Method for preparing mineralized protein films and applications thereof

Abstract

The present application relates to the field of biomaterials, in particular to a mineralized protein membrane and a preparation method and application thereof. The present application provides a mineralized protein membrane, comprising: a protein membrane and a mineral; the mineral comprises: a hydroxyapatite mineral. The mineralized protein membrane provided by the present application comprises an arthropod resilin hydrogel mineralization template and a hydroxyapatite mineral formed in situ by mineralization; the mineralized protein membrane has excellent mechanical properties, including a higher Young's modulus, breaking strength and flexibility. The mineralized protein membrane in the present application has biocompatibility and biological activity, can effectively isolate soft tissue cells, and can also effectively promote the repair and regeneration of bone defect sites.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomaterials, in particular to a mineralized protein membrane and a preparation method and application thereof. BACKGROUND

[0002] Guided bone regeneration (GBR) surgery is a classic method for treating bone injury diseases, which requires the use of a GBR membrane to prevent soft tissue from growing into the bone defect area. A GBR membrane with excellent barrier effect is the key to the success of bone injury disease treatment. According to clinical needs, the GBR membrane should first act as a barrier to cells, which can prevent the passage of soft tissue-related cells. In addition, the use of a GBR membrane with biological functionality can improve the speed of bone tissue regeneration, thereby shortening the treatment period. GBR membranes can be divided into two categories: non-absorbable and absorbable. Both have been widely used in clinical practice. Non-absorbable GBR membranes can provide a better barrier for bone regeneration and do not have the problem of mechanical performance degradation, but they need to be removed by surgery later, which can cause secondary damage. In contrast, absorbable GBR membranes have degradability, but their barrier ability gradually decreases, which affects the treatment effect. Therefore, there is an urgent need to develop new GBR membranes to solve the problem of balancing degradability and functionality.

[0003] Hydrogels, especially those based on biological macromolecules, have great potential in a variety of biomedical applications due to their degradability and tunable mechanical properties. However, in terms of mechanical properties, synthetic hydrogels still have a large gap compared to many natural hydrogels, such as cartilage and skin, which can maintain high strength, stiffness, and toughness under conditions of more than 50% water content. Although there have been many important breakthroughs in the field of synthetic hydrogels in recent years, especially double-network hydrogels that can even rival the toughness of natural skin. However, the toughness of synthetic hydrogels often comes from high elongation, and the stiffness is still poor, making it difficult to provide effective support in applications. In the context of GBR membrane use, support can provide space for bone regeneration, which is beneficial to improving the speed of bone regeneration. In order to better meet the application requirements of GBR, the stiffness of hydrogel materials needs to be improved. Biomimetic mineralization is an effective method to improve the stiffness of materials, but mineralization can easily produce the brittleness of materials. In order to prepare hydrogel materials with high toughness and stiffness, the strategy of enzymatic mineralization is used to realize the in-situ growth of amorphous mineralization in the hydrogel matrix, obtaining mineralized hydrogel materials with combined stiffness and toughness, and the inorganic mineralization also endows the material with biological activity. However, the biocompatibility problem of chemically synthesized materials limits further biomedical applications, so developing an enzymatic mineralization method based on protein hydrogels is expected to realize the preparation of new GBR membranes with excellent mechanical properties, biocompatibility, and biological activity. SUMMARY

[0004] In view of the above, the present application provides a mineralized protein membrane and a preparation method and application thereof.

[0005] In order to achieve the above-mentioned application purposes, the present application provides the following technical solutions.

[0006] The present application provides a mineralized protein membrane, comprising: a protein membrane and a mineral; the mineral comprises: a hydroxyapatite mineral.

[0007] In some embodiments of the present application, the protein membrane in the above-mentioned mineralized protein membrane comprises: elastin and functionalized or non-functionalized polyethylene glycol.

[0008] In some embodiments of the present application, the mass ratio of the elastin and the functionalized or non-functionalized polyethylene glycol in the above-mentioned mineralized protein membrane is 4:3.

[0009] In some embodiments of the present application, the elastin in the above-mentioned mineralized protein membrane has:

[0010] (1) an amino acid sequence as shown in SEQ ID NO: 1; or

[0011] (2) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence as shown in (1), and an amino acid sequence which is functionally identical or similar to the amino acid sequence as shown in (1); or

[0012] (3) an amino acid sequence which is at least 80% identical to the amino acid sequence as shown in (1) or (2).

[0013] In some embodiments of the application, the sequence of SEQ ID NO: 1 is: MGQGVGSGGRPSDSYGAPGGGNPVPGKGVPGKGVPGKGVPGKGVPGKGVPGVGSGGRPSDSYGAPGGGNPVPGKGVPGKGVPGKGVPGKGVPGKGVPGVGSGGRPSDSYGAPGGGNPVPGKGVPGKGVPGKGVPGKGVPGKGVPGVGSGGRPSDSYGAPGGGNPVPGKGVPGKGVPGKGVPGKGVPGKGVPGVGSGGRPSDSYGAPGGGNPVPGKGVPGKGVPGKGVPGKGVPGKGVPGVGSGGRPSDSYGAPGGGNPVPGKGVPGKGVPGKGVPGKGVPGKGVPGVGSGGRPSDSYGAPGGGNPVPGKGVPGKGVPGKGVPGKGVPGKGVPGVGSGGRPSDSYGAPGGGNPVPGKGVPGKGVPGKGVPGKGVPGKGVPGVGSGGRPSDSYGAPGGGNPVPGKGVPGKGVPGKGVPGKGVPGKGVPGVGSGGRPSDSYGAPGGGNPVPGKGVPGKGVPGKGVPGKGVPGKGVPGVGSGGRPSDSYGAPGGGNPVPGKGVPGKGVPGKGVPGKGVPGKGVPGVGSGGRPSDSYGAPGGGNPVPGKGVPGKGVPGKGVPGKGVPGKGVPGVGSGGRPSDSYGAPGGGNPVPGKGVPGKGVPGKGVPGKGVPGKGVPGVGSGGRPSDSYGAPGGGNPVPGKGVPGKGVPGKGVPGKGVPGKGVPGVGSGGRPSDSYGAPGGGNPVPGKGVPGKGVPGKGVPGKGVPGKGVPGVGSGGRPSDSYGAPGGGNPVPGKGVPGKGVPGKGVPGKGVPGKGVPGVGSGGRPSDSYGAPGGGNPVPGKGVPGKGVPGKGVPGKGVPGKGVPGVGSGGRPSDSYGAPGGGNPVPGKGVPGKGVPGKGVPGKGVPGKGVPGVGSGGRPSDSYGAPGGGNPVPGKGVPGKGVPGKGVPGKGVPGKGVPGVGSGGRPSDSYGAPGGGNPVPGKGVPGKGVPGKGVPGKGVPGKGVPGVGSGGRPSDSYGAPGGGNPVPGKGVPGKGVPGKGVPGKGVPGKGVPGWHHHHHH.

[0014] In some embodiments of the application, the resilin in the mineralized protein film described above comprises: arthropod resilin and positively charged resilin-like proteins.

[0015] In some embodiments of the application, the arthropod resilin in the mineralized protein film described above has an amino acid sequence as set forth in SEQ ID NO: 2: VGSGGRPSDSYGAPGGGNP.

[0016] In some embodiments of the present application, the positively charged elastin-like polypeptide in the mineralized protein film has an amino acid sequence as shown in SEQ ID NO: 3: VPGKGVPGKGVPGKGVPGKGVPGKG.

[0017] In some embodiments of the present application, the functionalization in the mineralized protein film comprises dialdehyde functionalization.

[0018] The present application also provides a preparation method of the mineralized protein film, wherein the mineral is in-situ mineralized in the protein film.

[0019] In some embodiments of the present application, the preparation method comprises the following steps:

[0020] S1: mixing the elastin, the functionalized or non-functionalized polyethylene glycol and alkaline phosphatase, and first cross-linking;

[0021] S2: second cross-linking the product after the first cross-linking, to obtain the protein film;

[0022] S3: mixing the protein film with a mineralization raw material, to obtain the mineralized protein film.

[0023] In some embodiments of the present application, the mineralization raw material in the preparation method comprises calcium glycerophosphate.

[0024] In some embodiments of the present application, the mineralization raw material in the preparation method is added in the form of a solution; the concentration of the solution of the mineralization raw material is 11 g / L.

[0025] In some embodiments of the present application, the solution of the mineralization raw material is obtained by mixing the mineralization raw material with a triethanolamine buffer.

[0026] In some embodiments of the present application, the mixing time in S3 of the preparation method is 6-24 h.

[0027] In some embodiments of the present application, the mixing time in S3 of the preparation method is 6 h, 12 h or 24 h.

[0028] In some embodiments of the present application, the elastin in the preparation method is added in the form of a solution, and the final concentration of the solution of the elastin is 20-200 mg / mL.

[0029] In some embodiments of the present application, the final concentration of the solution of the elastin in the preparation method is 100 mg / mL.

[0030] In some embodiments of the present application, the functionalized or non-functionalized polyethylene glycol in the above preparation method is added in the form of a solution, and the final concentration of the solution of the functionalized or non-functionalized polyethylene glycol is 50-300 mg / mL.

[0031] In some embodiments of the present application, the final concentration of the solution of the functionalized or non-functionalized polyethylene glycol in the above preparation method is 200 mg / mL.

[0032] In some embodiments of the present application, the first cross-linking in the above preparation method comprises: imine bond cross-linking; and the second cross-linking comprises: photo-cross-linking.

[0033] In some embodiments of the present application, the temperature of the first cross-linking in the above preparation method is 37℃, and the time is 2 h.

[0034] In some embodiments of the present application, the time of the second cross-linking in the above preparation method is 3 min, and the light intensity is 3.8 mW / cm 2 .

[0035] In some embodiments of the present application, the photo-cross-linking in the above preparation method comprises the steps of adding a photo-initiator and an oxidizing agent.

[0036] The photo-initiator comprises: trispyridine ruthenium; and the oxidizing agent comprises: a persulfate ion.

[0037] In some embodiments of the present application, the oxidizing agent in the above preparation method comprises: ammonium persulfate or potassium persulfate.

[0038] The present application also provides the use of the above mineralized protein membrane and / or the mineralized protein membrane obtained by the above preparation method in the preparation of a product for bone repair.

[0039] The present application also provides a product comprising: the above mineralized protein membrane and / or the mineralized protein membrane obtained by the above preparation method.

[0040] The present application provides a mineralized protein membrane for guiding bone regeneration, which is specifically composed of an arthropod resilin hydrogel mineralization template and a calcium phosphate mineralization formed in situ by mineralization, wherein the protein hydrogel is obtained by two-step chemical cross-linking of arthropod resilin, and the amino acid sequence of the arthropod resilin is MGQG[VGSGGRPSDSYGAPGGGNP(VPGKG)5VPG] 16WH6; the mineralized protein membrane can be used for promoting the repair and regeneration of bone defect sites. The mineralized protein membrane product described in the present application has excellent mechanical properties, including a higher Young's modulus, breaking strength and flexibility, and also has biocompatibility and bioactivity, can effectively isolate soft tissue cells, and can be used for promoting the repair and regeneration of bone defect sites. In addition, the preparation process of the present application is simple, does not require special equipment, has low cost, is green and environmentally friendly, and is conducive to the development and promotion of next-generation tissue barrier membranes and bone repair materials. BRIEF DESCRIPTION OF DRAWINGS

[0041] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or prior art description will be briefly introduced below.

[0042] Figure 1 SDS-PAGE gel electrophoresis chart of arthropod resilin prepared in Example 1;

[0043] Figure 2 Photos and scanning electron microscope charts of mineralized protein membranes prepared in different mineralization times in Example 1, 2 and 3; wherein: a shows the change chart of the appearance of the protein hydrogel with the increase of the mineralization time; b shows the change chart of the apparent mechanical properties of the mineralized hydrogel with the increase of the mineralization time; c shows the scanning electron microscope chart of the internal microstructure of the protein hydrogel with the increase of the mineralization time;

[0044] Figure 3 Dark field scanning transmission electron fiber microscope chart and corresponding element distribution analysis chart of the mineralized protein membrane prepared in Example 3 and mineralized for 24 h;

[0045] Figure 4 Transmission electron microscope characterization and selected area electron diffraction chart of the ultrathin section of the mineralized protein membrane prepared in Example 3 and mineralized for 24 h; wherein: a shows the transmission electron microscope chart of the ultrathin section sample of the mineralized protein hydrogel at low magnification; b shows the transmission electron microscope chart of the ultrathin section sample of the mineralized protein hydrogel at high magnification; c shows the high-resolution transmission electron microscope chart of the ultrathin section sample of the mineralized protein hydrogel and the corresponding lattice calibration; d shows the selected area electron diffraction chart of the ultrathin section sample of the mineralized protein hydrogel;

[0046] Figure 5 Mechanical property chart of the mineralized protein membrane prepared in different mineralization times in Example 1, 2 and 3; wherein: a shows the comparison chart of the typical stress-strain curves of the unmineralized and different mineralization time protein hydrogels; b shows the breaking strength statistical chart of the unmineralized and different mineralization time protein hydrogels; c shows the toughness statistical chart of the unmineralized and different mineralization time protein hydrogels; d shows the modulus statistical chart of the unmineralized and different mineralization time protein hydrogels;

[0047] Figure 6Figure showing the cell compatibility of mineralized protein films prepared in Examples 2 and 3 with different mineralization times;

[0048] Figure 7 Figure showing the guided bone defect repair regeneration of mineralized protein films prepared in Examples 2 and 3 with different mineralization times. DETAILED DESCRIPTION

[0049] The present application discloses a mineralized protein film and a preparation method and application thereof.

[0050] It should be understood that the expression "one or more of something" includes each of the objects recited after the expression separately as well as various combinations of two or more of the recited objects, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited objects should be understood to have the same meaning, unless otherwise understood from the context.

[0051] The terms "comprising", "having" or "including", including the use of their grammatical synonyms, should generally be understood to be open-ended and non-limiting, for example, not excluding other non-recited elements or steps, unless otherwise specifically stated or understood from the context.

[0052] It should be understood that the order of steps or the order of performing certain actions is not important, as long as the application remains operable. Furthermore, two or more steps or actions can be performed simultaneously.

[0053] The use of any and all examples, or exemplary language herein, for example, "such as" or "including", is intended merely to better illustrate the application and does not indicate a limitation on the scope of the application, unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the application.

[0054] Further, the numerical ranges and parameters setting forth the broadest scope of the application are approximations, and the numerical values set forth in the detailed description are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. Therefore, unless otherwise specified, all ranges disclosed herein are to be understood to be approximations, and the numerical values underlying any such ranges are intended to be precise. Herein, "about" or "approximately" means within 10%, 5%, 1%, or 0.5% of a stated value or range.

[0055] The present application provides a mineralized protein film which can be used to guide bone tissue repair and regeneration, comprising an arthropod resilin hydrogel mineralization template and hydroxyapatite mineralization formed in situ; the mineralized protein film has excellent biocompatibility and bioactivity.

[0056] The mineralized protein film provided by the embodiment of the present application has excellent mechanical properties, including high Young's modulus, breaking strength and flexibility, can effectively isolate soft tissue cells, and has good biocompatibility and bioactivity. In addition, the mineralized protein film provided by the present application can be used for biomedical applications and can promote the repair and regeneration of bone defect sites.

[0057] The arthropod resilin hydrogel in the embodiment of the present application plays the role of a mineralization template, which is a new mineralization template on a macro scale. Specifically, the mineralization template is a protein hydrogel network formed by cross-linking reaction of arthropod resilin and dialdehyde functionalized polyethylene glycol, and further secondary cross-linking of protein tyrosine radical reaction under visible light excitation. The macro scale referred to in the embodiment of the present application refers to what can be seen by the naked eye, compared to nanoscale mineralization.

[0058] In the embodiment of the present application, the mineralized protein film provided for promoting the repair and regeneration of bone defect sites is prepared by mineralizing the arthropod resilin hydrogel template; the mineralized template is a protein hydrogel obtained by secondary cross-linking of arthropod resilin and dialdehyde functionalized polyethylene glycol through tyrosine radical reaction under visible light excitation. That is, the mineralization template in the embodiment of the present application is composed of arthropod resilin hydrogel, and the main components are arthropod resilin and dialdehyde functionalized polyethylene glycol.

[0059] The mineralized protein film in the embodiment of the present application is composed of arthropod resilin hydrogel and hydroxyapatite formed in situ by mineralization, that is, hydroxyapatite can grow in the interior of the mineralization template. Hydroxyapatite, also known as hydroxyapatite, is a natural mineralization of calcium phosphate (Ca5(PO4)3(OH)), which is often written in the form of (Ca 10 (PO4)6(OH)2).

[0060] In the embodiment of the present application, the mineralization process of the arthropod resilin hydrogel is carried out under the guidance of alkaline phosphatase (purchased from Sigma-Aldrich Company). Specifically, during the preparation of the protein hydrogel, alkaline phosphatase is mixed with the arthropod resilin solution, and dialdehyde functionalized polyethylene glycol can also react with the lysine residues of alkaline phosphatase, so that alkaline phosphatase is covalently fixed in the protein hydrogel network. Alkaline phosphatase can hydrolyze calcium glycerophosphate mineralization solution to produce free phosphate and further form hydroxyapatite mineral with calcium ions in the solution, thereby completing the mineralization process of the protein hydrogel.

[0061] The arthropod resilin in the application is a recombinant protein obtained by fusing arthropod resilin and positively charged resilin-like protein expressed by a biosynthesis method, the amino acid sequence of the arthropod resilin block is VGSGGRPSDSYGAPGGGNP, the amino acid sequence of the positively charged resilin-like protein block is (VPGKG)5, and the host cell is an engineered Escherichia coli; a plasmid is constructed according to the amino acid sequence of the protein, and then the plasmid is introduced into the Escherichia coli for protein expression; and the amino acid sequence of the chimeric protein is MGQG[VGSGGRPSDSYGAPGGGNP(VPGKG)5VPG]. 16 WH6.

[0062] The application provides a preparation method of a mineralized protein film as described above, comprising the following steps:

[0063] S1, expressing a recombinant protein of arthropod resilin fused with positively charged resilin-like protein by an Escherichia coli host cell, referred to as arthropod resilin for short.

[0064] S2, dissolving the synthesized arthropod resilin and alkaline phosphatase in water, mixing with a dialdehyde functionalized polyethylene glycol aqueous solution, and placing to form an imine bond, which is the first step of cross-linking of the protein hydrogel.

[0065] S3, adding a ruthenium trispyridine aqueous solution to the mixed solution, and irradiating the mixed solution with visible light in the presence of a persulfate ion, to realize the second step of cross-linking of the protein hydrogel, and the mixed solution becomes gel-like.

[0066] S4, placing the cross-linked protein hydrogel overnight to fully cross-link, to form a protein film; and then placing the protein film in a glycerol calcium phosphate aqueous solution (triethanolamine as a buffer), to finally realize mineralization of the protein film.

[0067] Preferably, the concentration of the arthropod resilin solution is 20-200 mg / mL.

[0068] Preferably, the concentration of the dialdehyde functionalized polyethylene glycol aqueous solution is 50-300 mg / mL.

[0069] Preferably, the mass ratio of the arthropod resilin to the dialdehyde functionalized polyethylene glycol is 1:2-2:1, and the more preferable ratio is 4:3.

[0070] Preferably, the dosages of the alkaline phosphatase and ammonium persulfate are 0.1 mg and 0.5 mg (corresponding to 20 mg of arthropod resilin) respectively.

[0071] Preferably, the first step of cross-linking is performed at 37 ℃ for 2 h.

[0072] Preferably, the photo-crosslinking time is 3 min (the light intensity is about 3.8 mW / cm 2 ].

[0073] Preferably, the triethanolamine aqueous solution has a concentration of 0.2 M and a pH of 9.8.

[0074] Preferably, the mineralization solution has a concentration of 11 g / L (40% pure water and 60% triethylamine buffer).

[0075] Preferably, the mineralization time of the protein film is 6-24 h.

[0076] In the examples 1-3 and the effect examples of the present application, the raw materials and reagents used can be purchased from the market.

[0077] The present application will be further described below in combination with examples:

[0078] Example 1

[0079] (1) Prokaryotic cell expression of arthropod resilin. The amino acid sequence of the arthropod resilin expressed by recombination in the present application is MGQG[VGSGGRPSDSYGAPGGGNP(VPGKG)5VPG] 16WH6, the pET25b expression vector containing the sequence of the arthropod resilin gene was first transformed into E. coli BL21 (DE3) competent cells, and then further propagated and expressed. Specifically, 3 μL of the pET25b expression plasmid was added to the BL21 competent cells in an ice bath, and incubated for 30 min to allow the plasmid to approach the competent cells. Then, the expression vector was transformed into the BL21 competent cells by heat shock at 42°C for 90 s, and the ice bath was continued for 2 min to complete the plasmid transformation. After 400 μL of sterile LB medium (10 g / L of proteose peptone, 5 g / L of yeast powder, and 10 g / L of sodium chloride) was added to the transformed BL21 competent cells, the cells were cultured for about 45 min, followed by solid medium plating, and overnight culture in a 37°C incubator. A single colony on the solid medium was selected and picked into 20 mL of LB medium using a sterile gun tip, and cultured in a shaker for about 12 h (37°C, 220 rpm) to prepare a glycerol bacterial solution, which was stored in a -20°C refrigerator. Next, the protein expression was performed. First, a seed bacterial solution was prepared. 1 mL of the frozen glycerol bacteria and 200 μL of ampicillin solution (100 mg / mL) were added to 200 mL of LB medium, and cultured in a shaker for about 6 h until the OD value of the bacterial solution reached 2. The seed bacterial solution was added to a 4 L large flask for further culture (37°C, 220 rpm), and the large flask contained 1 L of sterile TB medium (12 g / L of proteose peptone, 24 g / L of yeast powder, 2.32 g / L of potassium dihydrogen phosphate, 12.54 g / L of dipotassium hydrogen phosphate, and 4 mL / L of glycerol) and 1 mL of ampicillin solution. After about 4 h of culture, the OD value of the bacterial solution was about 0.8, and then the protein induction expression was performed using an IPTG solution (1 M), and the culture temperature was changed to 28.5°C. The protein expression process was completed after overnight culture for about 12 h, and finally the bacterial cells were collected using a centrifuge (8000 rpm, 15 min) and stored in a -80°C refrigerator.

[0080] (2) Purification of arthropod resilin. Ni-chromatography column and ion exchange column were used for purification, and molecular sieve desalting column was used for desalting. First, the bacteria after expressing the protein above were resuspended in lysis solution (sodium chloride 500 mM, trisodium phosphate 50 mM, imidazole 20 mM, pH 7.4), and the final concentration of the bacterial solution was 100 g / L. DNAse and lysozyme were added and stirred for 1 h. Then the bacteria were broken by high-pressure crusher, and the E. coli fragments were removed by high-speed refrigerated centrifuge (14000 rpm, 30 min). The supernatant was retained for further purification. First, the target protein was purified by Ni affinity chromatography column and purification instrument, wherein the loading buffer was a mixed solution of sodium chloride 500 mM, trisodium phosphate 50 mM, imidazole 20 mM, pH 7.4, and the elution buffer was a mixed solution of sodium chloride 500 mM, trisodium phosphate 50 mM, imidazole 350 mM, pH 7.4. Gradient elution was used to obtain the preliminary purified target protein solution, which was further dialyzed and purified by cation exchange column. In the ion exchange process, the loading buffer was a mixed solution of sodium chloride 50 mM, trisodium phosphate 50 mM, pH 7.4, and the elution buffer was a mixed solution of sodium chloride 5 M, trisodium phosphate 50 mM, pH 7.4. Gradient elution was also used to obtain the target protein solution, and the excess salt in the protein solution was removed by desalting column. Finally, the protein sample was obtained by freeze-drying, and was stored in a refrigerator at -80°C.

[0081] (3) Preparation of protein hydrogel. Arthropod resilin hydrogel was obtained by two-step crosslinking. First, Schiff base reaction between dialdehyde functionalized polyethylene glycol and lysine residues in arthropod resilin occurred, and then radical reaction between tyrosine residues in arthropod resilin was initiated by light. Specifically, 20 mg of arthropod resilin was configured into a 100 mg / mL aqueous solution, and 15 mg of dialdehyde polyethylene glycol was configured into a 200 mg / mL aqueous solution. Then the arthropod resilin and polyethylene glycol solutions were mixed uniformly, and 0.1 mg of alkaline phosphatase (ALP) and 0.5 mg of ammonium persulfate were also added to the mixed solution. The mixed solution was placed in a 30°C oven for 2 h, and the preliminary pre-crosslinking of the protein and dialdehyde polyethylene glycol was completed. The viscosity of the solution increased compared with the initial state. Then 5 μL of tris (2-pyridyl) ruthenium (5 mg / mL) aqueous solution was added to the pre-crosslinking solution and mixed uniformly, and was poured into a polytetrafluoroethylene mold. Finally, white light flashlight was used for light crosslinking (light intensity was 0.5 mW / cm 2 ), and the sample was irradiated for 3 min according to the crosslinking degree, and the protein hydrogel was formed after light irradiation. The final crosslinking reaction was completed after overnight storage in the dark.

[0082] (4) Enzymatic mineralization of protein hydrogel. The mineralization of arthropod resilin hydrogel was performed under the induction of alkaline phosphatase. The mineralization buffer solution used in the present application was a triethanolamine aqueous solution (0.2 M, pH 9.8) for stabilizing the pH of the mineralization solution. Next, 11 g of glycerophosphate calcium was dissolved in 400 mL of pure water and 600 mL of the mineralization buffer solution, and after being fully stirred, the solution was filtered through a 0.4 μm filter membrane to remove insoluble substances in the mineralization solution, thereby completing the preparation of the mineralization solution. The pre-prepared arthropod resilin hydrogel was directly placed in the prepared mineralization solution for mineralization. A 30 mg sample of the hydrogel was used with 50 mL of the mineralization solution, and the mineralization time was 6 h.

[0083] Example 2

[0084] The same method as in Example 1 was used, except that the mineralization time of the protein hydrogel was changed to 12 h.

[0085] Evaluation of the bone regeneration effect of the mineralized protein membrane. In order to evaluate the barrier effect of the mineralized protein hydrogel in vivo, we used a rat skull defect model to compare the healing rates of animals treated with different barrier materials, including a blank control group, a protein hydrogel group, and a mineralized protein hydrogel group. Specifically, 6-week-old male SD rats were used for the experiment, and a skull drill was used to create two 5 mm diameter defect models in the skull area of each rat. All barrier materials were cut to a size of about 7 mm in diameter and covered on the skull defect site, and finally all rats were sutured and observed.

[0086] Example 3

[0087] The same method as in Example 1 was used, except that the mineralization time of the protein hydrogel was changed to 24 h.

[0088] Effect Example

[0089] Figure 1 SDS-PAGE gel electrophoresis analysis of arthropod resilin prepared in Example 1 of the present application Figure 1 a) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry Figure 1 b) results. In the gel electrophoresis diagram, there is a single band between the protein standards of 70-100 kDa, and no obvious impurity protein bands appear, indicating that the target protein after purification has a high purity Figure 1 a). The mass spectrometry results show that the molecular weight of the biosynthetic arthropod resilin is about 67 kDa.

[0090] Figure 2 Photos of mineralized protein membranes prepared in Examples 1, 2, and 3 of the present application Figure 2a and b) and scanning electron microscope images Figure 2 c). The unmineralized protein hydrogel is transparent and has flexible mechanical properties in the form of thin film, and the hydrogel has a very smooth and smooth micro-surface morphology after natural drying. The protein hydrogel changes from the initial transparent state to white opaque after 6 h of mineralization, indicating that inorganic mineralization rapidly forms inside the protein hydrogel under the induction of alkaline phosphatase, and the appearance of the protein hydrogel does not change further with the increase of mineralization time Figure 2 a). The stiffness of the protein hydrogel also changes significantly before and after mineralization. The protein hydrogel film before mineralization is very soft and has no self-supporting property, and the stiffness of the protein hydrogel gradually increases with the increase of mineralization time, and exhibits self-supporting property after 12 h of mineralization; when the mineralization time increases to 24 h, the stiffness of the protein hydrogel continues to increase, but still maintains very excellent flexibility, which can realize arbitrary bending and has no damage to the appearance of the material Figure 2 b). At the same time, we also use scanning electron microscopy to characterize the changes in micro-morphology during the mineralization of the protein hydrogel. As shown in Figure 2 c, after 6 h of mineralization, obvious mineral accumulation appears inside the protein hydrogel, and after 12 h of mineralization, inorganic matter gradually grows, but there are still a large number of pores for discontinuous mineralization. When the mineralization time continues to increase to 24 h, continuous mineralization structure is gradually formed in the protein hydrogel, and there is no large size inorganic matter Figure 2 c).

[0091] Figure 3 The dark field scanning transmission electron microscope image and the corresponding element distribution analysis diagram of the mineralized protein film prepared in Example 3 are shown. First, the protein hydrogel after 24 h of mineralization and drying is embedded with resin and ultra-thin sectioned, and then the element distribution of the ultra-thin sectioned sample is analyzed by transmission electron microscopy combined with X-ray energy spectrum. As shown in Figure 3 , in the sample area with obvious mineral formation, Ca and P elements have a very high proportion, and the outlines of the two elements are basically consistent with the mineralization area, which shows that the inorganic matter formed in situ mineralization is mainly calcium phosphate.

[0092] Figure 4 The transmission electron microscope characterization of the ultra-thin section of the mineralized protein film prepared in Example 3 and the selected electron diffraction pattern are shown. From the transmission electron microscope images of different magnifications, it can be seen that the inorganic mineral is in the form of small size flake, and has very high packing density and certain short-range order Figure 4 a). The inorganic mineral unit of the mineralized hydrogel is about 20 nm long and has a large aspect ratio Figure 4b). The formation of small-sized inorganic matter during mineralization is due, on the one hand, to the presence of triethanolamine in the mineralization solution, which can form complexes with the mineralization precursors, thus effectively controlling the growth process of the minerals. On the other hand, the double cross-linked network of the protein hydrogel may exert a certain confinement effect on the minerals, preventing the fusion growth between minerals. The presence of lattice fringes can be faintly observed using high-resolution transmission electron microscopy, indicating that the calcium phosphate formed by in-situ mineralization has certain crystalline characteristics, but its crystallinity is poor. Figure 4 c). Furthermore, two distinct diffraction rings can be observed in the selected area electron diffraction pattern of the mineralized protein hydrogel ultrathin section sample. Combined with lattice size measurements using high-resolution transmission electron microscopy, it can be determined that the formed inorganic mineral is poorly crystallized hydroxyapatite (c). Figure 4 d). The study of the fine microstructure of mineralized protein hydrogels further explains why protein hydrogels can maintain good flexibility even with high mineral content.

[0093] Figure 5 The figures show the mechanical properties of mineralized protein films prepared for different mineralization times in Examples 1, 2, and 3. Typical stress-strain curves of unmineralized protein hydrogels and protein hydrogels with different mineralization times are shown below. Figure 5 As shown in Figure a, the mechanical strength of the protein hydrogel gradually increases with increasing mineralization time, while the corresponding elongation at break gradually decreases. However, the highly mineralized protein hydrogel can still maintain an elongation at break of over 15%. Subsequently, we statistically analyzed and calculated the mechanical strength of the protein hydrogel (based on three sets of parallel mechanical test results). Specifically, the fracture strength of the unmineralized protein hydrogel was 154.2 ± 2.6 kPa, and the toughness was 84.0 kJ / m. 3 The modulus is only about 0.35 MPa. Figure 5 (bd). Hydrogel materials, due to their high water content, typically exhibit very soft mechanical properties, resulting in generally low modulus and difficulty in providing strong support. As the protein hydrogel undergoes in-situ mineralization, inorganic matter significantly fills the matrix, gradually increasing its mechanical properties. After 6 hours of mineralization, the hydrogel's fracture strength was 671.0 ± 33.6 kPa, its modulus increased to 2.0 ± 0.3 MPa, and its toughness also slightly improved to approximately 121 kJ / m. 3 ( Figure 5 (bd). Comparison reveals that short-term mineralization can increase the modulus of protein hydrogels by more than 5 times. With continued mineralization, the mechanical strength of the protein hydrogel further improves; after 12 h and 24 h of mineralization, the fracture strength increases to 1.8 ± 0.3 MPa and 3.2 ± 0.2 MPa, respectively. The fracture strength of the highly mineralized protein hydrogel is superior to most previously reported hydrogel materials. Figure 5b). More importantly, the modulus of the protein hydrogel increased to 43.5 ± 8.3 MPa and 145.3 ± 44.5 MPa after 12 h and 24 h mineralization, respectively, which is a high modulus characteristic that is difficult to achieve for traditional synthetic high polymer hydrogel materials Figure 5 d). At the same time, the toughness of the protein hydrogel also gradually increased with the increase of mineralization time, and the toughness of the protein hydrogel increased to about 367 kJ / m 3 and 457 kJ / m 3 ( Figure 5 c).

[0094] Figure 6 Figure 2 shows the cell compatibility of the mineralized protein films prepared in Examples 2 and 3. To evaluate the cell compatibility of the materials, we used the live / dead cell staining assay to test the effects of cell viability and proliferation of the mineralized 12 h protein film and the mineralized 24 h protein film. Fluorescence microscopy observation showed that no dead cells were observed in the mineralized hydrogel group, and there was no significant difference compared with the blank control group and the polytetrafluoroethylene film group, which indicated that these mineralized hydrogels had good cell compatibility. The specific experimental steps are as follows: L929 fibroblasts were incubated with polytetrafluoroethylene film, unmineralized protein film, mineralized 12 h protein film and mineralized 24 h protein film extract at 37°C, 5% CO2 for 24 hours. After incubation, 1 µL of Calcein-AM dye (1 mg / mL aqueous solution) and 1 µL of PI dye (1 mg / mL DMSO solution) were added to the cell culture medium, and then incubated at 37°C for 15 minutes. After replacing the supernatant with fresh DMEM, the images were observed using a focused microscope at 488 nm and 561 nm excitation wavelengths.

[0095] Figure 7To evaluate the effect of mineralized protein membranes with different mineralization time on guided bone regeneration, we used the rat calvarial critical-size bone defect model. Specifically, we used male SD rats weighing 200-250 g (6-7 weeks old) for in vivo studies. To construct the calvarial critical-size bone defect model, we exposed the rat skull and used a stainless steel skull drill to construct bilateral full-thickness circular defects (5 mm in diameter). Subsequently, polytetrafluoroethylene membranes, collagen membranes, non-mineralized protein membranes, mineralized 12 h protein membranes, and mineralized 24 h protein membranes were implanted into the bone defect area, while the blank control group was not implanted with any material after the defect was created. The skin incision was closed using 4-0 suture. At 12 weeks after surgery, the rats were sacrificed, and their skulls were dissected and fixed in a 4% paraformaldehyde solution for 48 hours. To further evaluate the in vivo osteogenesis effect of each group, we used micro-CT to compare and analyze the osteogenesis area. The results of the analysis showed that the mineralized 12 h protein membrane had the largest area of new bone, followed by the mineralized 24 h protein membrane and the collagen membrane, and the non-mineralized protein membrane also showed some osteogenic effect, while the polytetrafluoroethylene membrane and the blank control group had the least amount of new bone. By comparison, it can be proved that the mineralized 12 h protein membrane has excellent effect on promoting bone regeneration, and the effect is better than the current market polytetrafluoroethylene membrane and collagen membrane products.

[0096] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, several improvements and refinements can be made, and these improvements and refinements should also be considered within the scope of protection of the present application.

Claims

1. A mineralized protein film characterized in that, The application relates to a mineralized protein membrane and a preparation method thereof. The mineralized template of the arthropod resilin hydrogel and the hydroxyapatite mineralization product formed in situ; The mineralized template of the arthropod resilin hydrogel is formed by cross-linking reaction of recombinant arthropod resilin and dialdehyde functional polyethylene glycol, and further secondary cross-linking of the protein hydrogel network formed by radical reaction of tyrosine of the protein under visible light excitation; The recombinant arthropod resilin is obtained by biosynthesis and fusion expression of arthropod resilin and positive electric elastin; The amino acid sequence of the recombinant resilin is MGQG[VGSGGRPSDSYGAPGGGNP(VPGKG)5VPG] 16 WH6.

2. The method for preparing the mineralized protein membrane as described in claim 1, characterized in that, The mineralized protein membrane is obtained by in-situ mineralization of the mineralization product in the protein membrane.

3. The production method according to claim 2, wherein The application further provides a preparation method of the mineralized protein membrane. S1: mixing the recombinant arthropod resilin, the dialdehyde functional polyethylene glycol and alkaline phosphatase, and first cross-linking; S2: second cross-linking of the product after the first cross-linking to obtain the protein membrane; S3: mixing the protein membrane with mineralization raw materials to obtain the mineralized protein membrane.

4. The production method according to claim 3, wherein The mineralization raw materials include glycerol calcium phosphate.

5. The production method according to claim 4, wherein The mixing time in S3 is 6-24 hours.

6. The mineralized protein membrane of claim 1 and / or the mineralized protein membrane obtained by the preparation method of any one of claims 2-5 is applied to preparation of a bone repair product.

7. A bone repair product characterised in that, The application further provides a mineralized protein membrane. The mineralized protein membrane of claim 1 and / or the mineralized protein membrane obtained by the preparation method of any one of claims 2-5.

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