applications in tissue engineering and wound dressings, particularly for preparing biomaterials with properties that improve wound healing, bone repair, and tissue regeneration. Traditional methods for preparing such materials primarily involve adding cofactory active molecules (such as FGF and PDGF-BB) during processing; these materials have been proven to significantly enhance cell proliferation and promote bone formation. In recent years, by overexpressing artificially designed active proteins in silk, genetic engineering techniques have enabled the efficient preparation of various genetically engineered functional silk materials. These proteins encompass human cytokines (such as FGF1, TGF-β, CTGF, and PDGF), antibodies (such as CD20), and other functional molecules (such as glucose oxidase and lactoferrin). Crucially, using these genetically engineered silk materials as raw materials, various functional biomaterials (such as hydrogels and nanocarriers) can be successfully prepared without the addition of cofactory active molecules. These materials possess properties that promote cell growth and wound healing, inhibit inflammation, promote bone regeneration, and treat colitis. Therefore, genetically engineered silk materials hold immense potential for the future manufacture of various functional medical biomaterials.

[0003] However, the practical application of these genetically engineered silk materials still faces many challenges. The primary challenge is how to prevent the inactivation of active proteins within the silk fibers during the preparation of functional biomaterials. Traditional extraction methods, such as high-temperature and high-pressure processes, acid-base treatments, and strong oxidant treatments, have been proven to easily damage the structure of active proteins or degrade and inactivate them due to their harsh extraction conditions or the presence of strong oxidants. Based on this, researchers have attempted to use the glutathione redox system to restore the structure and bioactivity of target proteins, but this method is complex and inefficient, restoring only about one-third of the target protein activity. The second challenge is preparing stable biomaterials without compromising the functionality of active proteins. Traditional methods for stabilizing silk protein biomaterials mainly use chemical cross-linking reagents, including glutaraldehyde, genipin, and the EDC-NHS system. While these methods can significantly improve the stability of biomaterials to some extent by forming intramolecular or intermolecular covalent networks, the connection and destruction of corresponding groups in the active target proteins can also lead to a decrease in the bioactivity of the active proteins in the biomaterials.

[0004] Besides chemical crosslinking, physical crosslinking strategies are also widely used to enhance the stability of biomaterials. Physical crosslinking primarily utilizes physical mechanisms such as cooling, heating, light exposure, and pressure to construct transient physical interactions (including hydrogen bonds, van der Waals forces, and hydrophobic interactions) between polymer chains within the biomaterial, thereby achieving crosslinking and curing. Examples of such materials include chitosan-based thermosensitive hydrogels, chemically synthesized ABC block copolymers, and sericin / agarose composite gel materials. Compared to natural chitosan molecules, carboxychitosan has been shown to have superior water solubility, and its dissolution process does not require acidic solutions in the preparation of various medical biomaterials. Therefore, this material has been applied to the preparation of various medical biomaterials, including carboxychitosan / poloxamer nanoparticles loaded with voriconazole for ocular drug delivery, polyvinyl alcohol / carboxychitosan hydrogels for promoting dynamic skin wound healing, and ultraporous polyquaternary ammonium salt / carboxychitosan composite hydrogel spheres for sepsis treatment, which possess anticoagulant, antibacterial, and endotoxin clearance functions.

[0005] Previous studies have shown that sericin and its degraded peptides possess various bioactivities, including promoting cell proliferation, antioxidation, anti-inflammation, and wound healing. Among them, short peptides of hydrolyzed sericin exhibit stronger bioactivity, making them an ideal natural active substance for developing functional biomaterials. Based on this, this study aims to develop a simple method for preparing an active ingredient containing both active sericin and cytokine proteins from genetically engineered silk materials. This active ingredient will then be used to develop functional biomaterials based on carboxychitosan for use in the biomedical field. Summary of the Invention

[0006] In view of this, one objective of the present invention is to provide an SS / CTGF composite biomaterial based on carboxy chitosan; a second objective of the present invention is to provide a method for preparing the SS / CTGF composite biomaterial based on carboxy chitosan; and a third objective of the present invention is to provide the application of the SS / CTGF composite biomaterial based on carboxy chitosan in the preparation of biomedical materials that promote cell proliferation and / or wound healing.

[0007] To achieve the above objectives, the present invention provides the following technical solution: A carboxychitosan-based SS / CTGF composite biomaterial is disclosed, comprising sericin SS / CTGF containing exogenous connective tissue growth factor, carboxychitosan, and β-glycerophosphate. The carboxychitosan is dissolved in the SS / CTGF to obtain an SS / CTGF / carboxychitosan solution, and the β-glycerophosphate is dissolved in water and mixed with the SS / CTGF / carboxychitosan solution. The composite biomaterial is obtained by induction at a temperature above 37°C.

[0008] Preferably, the sericin SS / CTGF containing exogenous connective tissue growth factor of the present invention is prepared by the following method: silkworm cocoons expressing biologically active human connective tissue growth factor are crushed and first dissolved in an alkaline aqueous solution with a pH of 13. The supernatant is collected by centrifugation to obtain a primary sericin extract, and then acid is added to neutralize it to obtain SS / CTGF.

[0009] Preferably, the alkaline aqueous solution of the present invention is an aqueous solution of sodium hydroxide and an aqueous solution of sodium carbonate.

[0010] Preferably, the acid solution used for acid neutralization in this invention is a hydrochloric acid solution.

[0011] 2. The preparation method of the SS / CTGF composite biomaterial based on carboxylated chitosan includes the following steps: (1) The silkworm cocoons expressing biologically active human connective tissue growth factor were crushed; (2) Immerse the powder obtained in step (1) in an alkaline aqueous solution and stir and extract for 1 to 48 hours at 40 to 80°C; centrifuge and take the supernatant to obtain primary sericin extract; add acid solution to the primary sericin extract for neutralization to obtain sericin SS / CTGF containing exogenous connective tissue growth factor. (3) Dissolve carboxy chitosan in the sericin containing exogenous connective tissue growth factor to obtain SS / CTGF / carboxy chitosan solution; then dissolve β-glycerophosphate in water and mix it with the SS / CTGF / carboxy chitosan solution, and induce the composite biomaterial at a temperature above 37°C.

[0012] 3. The application of the SS / CTGF composite biomaterial based on carboxylated chitosan in the preparation of biomedical materials that promote cell proliferation and / or wound healing.

[0013] Preferably, the biomedical material is used to prepare a drug delivery system or a tissue engineering scaffold.

[0014] The beneficial effects of this invention are as follows: This invention provides an SS / CTGF composite biomaterial based on carboxylated chitosan, which has the following outstanding advantages compared to the prior art: Successfully prepared a highly active composite functional substance: The "alkali extraction-acid protection" method utilizes a strong alkali to rapidly dissolve the sericin layer of silkworm silk and immediately neutralize it to stabilize the protein conformation. This effectively overcomes the problem of denaturation and degradation of cytokines such as CTGF that easily occurs when extracting functional proteins from genetically engineered silkworm silk using traditional extraction methods (such as the sodium carbonate method). This method successfully prepared an SS / CTGF substance containing both active sericin and intact CTGF protein, maximizing the preservation of biological activity and laying the foundation for the development of functional biomaterials.

[0015] The biomaterial exhibits a novel structure and superior performance: the CH-SS / CTGF biomaterial prepared from this active substance spontaneously forms a unique core-shell structure of "loose porous core-dense shell" within a thermosensitive gel network formed by carboxychitosan and β-glycerophosphate. This structure not only endows the material with tunable mechanical properties (achieved by controlling water content), but its porous core also provides ideal space for loading the active substance, while the dense shell can regulate drug release kinetics.

[0016] Featuring intelligent and efficient protein release properties: Thanks to its core-shell structure, the CH-SS / CTGF hydrogel achieves rapid, phased release of the CTGF protein. The rapid dissolution of the outer shell results in an initial burst of release, while the gradual dissolution of the core releases subsequent drugs. This release mode is particularly suitable for applications requiring rapid initiation of the repair process, such as wound healing. Furthermore, this release behavior is unaffected by lysozyme, making it more stable in the in vivo environment.

[0017] Significantly enhanced biological function: Experiments have confirmed that the active SS / CTGF substance and its composite hydrogel prepared in this invention exhibit significantly enhanced effects in promoting the proliferation and migration of fibroblasts (NIH / 3T3) compared to the control group containing only sericin. This is attributed to the synergistic effect of sericin and CTGF. Sericin provides a good cell adhesion and growth matrix, while CTGF specifically activates signaling pathways related to tissue repair and proliferation, and the combination of the two produces a synergistic effect.

[0018] 5) Excellent biocompatibility and broad application prospects: Cytotoxicity and inflammatory response assessments showed that the CH-SS / CTGF biomaterial was non-toxic to normal cells, inducing only a mild inflammatory response, demonstrating excellent biocompatibility. Combined with its injectable / shape-adjustable, highly reparative, and biodegradable properties, it is a highly promising new biomedical material with significant application value in acute and chronic wound dressings, drug delivery systems, and soft tissue engineering scaffolds. Attached Figure Description

[0019] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the following figures are provided for illustration: Figure 1A schematic diagram illustrating the process of preparing CH-SS / cytokine composite biomaterials using silk materials (A: genetically engineered silkworm cocoon (containing active proteins such as cytokines); B: silkworm cocoon powder; C: active substances containing sericin and cytokines; D: liquid CH-SS / cytokine mixture; E: solid CH-SS / cytokine biomaterial; F: various CH-SS / cytokine composite biomaterials; Note: red circles, yellow circles, green diamonds, and black lines represent cytokines, sericin, β-glycerophosphate, and carboxychitosan molecules, respectively; dashed lines represent hydrogen bonds and interactions between carboxychitosan and sericin).

[0020] Figure 2 This study investigated the effects of urea extraction, sodium carbonate extraction, and the proposed "alkali extraction-acid protection" method on the extraction of total sericin from silk. Urea-WT silk and Urea-CTGF silk were extracted using the urea method, respectively, yielding total sericin samples from WT and CTGF silk. Alkali-acid-CTGF silk and Na2CO3-CTGF silk were extracted using the "alkali extraction-acid protection" method and the sodium carbonate method, respectively, yielding total sericin samples from CTGF silk.

[0021] Figure 3 SS / CTGF substances can promote the proliferation and growth of NIH / 3T3 cells (A: Detection of sericin and CTGF proteins in SS / CTGF substances; where SS refers to sericin samples extracted from natural silk materials; SS / CTGF-1, -2, and -3 refer to SS / CTGF substances extracted from CTGF-functionalized genetically engineered silk materials; CTGFstd is commercially available CTGF protein; B: After 3 days of culture, the growth status of NIH / 3T3 cells was detected using the LIVE / DEAD method; C: After 1 and 3 days of culture, the viability of NIH / 3T3 cells was detected using the AlamarBlue method. Green and red fluorescence signals represent live and dead cells, respectively. In the cell proliferation experiment, the total content of SS and SS / CTGF proteins was 187.5 µg / ml. Scale bar: 200 µm).

[0022] Figure 4Characteristics of CH-SS / CTGF hydrogel biomaterials (A: Morphology of CH-SS / CTGF hydrogel at 4℃ and 37℃; B: SEM sampling diagram; OL represents the outer surface; IL-1, IL-2, and IL-3 represent internal regions with gradually increasing depth; C: SEM results of CH-SS / CTGF hydrogel; yellow and blue arrows point to the layered and dense structure inside; green and purple arrows point to the granular structure of the inner and outer surfaces; scale bar: 100 µm; D: Carboxychitosan powder, lyophilized-CH hydrogel, CH-SS / CTGF hydrogel, and SS / CTGF material at 400-4000 cm⁻¹) -1 (FTIR spectra within the range).

[0023] Figure 5 The relationship between water content and induction time of CH-SS / CTGF hydrogel at 37℃ is shown.

[0024] Figure 6 Mechanical properties of CH-SS / CTGF hydrogel biomaterials (A: Schematic diagram of mechanical property testing of CH-SS / CTGF hydrogel; BC: Mechanical properties of CH-SS / CTGF hydrogels with water contents of 20%, 30%, and 45% were tested using the gravimetric crushing method; D: Stress-strain curve of CH-SS / CTGF hydrogel with 45% water content).

[0025] Figure 7 The stability of CH-SS / CTGF hydrogel biomaterials (A: Thermogravimetric analysis (TGA) results of CH-SS / CTGF hydrogel; B: Schematic diagram of CH-SS / CTGF hydrogel degradation experiment; CD: Stability of CH-SS / CTGF hydrogel in phosphate-buffered saline (PBS) at different temperatures (4℃, 37℃, 65℃) and different pH values ​​(4.0, 7.4, 11.0); E: Stability of CH-SS / CTGF hydrogel in phosphate-buffered saline (PBS) with or without 10 U / mL lysozyme).

[0026] Figure 8 Morphological characteristics of CH-SS / CTGF hydrogel after incubation in PBS solution; Scale bar: 1 cm.

[0027] Figure 9The release behavior of sericin and CTGF protein in CH-SS / CTGF hydrogel biomaterials is shown in the figure. (A: Schematic diagram of protein release experiment in CH-SS / CTGF hydrogel; B: Cumulative release curve of sericin and CTGF protein from CH-SS / CTGF hydrogel; CD: Detection results of CTGF protein release from CH-SS / CTGF hydrogel in PBS solution with / without 10 U / mL lysozyme; E: Cumulative release curve of CTGF protein in CH-SS / CTGF hydrogel).

[0028] Figure 10 To promote the proliferation of NIH / 3T3 cells using CH-SS / CTGF hydrogel biomaterials (A: Growth of NIH / 3T3 cells on CH-SS and CH-SS / CTGF hydrogel surfaces after 1 day of co-culture; B: LIVE / DEAD staining results of NIH / 3T3 cells on CH-SS and CH-SS / CTGF hydrogel surfaces after 3 days of co-culture; CD: Growth of NIH / 3T3 cells after 1-3 days of co-culture using AlamarBlue and CCK-8 assays; E: In vitro wound healing assay, scale bar: 200 µm).

[0029] Figure 11 The CH-SS / CTGF hydrogel biomaterial exhibited good biocompatibility (A: AlamarBlue analysis of NIH / 3T3 cell growth on TCP and CH-SS / CTGF hydrogel surfaces; B: Morphology of NIH / 3T3 cells after 7 days of culture on TCP and CH-SS / CTGF hydrogel surfaces; C: LIVE / DEAD staining results of NIH / 3T3 cells after 7 days of culture on TCP and CH-SS / CTGF hydrogel surfaces; Scale bar: 400 μm; D-F: Expression levels of inflammatory markers iNOS and COX-2 in Raw264.7 cells after 2 days of co-culture with CH-SS and CH-SS / CTGF hydrogels, LPS concentration was 100 ng / mL, TCP was a tissue culture plate). Detailed Implementation

[0030] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0031] In this invention, mouse embryonic fibroblast cell line NIH / 3T3 (CL-0171, Procell, China) and mouse macrophage cell line Raw264.7 (CL-0190, Procell, China) were selected for cell proliferation and cytotoxicity analysis. They were cultured in Dulbecco's Modified Eagle Medium (11995065, DMEM, Gibco, USA) and Roswell Park Memorial Institute 1640 Medium (22400089, RPMI, Gibco, USA), respectively. These media were supplemented with 10% fetal bovine serum albumin (FBS, Gibco, USA), 50 mg / ml streptomycin (Gibco, USA), and 100 mg / ml ampicillin (Gibco, USA).

[0032] Example 1: Preparation of active SS / CTGF substances The preparation method of the active SS / CTGF substance is as follows: First, CTGF-functionalized silkworm cocoons (see Chinese Patent Publication No. CN111424035A), prepared by tissue-specific overexpression of human connective tissue growth factor gene in the sericin layer of silkworm silk, were ground into fine powder in liquid nitrogen using an IKA® A11 alkaline grinding chamber (Merck, Germany). Then, 1 gram of this silkworm powder was immersed in 1 L of alkaline aqueous solution with a pH of 13 and extracted by stirring at 50°C for 3 hours. Next, the mixture was centrifuged at 12,000 rpm for 10 minutes, and the resulting supernatant was the primary sericin extract. Finally, the pH was adjusted to 7.4 by adding 0.1 M hydrochloric acid solution dropwise to the primary sericin extract to neutralize it, thereby obtaining the active SS / CTGF substance, in which the sericin content was 7.5 mg / mL and the CTGF content was 2.81 μg / mL. This method is named the "alkali extraction-acid protection" method, and the specific extraction process is as follows. Figure 1 As shown in the figure. Simultaneously, sericin was extracted using the urea method and the sodium carbonate method, respectively. The extraction results were analyzed by SDS-PAGE, and the results are shown in the figure. Figure 2 As shown in the figure. The results show that the sericin extracted by the urea method can better maintain the molecular weight of sericin; the sericin extracted by the sodium carbonate method will degrade and the molecular weight will decrease; the total sericin extracted by the "alkali extraction-acid protection" method proposed in this study has a wider molecular weight distribution, and although some degradation occurs, the molecular weight distribution is significantly better than that of the sericin extracted by the sodium carbonate method.

[0033] Testing revealed that, using the "alkali extraction-acid protection" method, the contents of sericin and CTGF in the active SS / CTGF substance were 7.5 mg / mL and 2.81 µg / mL, respectively. Figure 3(A) After co-culturing SS containing sericin and an active substance containing both sericin and CTGF (SS / CTGF) with NIH / 3T3 cells, the live / dead cell staining results showed that, compared with the Null group, the number of NIH / 3T3 cells emitting green fluorescent signals was significantly increased in the SS group, with the SS / CTGF group showing the highest number of cells observed. Figure 3 (B). Cell viability assay results showed that after co-culturing with SS and SS / CTGF for 1 and 3 days, the cell number in the SS group was significantly higher than that in the Null group, while the cell number in the SS / CTGF group was significantly higher than that in both the Null and SS groups. Figure 3 These results indicate that both sericin and CTGF in the SS / CTGF material exhibit cell proliferation-promoting activity.

[0034] Example 2: Preparation of CH-SS / CTGF biomaterials The preparation method of CH-SS / CTGF biomaterials is based on literature reports. The specific steps are as follows: 500 mg of carboxychitosan (catalog number C105800-100g, Aladdin) was dissolved in 10 mL of active SS / CTGF solution and placed at a constant temperature of 4°C. Next, 2 g of β-glycerophosphate (catalog number 35675-100GM, Sigma-Aldrich, USA) was dissolved in 4 mL of water and mixed with 10 mL of SS / CTGF / carboxychitosan solution to form a composite system. This mixture was injected into a specially designed mold and reacted at a constant temperature above 37°C for more than 2 hours to obtain CH-SS / CTGF biomaterials in different forms (e.g., hydrogels, membrane structures). Figure 1 F).

[0035] The water content of CH-SS / CTGF biomaterials decreases with increasing temperature and longer induction time. To determine the water content, a CH-SS / CTGF biomaterial sample weighing m1 g was dried to a constant weight (m2 g) in an oven at 60 °C. The water content was then calculated using the following formula: Water content (%) = 100 × [(m1 - m2) / m1].

[0036] Example 3: Characterization of CH-SS / CTGF composite hydrogel biomaterial Next, the basic properties of the CH-SS / CTGF hydrogel biomaterial were analyzed. Thermosensitive testing showed that the CH-SS / CTGF mixture, which was liquid at 4°C, transformed into a solid gel within one hour at 37°C. Figure 4(A), indicating that its morphology is temperature-dependent. SEM results show that the CH-SS / CTGF hydrogel has a loose and porous "core" interior and a dense, pitted "shell" exterior, with the internal structure gradually transitioning from a layered porous structure to a dense structure. Figure 4 (B - C), with a maximum pore size of 185.6 ± 13.88 micrometers.

[0037] FTIR results showed that no characteristic absorption peaks were found in the carboxychitosan sample, indicating that its molecules were in a disordered state. In the lyophilized carboxychitosan hydrogel sample (Lyophilized -CH), several key absorption peaks were observed, specifically: the P-O and C-O stretching vibration peaks of sodium β-glycerophosphate (900-1200 cm⁻¹). -1 The peak of the C=O interaction between carboxylated chitosan molecules and sodium β-glycerophosphate (1500-1700 cm⁻¹). -1 ) and O-H group peaks (3300 - 3500 cm⁻¹) -1 ); The methyl group peak of carboxylated chitosan molecules (500-800 cm⁻¹) -1 and 2800-3000 cm -1 ); Molecular chain ordering enhancement peak (1300-1500 cm⁻¹) -1 These results indicate that during the gelation of the liquid CH-SS / CTGF mixture, numerous hydrogen bonds and electrostatic interactions formed between carboxychitosan and sodium β-glycerophosphate. These hydrogen bonds and electrostatic interactions constructed a complex cross-linked network, constituting the basic structure of the carboxychitosan-based biomaterial. Comparison of CH-SS / CTGF and SS / CTGF active substance samples revealed that although the active SS / CTGF substance did not exhibit obvious characteristic peaks, it significantly enhanced and broadened the characteristic peaks of carboxychitosan molecules in the CH-SS / CTGF sample. Figure 4 (D). This indicates that SS / CTGF enhances the hydrogen bonding and electrostatic interactions within the CH-SS / CTGF composite hydrogel without directly participating in hydrogel formation.

[0038] Example 4: Mechanical properties of CH-SS / CTGF composite hydrogel biomaterials Since the mechanical properties of CH-SS / CTGF biomaterials are closely related to their water content, the water content variation of the CH-SS / CTGF hydrogel biomaterial was first analyzed. The results showed that the water content of the hydrogel continuously decreased over 80 hours at 37℃, reaching a peak of 45% after 1 hour. Figure 5Subsequently, the mechanical properties of the CH-SS / CTGF hydrogel biomaterial were tested using the gravimetric crushing method. The results showed that the CH-SS / CTGF hydrogels with water contents of 45% and 30% completely fractured when subjected to a weight of 50 g and 100 g, respectively, while the hydrogel with a water content of 20% remained intact when subjected to a weight of 200 g. Figure 6 (A - B). Hardness analysis showed that the hydrogel with a water content of 20% had the highest hardness, followed by hydrogels with water contents of 30% and 45%. Figure 6 These results indicate a significant correlation between water content and the mechanical strength of CH-SS / CTGF hydrogel biomaterials. Furthermore, the stress strength, strain strength, and Young's modulus of the CH-SS / CTGF hydrogel with a water content of 45% were 0.07 ± 0.01 kPa, 83.26 ± 2.90%, and 0.17 ± 0.03 kPa, respectively. Figure 6 (D), indicating that this hydrogel biomaterial possesses extremely high flexibility. In summary, by adjusting the water content of CH-SS / CTGF biomaterials, biomaterials with different mechanical properties can be prepared to meet the needs of various medical applications.

[0039] Example 5: Stability of CH-SS / CTGF hydrogel biomaterial The stability assessment method for CH-SS / CTGF hydrogel biomaterials was slightly modified from the previously reported method. In short, 500 mg of hydrogel sample was immersed in 1.5 mL of PBS and placed at different temperatures (4℃, 37℃, 65℃), pH values ​​(4.0, 7.4, 11.0), and with or without the addition of 10 U / mL lysozyme. After several hours, the remaining hydrogel was dried and weighed (Wx). The residual rate was calculated using the formula: Residual rate (%) = 100 × (Wx / 100). Each time point was tested three times. The stability results showed that the degradation process of CH-SS / CTGF hydrogel could be divided into three stages: 40-70℃, 70-250℃, and 250-500℃. Figure 7 (A), corresponding to water release, dissociation of the hydrogen bond network between carboxychitosan and sodium β-glycerophosphate and sericin, and thermal decomposition of the CH-SS / CTGF hydrogel components, respectively. Stability tests showed that the CH-SS / CTGF hydrogel degraded within approximately 120 minutes under different temperatures (4℃, 37℃, 65℃) and pH values ​​(4.0, 7.4, 11.0). The degradation rate was faster at pH 4.0 and temperature 4℃. Figure 7 B - D Figure 8It is worth mentioning that the stability of the CH-SS / CTGF hydrogel is not affected by the presence of 10 U / mL lysozyme in PBS. Figure 7 In summary, CH-SS / CTGF hydrogel biomaterials exhibit relatively low stability and rapid dissolution in aqueous solutions.

[0040] Example 6: Release behavior of sericin and CTGF proteins from CH-SS / CTGF hydrogel biomaterials The method for analyzing the release behavior of sericin and CTGF from the hydrogel was as follows: 500 mg of CH-SS / CTGF composite hydrogel sample was placed in an experimental bottle, and then 1.5 mL of PBS buffer (pH 7.4) with or without 10 U / mL lysozyme was added to each bottle, and the sample was incubated at 37°C. Figure 9 (A). After a period of time, the supernatant was carefully collected and stored at -40°C. The total protein content, including sericin and CTGF, was determined using a Nano-300 microspectrophotometer (Shanghai Yongneng, China). The CTGF protein content was quantified by Western blotting using primary anti-CTGF antibody (86641T, Cell Signaling Technology, USA) and secondary anti-rabbit IgG antibody (A01827-200, Genscript, China). The results showed that the total protein content, including sericin and CTGF, increased sharply within 20 minutes after release from PBS buffer and remained relatively stable for the following 2 hours, although it may decrease slightly due to protein degradation. Figure 9 B). Western blot analysis showed that CTGF protein was effectively released from CH-SS / CTGF hydrogel regardless of the addition of 10 U / mL lysozyme, with the release mainly concentrated in the first 20 minutes and the last 30 minutes. Figure 9 The phased release of CTGF protein is related to the core-shell structure of the CH-SS / CTGF hydrogel. This is because the burst release of CTGF protein only occurs after the outer shell has completely dissolved, at which point the CTGF protein stored in the core is released in a single rapid pulse. Overall, 1.0 g of CH-SS / CTGF hydrogel biomaterial released a total of 3.76 ± 0.33 mg of total protein and 68.04 ± 5.48 ng of CTGF protein.

[0041] Example 7: Cell proliferation promoting activity of CH-SS / CTGF hydrogel biomaterial The ability of CH-SS / CTGF hydrogel to promote NIH / 3T3 cells was assessed by co-culturing the cells with DMEM containing 0.5% fetal bovine serum (FBS) for several days. The results showed that the CH-SS group had a higher number of NIH / 3T3 cells compared to the null control group. The CH-SS / CTGF group showed the highest cell counts observed after 1 and 3 days of co-culture. Figure 10 A). The LIVE / DEAD cell experiment results showed that the CH-SS / CTGF group had the highest number of live NIH / 3T3 cells with green fluorescence, followed by the CH-SS group and the blank group Null (…). Figure 10 B). AlamarBlue experimental results further confirmed that after 1 and 3 days of co-culture, the fluorescence signal intensity of the CH-SS / CTGF group was significantly higher than that of the CH-SS group, and the fluorescence signal intensity of the CH-SS group was higher than that of the blank group Null. Figure 10 The CCK-8 assay results showed that the viability of NIH / 3T3 cells in the CH-SS group was significantly higher than that in the blank group Null, and the viability of cells in the CH-SS / CTGF group was further increased after 1, 2 and 3 days of co-culture. Figure 10 These results all indicate that CH-SS / CTGF hydrogel biomaterials can effectively promote cell proliferation.

[0042] Furthermore, in vitro wound healing experiments showed that, compared with the untreated Null control group, CH-SS hydrogel significantly promoted the migration of newly generated cells to the scratch area after 24 hours; the wound closure effect of the CH-SS / CTGF group was superior to that of the CH-SS group. This indicates that CH-SS / CTGF hydrogel biomaterials have superior efficacy in wound healing.

[0043] Example 8: Biocompatibility of CH-SS / CTGF hydrogel biomaterial After co-culturing CH-SS / CTGF hydrogel with NIH / 3T3 and Raw264.7 cells for a period of time, their cytotoxicity and cytoinflammatory effects were assessed. The results showed that, similar to the control TCP group, NIH / 3T3 cells cultured on the hydrogel surface exhibited significant proliferation and growth on days 1, 3, 5, and 7. Figure 11 After 7 days of co-culture, almost all cells maintained normal morphology and emitted green fluorescence signals in the LIVE / DEAD cell staining experiment (A). Figure 11(BC), which means that the CH-SS / CTGF hydrogel has low toxicity. Raw264.7 cell experiments showed that the expression levels of inflammatory markers iNOS and COX-2 in the CH-SS / CTGF group were similar to those in the control TCP group, and significantly lower than those in the positive control LPS group after 2 days of co-culture (BC). Figure 11 (DF), indicating that CH-SS / CTGF hydrogel only elicits a mild inflammatory response. In conclusion, CH-SS / CTGF hydrogel biomaterials possess good biocompatibility.

[0044] Genetically engineered silk materials endowed with human cytokines show great potential in the preparation of functional biomaterials for future medical applications. To overcome the key challenge of cytokine inactivation during the processing of such silk materials, this study developed an "alkali extraction + acid protection" method to prepare an active SS / CTGF substance containing both CTGF cytokines and sericin from genetically engineered silk materials containing CTGF. Subsequently, using carboxylated chitosan as a matrix, a novel CH-SS / CTGF composite biomaterial with a core-shell structure was prepared. Results showed that compared to sericin alone, the SS / CTGF substance was more effective in promoting cell proliferation and growth. This core-shell structured CH-SS / CTGF biomaterial possesses adjustable mechanical properties, rapid release of active substances, enhanced cell proliferation activity, and good biocompatibility. These characteristics make it promising for applications in wound healing, drug delivery systems, and other regenerative medicine fields.

[0045] The above-described embodiments are merely preferred embodiments provided to fully illustrate the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are all within the scope of protection of the present invention. The scope of protection of the present invention is defined by the claims.

Claims

1. A SS / CTGF composite biomaterial based on carboxylated chitosan, characterized in that: The composite biomaterial is composed of sericin SS / CTGF containing exogenous connective tissue growth factor, carboxylated chitosan, and β-glycerophosphate. The carboxylated chitosan is dissolved in the SS / CTGF to obtain an SS / CTGF / carboxylated chitosan solution. The β-glycerophosphate is dissolved in water and then mixed with the SS / CTGF / carboxylated chitosan solution. The composite biomaterial is obtained by induction at a temperature above 37°C.

2. The SS / CTGF composite biomaterial based on carboxylated chitosan according to claim 1, characterized in that: The sericin SS / CTGF containing exogenous connective tissue growth factor was prepared by the following method: silkworm cocoons expressing biologically active human connective tissue growth factor were crushed, and the sericin was first dissolved in an alkaline aqueous solution with a pH of 13. The supernatant was collected by centrifugation to obtain a primary sericin extract, and then acid was added to neutralize it to obtain SS / CTGF.

3. The SS / CTGF composite biomaterial based on carboxyl chitosan according to claim 2, characterized in that: The alkaline aqueous solution is an aqueous solution of sodium hydroxide and an aqueous solution of sodium carbonate.

4. The SS / CTGF composite biomaterial based on carboxyl chitosan according to claim 2, characterized in that: The acid solution used for neutralization is hydrochloric acid solution.

5. The method for preparing the SS / CTGF composite biomaterial based on carboxylated chitosan according to any one of claims 1 to 4, characterized in that: Includes the following steps: (1) The silkworm cocoons expressing biologically active human connective tissue growth factor were crushed; (2) Immerse the powder obtained in step (1) in an alkaline aqueous solution and stir and extract for 1 to 48 hours at 40 to 80°C; centrifuge and take the supernatant to obtain primary sericin extract; add acid solution to the primary sericin extract for neutralization to obtain sericin SS / CTGF containing exogenous connective tissue growth factor. (3) Dissolve carboxy chitosan in the sericin containing exogenous connective tissue growth factor to obtain SS / CTGF / carboxy chitosan solution; then dissolve β-glycerophosphate in water and mix it with the SS / CTGF / carboxy chitosan solution, and induce the composite biomaterial at a temperature above 37°C.

6. The use of the SS / CTGF composite biomaterial based on carboxy-chitosan as described in any one of claims 1 to 4 in the preparation of biomedical materials that promote cell proliferation and / or wound healing.

7. The application according to claim 6, characterized in that, The biomedical materials are used to prepare drug delivery systems or tissue engineering scaffolds.

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