Silk fibroin glaze, preparation method and application thereof

Abstract

This invention provides a silk fibroin glaze with a core-shell structure in which soluble silk fibroin encapsulates insoluble silk fibroin. The silk fibroin glaze of this invention is prepared by hydrolyzing silkworm silk into soluble and insoluble forms of silk fibroin, followed by further mixing the two types of silk fibroin. Based on a homologous similarity mechanism, the soluble protein tightly adheres to and encapsulates the insoluble silk fibroin, resulting in a core-shell structured micron-sized silk fibroin composite material (silk fibroin glaze). The unique core-shell structure of this composite micron-sized silk fibroin material (silk fibroin glaze) gives it both the rigidity of the core and the flexibility of the shell. Its micron-sized dimensions make it particularly suitable for spreading and filling gaps / shaping, achieving malleability in a wet state and high-strength curing in a dry state. This provides an innovative biomaterial for dentinal tubule occlusion and hard tissue repair, with enormous application potential.

Description

Technical Field

[0001] This invention belongs to the field of hard tissue repair, specifically relating to a silk fibroin glaze, its preparation method, and its application. Background Technology

[0002] Hard tissue repair materials, as a key means of replacing, repairing or reconstructing the function of damaged hard tissues, have huge market demand and application prospects in fields such as orthopedics, dentistry and craniofacial surgery.

[0003] Due to significant limitations in bone grafting technology, synthetic materials are currently the primary means of hard tissue repair in clinical practice. However, existing materials in the field of hard tissue repair still face key challenges, including insufficient biocompatibility, susceptibility to infection, and the difficulty in balancing mechanical properties with osteogenic activity. Most commercially available materials currently cannot simultaneously achieve an integrated structure that combines rigid core support with a flexible outer shell, especially high-end composite materials that can simultaneously satisfy both biocompatibility and osteogenic induction functions. This is currently a gap in the domestic market for high-end repair materials.

[0004] As a natural high-molecular-weight protein extracted from silkworm silk, silk fibroin has shown great potential in the field of biomedical materials due to its excellent biocompatibility, controllable biodegradability, good mechanical properties, and ease of processing and modification. In particular, soluble regenerated silk fibroin solutions can be easily constructed into various forms (such as porous scaffolds, hydrogels, and films) through physical or chemical cross-linking methods, providing flexible solutions for different hard tissue repair scenarios. However, pure silk fibroin materials still face challenges when applied to hard tissue repair; their mechanical strength is still insufficient, and there is room for improvement in their biocompatibility and osteogenic induction function.

[0005] Therefore, there is an urgent need in this field to develop a high-end composite material that combines a rigid framework with a flexible interface, and can simultaneously meet the requirements of biosafety and osteogenic induction. Summary of the Invention

[0006] This invention aims to develop a high-end composite material that combines a rigid framework with a flexible interface, and can simultaneously meet the requirements of biocompatibility and osteogenic induction. Specifically, it relates to a silk fibroin glaze, its preparation method, and its application.

[0007] In a first aspect of the present invention, a silk fibroin glaze is provided, the silk fibroin glaze comprising:

[0008] (Z1) Soluble silk fibroin,

[0009] (Z2) Insoluble silk fibroin;

[0010] The soluble silk fibroin has a molecular weight of 1-100,000 Daltons, and the insoluble silk fibroin has a fiber length of 2-15 micrometers and a fiber diameter of 1-4 micrometers.

[0011] Furthermore, in the silk fibroin glaze, the mass ratio of soluble silk fibroin to insoluble silk fibroin is 1:9-9:1;

[0012] Furthermore, in the silk fibroin glaze, the soluble silk fibroin encapsulates the insoluble silk fibroin, thereby forming a core-shell structure.

[0013] In another preferred embodiment, the silk fibroin enamel has a length of 2-15 micrometers and a fiber diameter of 1-4 micrometers.

[0014] In another preferred embodiment, the mass ratio of soluble silk fibroin to insoluble silk fibroin in the silk fibroin glaze is 3:7 to 7:3.

[0015] In another preferred embodiment, the mass ratio of soluble silk fibroin to insoluble silk fibroin in the silk fibroin glaze is 4-6:6-4.

[0016] In another preferred embodiment, the infrared spectrum of the soluble silk fibroin is in the range of 1650-1655 cm⁻¹. -1 (Amide I) and 1540-1555cm -1 It has an absorption peak at (amide II); the insoluble silk fibroin has an absorption peak at 1615-1635 cm⁻¹. -1 (Amide I) and 1525-1540cm -1 (Amide II).

[0017] In a second aspect of the present invention, a method for preparing silk fibroin enamel as described in the first aspect is provided, the method comprising the steps of:

[0018] (1) Mix mulberry silk with sodium carbonate aqueous solution, boil, separate the silk and wash with water; repeat the above boiling and washing steps 2-4 times to obtain crude silk fibroin fiber.

[0019] (2) Mix the crude silk fibroin fiber obtained in step (1) with sodium carbonate aqueous solution, boil it, separate the silk and wash it with water. Repeat the above boiling and washing steps 1-3 times to perform the second elution procedure to obtain short silk fibroin fiber.

[0020] (3) Dry the short silk fibroin fibers obtained in step (2) to obtain insoluble silk fibroin;

[0021] (4) Add LiBr solution to the insoluble silk fibroin obtained in step (3), dissolve and then dialyze to obtain a silk fibroin solution, which is soluble silk fibroin;

[0022] (5) Take the soluble silk fibroin obtained in step (4) and the insoluble silk fibroin obtained in step (3), stir and mix them, and after fully combining and crystallizing, dry them to obtain the silk fibroin glaze as described in claim 1.

[0023] In another preferred embodiment, in step (1), the concentration of the sodium carbonate solution is 0.15-0.25 wt%; and / or

[0024] In step (2), the concentration of the first sodium carbonate solution is 0.05-0.15 wt%.

[0025] In another preferred embodiment, in step (1), the mass-to-volume ratio of the silkworm silk to the first sodium carbonate solution is 1:90-110; and / or

[0026] In step (2), the mass-to-volume ratio of the silkworm silk to the second sodium carbonate solution is 1:40-60.

[0027] In another preferred embodiment, in step (1), the elution time of the first elution procedure is 1-2 hours; and / or

[0028] In step (2), the elution time of the first elution procedure is 0.5-1.2h.

[0029] In another preferred embodiment, the first silk fibroin fiber has a fiber length of 12-15 micrometers; and / or

[0030] The second silk fibroin fiber has a fiber length of 2-10 micrometers.

[0031] In another preferred embodiment, the washing in step (1) includes the steps of: first washing with hot water at 50-70°C, then washing with room temperature water; and / or

[0032] The washing process described in step (2) includes the following steps: first washing with hot water at 50-70℃, and then washing with room temperature water.

[0033] In another preferred embodiment, the molar concentration of the LiBr solution in step (4) is 8.5-9.5 M.

[0034] In another preferred embodiment, in step (4), the mass-to-volume ratio of the insoluble silk fibroin to the LiBr solution is 1:3.5-4.5.

[0035] In another preferred embodiment, the dialysis time in step (4) is 40-55 hours.

[0036] In another preferred embodiment, the mass ratio of soluble silk fibroin to insoluble silk fibroin in step (5) is 90:10.

[0037] In another preferred embodiment, the mass ratio of soluble silk fibroin to insoluble silk fibroin in step (5) is 5:2-12.

[0038] In another preferred embodiment, the drying method described in step (5) is selected from the group consisting of: low-temperature powder spraying drying, room temperature vacuum drying, or freeze drying.

[0039] In a third aspect of the invention, a use is provided for silk fibroin glaze as described in the first aspect, for preparing hard tissue repair materials.

[0040] In a fourth aspect of the invention, a hard tissue repair material is provided, said material comprising silk fibroin glaze as described in the first aspect.

[0041] In another preferred embodiment, the hard tissue repair material is an oral hard tissue repair material.

[0042] In another preferred embodiment, the oral hard tissue repair material is selected from the group consisting of: dentinal tubule damage repair materials and caries biomimetic remineralization materials.

[0043] In a fifth aspect of the invention, a medical device is provided, wherein the medical device contains or is coated with silk fibroin glaze as described in the first aspect.

[0044] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0045] Figure 1 The infrared spectrum of soluble silk fibroin is shown.

[0046] Figure 2 The infrared spectrum of insoluble silk fibroin is shown.

[0047] Figure 3 The infrared spectrum of silk fibroin enamel is shown.

[0048] Figure 4 The image shows a scanning electron microscope image of insoluble silk fibroin fibers.

[0049] Figure 5 A scanning electron microscope image of silk fibroin enamel (E2) is shown.

[0050] Figure 6 A scanning electron microscope image of silk fibroin enamel (E3) is shown.

[0051] Figure 7 High-resolution laser confocal microscopy images of fluorescently labeled bioglaze (E2) are shown.

[0052] Figure 8 This image shows a low-magnification, large-field fluorescence confocal microscopy image of fluorescently labeled bioglaze (E2).

[0053] Figure 9 High-resolution transmission electron microscopy images of bioglaze (E2) labeled with metal nanoparticles are shown.

[0054] Figure 10 The image shows a scanning electron microscope image of a blank dentinal tubule.

[0055] Figure 11 The image shows a scanning electron microscope image of a damaged tooth radiograph repaired with silk fibroin enamel.

[0056] Figure 12 Scanning electron micrographs showing the repair effects of different repair materials are displayed. Detailed Implementation

[0057] Through extensive and in-depth research and numerous experimental screenings, the inventors have unexpectedly developed a silk fibroin glaze for the first time. This glaze possesses a core-shell structure in which soluble silk fibroin encapsulates insoluble silk fibroin. The unique core-shell structure of this composite silk fibroin micron-material (silk fibroin glaze) gives it both the rigidity of the core and the flexibility of the shell. Its micron-sized dimensions make it particularly suitable for spreading and filling gaps / shaping, achieving malleability in a wet state and high-strength curing in a dry state. Furthermore, this core-shell micron-type composite protein material (silk fibroin glaze) exhibits excellent biocompatibility, stability, and adhesion, making it a high-end restorative material for damaged hard tissue repair. It provides an innovative biomaterial for dentinal tubule occlusion and hard tissue repair, filling a gap in the domestic high-end restorative materials field and possessing strong industrial competitiveness in the global market. Based on this, the inventors completed this invention.

[0058] Terminology Explanation

[0059] The silk fibroin enamel of the present invention

[0060] In this invention, the terms "silk fibroin glaze", "bio-glaze", and "composite silk fibroin micron material" can be used interchangeably, all referring to a core-shell structured composite material in which soluble silk fibroin encapsulates insoluble silk fibroin using the preparation process of this invention.

[0061] The composite silk fibroin micron material of this invention has a special core-shell structure, which gives it both the rigidity of the core and the flexibility of the shell. In addition, its micron-sized dimensions make it particularly suitable for spreading and filling gaps / shaping. For example, it can be directly applied to seal dental tubules and repair hard tissues such as bones. Its working principle is similar to industrial reinforced concrete, with both the skeleton of steel bars (core protein) and cement (soluble protein) that acts as a binder. It can be spread and shaped in a wet state and can maintain its shape after drying, making it strong and glossy.

[0062] The core-shell micron composite protein material of this invention has good biocompatibility, stability and adhesion. It can be used as a high-end repair material for the repair of damaged hard tissues. It can not only fill the gap in the domestic high-end repair material category, but also promote related products to the global market, and has a very good industrial prospect.

[0063] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0064] 1. The composite silk fibroin micron material of the present invention has a special core-shell structure, which gives it both the rigidity of the core and the flexibility of the shell. In addition, its micron-sized size makes it particularly suitable for spreading and filling gaps / shaping, achieving the characteristics of being malleable in a wet state and highly strong curing in a dry state, providing an innovative biomaterial solution for dentinal tubule occlusion and hard tissue repair.

[0065] 2. The core-shell micron composite protein material of the present invention has excellent biocompatibility, stability and adhesion, and is a high-end repair material for the repair of damaged hard tissues. It not only fills the gap in the field of high-end repair materials in China, but also has strong industrial competitiveness to enter the global market.

[0066] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise stated.

[0067] Example 1: Preparation of bio-glaze (core-shell structured micron-sized silk fibroin composite material)

[0068] In this embodiment, silk is first hydrolyzed using different processes to obtain soluble and insoluble silk fibroin. Then, the two types of silk fibroin are further mixed and prepared, with the soluble protein tightly adhering to and encapsulating the insoluble silk fibroin to form a core-shell structured micron-sized silk fibroin composite material. The specific preparation process is shown below:

[0069] (1) Silk digestion and first degumming: Weigh a certain amount of shredded mulberry silk, add it to a 0.2% sodium carbonate solution and boil it at a bath ratio of 1:100. After half an hour, take out the silk and wash it with hot water at 60℃, then wash it with room temperature water. Repeat the above steps three times, for a total of 1.5 hours of washing, to remove as much sericin as possible and obtain longer fibers, mostly 12-15 micrometers in length, the first silk fibroin fiber;

[0070] (2) Second degumming: The first silk fibroin fiber washed in step (1) is added to a sodium carbonate solution with a concentration of 0.1% and boiled at a bath ratio of 1:50. After half an hour, the silk is taken out and washed with hot water at 60°C and then with room temperature water. The above steps are repeated twice for a total of 1 hour to remove as much sericin as possible and obtain the second silk fibroin fiber with shorter fibers and a length of 2-10 micrometers.

[0071] (3) Place the second silk fibroin fiber obtained in step (2) into a 40°C oven and dry it until its mass remains unchanged to obtain insoluble silk fibroin, the infrared spectrum of which is shown below. Figure 2 As shown, its scanning electron microscope image is as follows: Figure 4 As shown;

[0072] (4) Take an appropriate amount of the insoluble silk fibroin obtained in step (3) above, add it to 9.0M LiBr solution, bath ratio 1:4, dissolve in hot water at 60℃ for 4 hours, allow to cool naturally, then transfer to a dialysis bag and dialyze for 48 hours, changing the water every 4 hours until the insoluble silk fibroin dissolves, obtaining a silk fibroin solution, which is the soluble silk fibroin. Its infrared spectrum is shown below. Figure 1 As shown;

[0073] (5) Take the soluble silk fibroin obtained in step (4) and the insoluble silk fibroin obtained in step (3) in different proportions, and stir at room temperature for more than 2 hours to allow the soluble and insoluble silk fibroin to fully combine and crystallize. Then, perform low-temperature spray drying to obtain a micron-sized silk fibroin composite material with a core-shell structure, namely bio-glaze (silk fibroin glaze) E1, whose infrared spectrum is as follows. Figure 3 As shown.

[0074] In addition, bio-glazes can also be prepared by fully combining and crystallizing two types of silk fibroin, followed by vacuum drying at room temperature or freeze drying.

[0075] Depend on Figure 1 As can be seen, the characteristic absorption peak of the soluble silk fibroin prepared by this invention is 1651.64 cm⁻¹. -1 and 1542.06cm -1 This characteristic peak is located at the main characteristic absorption peak of soluble silk fibroin at 1650-1655 cm⁻¹. -1 Amide I and 1540-1555cm-1 Within the range of amide II.

[0076] Depend on Figure 2 As can be seen, the characteristic absorption peak of the insoluble silk fibroin prepared by this invention is 1632.15 cm⁻¹. -1 and 1518.45cm -1 This characteristic peak is located at the main characteristic absorption peak of insoluble silk fibroin, 1615-1635 cm⁻¹. -1 Amide I, 1525-1540cm -1 Within the range of amide II.

[0077] Depend on Figure 3 As can be seen, the infrared characteristic peaks of the core-shell structured micron-sized silk fibroin composite material prepared by the invention are basically located near the positions of characteristic amide I and amide II, and the peak width has become wider, which also confirms that the composite silk fibroin glaze of the present invention is a composite material formed by two structures: soluble silk fibroin and insoluble silk fibroin.

[0078] Example 2: Preparation of bio-glaze (core-shell structured micron-sized silk fibroin composite material)

[0079] In this embodiment, soluble silk fibroin and insoluble silk fibroin are mixed in different proportions to prepare bio-glazes. The preparation method is the same as in Example 1, wherein the mass ratios of soluble silk fibroin and insoluble silk fibroin described in step (5) are 2:8, 3:7, 5:5, and 7:3, respectively, to obtain bio-glazes (silk fibroin glazes) E1, E2, E3, and E4.

[0080] Example 3: Characterization of the bio-glaze (silk fibroin glaze) prepared in this invention

[0081] 3.1 Scanning electron microscopy data: Characterizing material morphology

[0082] The bio-glazes (silk fibroin glazes) E2 and E3 prepared in this invention were characterized by scanning electron microscopy, and the results are as follows: Figure 5 , Figure 6 As shown.

[0083] As shown in the figure, the silk fibroin glaze prepared by this invention has a length of 2-15 μm. The material has uniform size and regular morphology. Its particles are highly monodisperse, with smooth surface and clear outline. There is no obvious agglomeration, which shows excellent dispersibility and flowability.

[0084] 3.2 Laser confocal microscopy data

[0085] The core-shell structure of the bio-glaze (silk fibroin glaze) prepared in this invention was confirmed by fluorescent labeling, and the results are as follows: Figure 7 , Figure 8As shown.

[0086] exist Figure 7 The images show high-resolution laser confocal microscopy images of fluorescently labeled bioglazes. Insoluble proteins are labeled with FITC green fluorescence, and soluble proteins are labeled with Cy5 red fluorescence. In Figure a, Transmission mode is used, where areas where red and green light overlap appear orange or yellow. In Figure b, Scan mode is used, with a scale bar of 2 micrometers. As can be seen from the figures, this data clearly shows that soluble proteins (red) are almost uniformly deposited on the surface of insoluble proteins (green), forming a well-defined core-shell structure.

[0087] exist Figure 8 The images show low-magnification, large-field fluorescence confocal microscopy images of fluorescently labeled bioglazes. Insoluble proteins are labeled with FITC green fluorescence, and soluble proteins are labeled with Cy5 red fluorescence. Image a shows the green fluorescence channel, image b shows the red fluorescence channel, and image c shows the overlay mode. Scale bar: 20 μm. The figures demonstrate that the yield of the core-shell structure, where soluble proteins (red) encapsulate insoluble proteins (green), is very high. This indicates that the basic composition and structure of bioglazes is a core-shell structure formed by soluble proteins encapsulating insoluble proteins.

[0088] 3.3 High-resolution transmission electron microscopy data

[0089] The core-shell structure and other fine structures of the bio-glaze (silk fibroin glaze) prepared in this invention were confirmed by metal labeling. Figure 9 As shown.

[0090] exist Figure 9In the image, high-resolution transmission electron microscopy (HRTEM) images of bioglaze labeled with metal nanoparticles are shown. Insoluble proteins are labeled with 8 nm gold (Au) nanoparticles, while soluble proteins are labeled with 3–5 nm CdTe cadmium telluride quantum dots. Image a shows the HRTEM transmission mode, revealing that the surface of the rod-shaped proteins is not smooth; grooves easily lead to the aggregation of labeled nanoparticles. The metal nanoparticles show lower contrast (darker) in the HRTEM mode, as indicated by the red dashed box, demonstrating successful labeling of the proteins. Image b shows the STEM high-angle scattering dark-field mode, where high atomic number metal elements show whiter contrast than the C, H, O, and N elements that constitute the proteins, as indicated by the green dashed box, further demonstrating successful labeling of the proteins by heavy metal elements. The labeling is also reflected in the C-map surface scan energy dispersive spectroscopy; d, e, and f are the distribution images of Au (representing the insoluble protein part), Cd, and Te (representing the soluble protein) in the sample under the surface scan mapping mode, respectively. g is the overlapping image of d, e, and f. The high overlap of the distribution of these elements indicates that the soluble protein is basically uniformly and tightly wrapped and deposited on the surface of the insoluble protein. Since the insoluble protein is not a porous structure, the accumulation of a large number of labeled nanoparticles in the grooves on the surface also indicates that it is a surface wrapping mode, not internal penetration, forming a core-shell structure.

[0091] Example 4: Application Performance Test: Effective Sealing of Damaged Dental Tubules

[0092] Experimental methods:

[0093] (1) Demineralize 5mm*5mm bovine tooth fragments to expose the dentinal tubules.

[0094] Soak the dental slide in a 40% phosphoric acid (H3PO4) solution for 10 minutes with shaking; rinse with deionized water, then soak in a 5% sodium hypochlorite solution for 5 minutes with shaking; rinse with deionized water, change the water, and sonicate for 20 minutes for later use.

[0095] (2) Dental tubule occlusion test

[0096] Take the bio-glazes (silk fibroin glazes) E1, E2, E3, and E4 prepared in different proportions according to this invention, prepare 100 ml of 10% sample solution, stir at 500 rpm, half of the dental slide is attached with sealant tape, and stirred for 1 hour before removal. Weigh 15 g of deionized water, rinse for 10 seconds, and send to a scanning electron microscope. The results are as follows. Figure 10 , Figure 11 As shown.

[0097] like Figure 10 As shown in the blank dentinal tubule scanning electron microscope image, the rough morphology of the dentinal tubule surface with small pores has an open diameter of about 5 micrometers. Theoretically, spherical or rod-shaped structures with particle sizes around this size have a good gap-filling effect.

[0098] The results of repairing damaged dental radiographs using various samples in this invention are as follows: Figure 11 As shown in the figure, where a is sample E1, b is sample E2, c is sample E3, and d is sample E4. From the figure, we can see that:

[0099] a) Soluble:Insoluble protein = 2:8 (E1) If the proportion of soluble protein is too low, the composite protein material will not adhere well to the dental radiograph and the filling effect will be poor.

[0100] b) Soluble: Insoluble protein = 3:7 (E2). The increased proportion of soluble protein causes the insoluble protein to clump together and adhere to the dental radiograph, which has a restorative effect. However, some aggregation occurs, and the spreading effect of the composite protein material on the dental radiograph does not achieve the most ideal restorative effect.

[0101] c) Soluble: Insoluble protein = 5:5 (E3). The ratio of soluble protein is just right so that the insoluble protein can be linked together and spread like a film. The rod-shaped particles of insoluble protein that do not clump together fit the pores of the exposed dentinal tubules well in terms of size and morphology, resulting in the best restoration effect.

[0102] d) Soluble:Insoluble protein = 7:3 (E4) The resulting composite protein material has a certain gap-filling effect and a repair effect, but the proportion of insoluble protein that plays a role is low, and it is only scattered on the dental radiograph in the form of single particles, so the repair effect is limited.

[0103] As the above analysis shows, the bio-enamel (silk fibroin enamel) prepared by this invention has a filling and sealing repair effect on damaged dentinal tubules. In particular, the silk fibroin enamel prepared with a mass ratio of soluble silk fibroin to insoluble silk fibroin of 5:2-12 exhibits better repair effects on damaged dentinal tubules, especially the silk fibroin enamel with a mass ratio of 5:5, which shows the best repair effect. Therefore, the ratio of the two proteins (soluble silk fibroin and insoluble silk fibroin) forming the silk fibroin enamel mainly affects the following repair properties of the enamel:

[0104] 1) Adhesion (adhesion) performance: If the proportion of soluble protein is too low, the adhesion will be poor.

[0105] 2) Spreading performance: Only a suitable proportion of soluble protein can make insoluble protein evenly dispersed without clumping, thus playing a role in smoothing, spreading and filling gaps.

[0106] 3) Repair strength: Insoluble protein is the actual interstitial material, and its relative concentration, morphology and size ultimately determine the strength and quality of the repair surface.

[0107] In summary, this invention prepares a composite silk fibroin micromaterial (silk fibroin) with a special core-shell structure by mixing soluble and insoluble silk fibroin in a specific ratio. This gives the material the rigidity of the core and the flexibility of the shell. Furthermore, its micron-sized dimensions make it particularly suitable for spreading and filling gaps / shaping, resulting in excellent restorative effects for dentinal tubule occlusion and hard tissue repair. This provides an innovative biomaterial for the field of high-end restorative materials in China.

[0108] Comparative experiments on the repair effects of soluble and / or insoluble silk fibroin combined with other rigid materials

[0109] In this embodiment, a composite material for hard tissue repair is prepared by combining common hydroxyapatite as a rigid material with soluble and / or insoluble silk fibroin, as shown in Table 1:

[0110] Table 1 Preparation of different remedial materials

[0111]

[0112]

[0113] like Figure 12 As shown, conventional restorative materials (C1-C3) all have significant limitations in the repair of damaged dentinal tubules: hydroxyapatite, silica, and regenerated silica materials cannot effectively fit the dentinal tubule pores, resulting in problems such as particle scattering, poor adhesion, or no visible changes before and after restoration; although the various composites of silk fibroin and hydroxyapatite have a certain degree of adhesion and play a certain role in filling gaps, they still fail to achieve morphological fit with the pores, and the problem of particle dispersion still exists. In contrast, the present invention uses a protein enamel material formed by a 5:5 mass ratio of soluble and insoluble silk fibroin, which exhibits superior restorative performance—the soluble protein acts as a bonding matrix, enabling the insoluble protein to form a continuous film structure that is evenly spread on the damaged surface. Its rod-shaped particles precisely match the dentinal tubule pores in size and morphology, achieving a tight fit and thus obtaining the best restorative effect.

[0114] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A silk fibroin glaze, characterized in that, The silk fibroin enamel comprises: (Z1) Soluble silk fibroin, (Z2) Insoluble silk fibroin; The soluble silk fibroin has a molecular weight of 1-100,000 Daltons, and the insoluble silk fibroin has a fiber length of 2-15 micrometers and a fiber diameter of 1-4 micrometers. Furthermore, in the silk fibroin glaze, the mass ratio of soluble silk fibroin to insoluble silk fibroin is 1:9-9:1; Furthermore, in the silk fibroin glaze, the soluble silk fibroin encapsulates the insoluble silk fibroin, thereby forming a core-shell structure.

2. The silk fibroin enamel as described in claim 1, characterized in that, The infrared spectrum of the soluble fibroin protein has absorption peaks at 1650-1655 cm -1 (amide I) and 1540-1555 cm -1 (amide II); the insoluble fibroin protein has absorption peaks at 1615-1635 cm -1 (amide I) and 1525-1540 cm -1 (amide II).

3. The method for preparing silk fibroin enamel as described in claim 1, characterized in that, The method includes the following steps: (1) Mix mulberry silk with sodium carbonate aqueous solution, boil, separate the silk and wash with water; repeat the above boiling and washing steps 2-4 times to obtain crude silk fibroin fiber. (2) Mix the crude silk fibroin fiber obtained in step (1) with sodium carbonate aqueous solution, boil it, separate the silk and wash it with water. Repeat the above boiling and washing steps 1-3 times to perform the second elution procedure to obtain short silk fibroin fiber. (3) Dry the short silk fibroin fibers obtained in step (2) to obtain insoluble silk fibroin; (4) Add LiBr solution to the insoluble silk fibroin obtained in step (3), dissolve and then dialyze to obtain a silk fibroin solution, which is soluble silk fibroin; (5) Take the soluble silk fibroin obtained in step (4) and the insoluble silk fibroin obtained in step (3), stir and mix them, and after fully combining and crystallizing, dry them to obtain the silk fibroin glaze as described in claim 1.

4. The method as described in claim 3, characterized in that, In step (1), the concentration of the sodium carbonate solution is 0.15-0.25 wt%; and / or In step (2), the concentration of the first sodium carbonate solution is 0.05-0.15 wt%.

5. The method as described in claim 3, characterized in that, In step (1), the mass-to-volume ratio of the silkworm silk to the first sodium carbonate solution is 1:90-110; and / or In step (2), the mass-to-volume ratio of the silkworm silk to the second sodium carbonate solution is 1:40-60.

6. The method as described in claim 3, characterized in that, In step (1), the elution time of the first elution procedure is 1-2 hours; and / or In step (2), the elution time of the first elution procedure is 0.5-1.2h.

7. The method as described in claim 3, characterized in that, The first silk fibroin fiber has a fiber length of 12-15 micrometers; and / or The second silk fibroin fiber has a fiber length of 2-10 micrometers.

8. The method as described in claim 3, characterized in that, The washing process described in step (1) includes the following steps: first washing with hot water at 50-70℃, then washing with room temperature water; and / or The washing process described in step (2) includes the following steps: first washing with hot water at 50-70℃, and then washing with room temperature water.

9. The method as described in claim 3, characterized in that, The molar concentration of the LiBr solution mentioned in step (4) is 8.5-9.5M.

10. The use of a silk fibroin enamel as described in claim 1, characterized in that, Used to prepare materials for hard tissue repair.

11. A hard tissue repair material, characterized in that, The material described includes silk fibroin glaze as described in any one of claims 1-2.

12. A medical device, characterized in that, The medical device contains or is coated with silk fibroin glaze as described in any one of claims 1-2.

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