Controllable injectable silk fibroin extracellular matrix gel material and preparation method thereof

Abstract

The application discloses a preparation method of controllable silk fibroin extracellular matrix gel material for injection. The composite filler is composed of raw materials in the following mass ratio: 5-50 parts of silk fibroin, 0.01-5 parts of cellulose, and 0.7-10 parts of hyaluronate. The preparation method comprises the following steps: (1) preparation of silk fibroin microfiber gel: adding silk fibroin aqueous solution into cellulose to prepare a gel, crushing, and sieving to obtain silk fibroin microfiber gel; (2) preparation of extracellular matrix gel material: mixing hyaluronate with the silk fibroin microfiber gel, physically cross-linking the mixture, and forming silk fibroin extracellular matrix gel material. The modified silk fibroin hydrogel prepared by the method can promote in-situ tissue regeneration after injection, the molecular weight and cross-linking degree of the modified silk fibroin hydrogel are controllable, the structure of the modified silk fibroin hydrogel is similar to the structure of extracellular matrix (ECM), the degradation process of the modified silk fibroin hydrogel is matched with the in-situ tissue regeneration process, the mechanical property, degradation property and biocompatibility of the modified silk fibroin hydrogel are improved, no chemical cross-linking agent is used, and the modified silk fibroin hydrogel is injectable, so the modified silk fibroin hydrogel has a good application prospect in the field of medical and beauty injection.

Description

Technical Field

[0001] This invention relates to medical cosmetic filler materials, and particularly to a controllable injectable extracellular matrix gel material and its preparation method. Background Technology

[0002] Tissue regeneration can be achieved through various methods, such as intracellular / extracellular polymerization, which involves synthesizing functional polymers in situ on living cells, or implanting scaffolds prepared in vitro, seeded with cells, and cultured before in vivo regeneration. However, these methods have significant limitations. Intracellular / extracellular polymerization is a relatively new and immature field, and the in situ synthesis of functional polymers requires complex techniques. Furthermore, in vitro tissue engineering requires complex cell culture conditions to obtain functional tissues, and autocrine and paracrine signaling effects are difficult to reproduce in vitro.

[0003] In situ tissue engineering is an effective tissue engineering regeneration method that combines the tissue's natural regenerative capacity with engineered biomaterials. In this process, biomaterials provide a structural framework to promote the attachment and migration of host stem cells and progenitor cells, and to drive these cells to differentiate into specific cell types, thereby achieving the purpose of repair or regeneration.

[0004] The ultimate goal of tissue regeneration is to achieve cell migration and proliferation through specific methods, remodeling the extracellular matrix (ECM) to influence cell function, such as reorganizing the cytoskeleton, activating integrins, and promoting normal gene expression. The ECM possesses a unique spatial organization; distinctive cell and ECM arrangements can be observed in directional tissues such as muscle and nerves. In situ tissue engineering guides cell regeneration by designing scaffolds with near-ECM structures, thereby creating artificial ECM morphologies. This enables guided differentiation, cell encapsulation, in situ regeneration and protection, ECM remodeling, and tissue repair.

[0005] In in situ tissue regeneration, the scaffold provided by biomaterials should facilitate cell and tissue penetration after implantation. Subsequently, the scaffold should be degraded and completely reabsorbed. A key point in this process is that the scaffold needs to undergo controlled degradation to guide in situ tissue regeneration. This process requires balancing the degradation rate of the scaffold with the regeneration rate of the tissue to achieve structural and functional regeneration.

[0006] In-situ tissue-engineered biomaterials have a wide range of raw material sources. These materials require biological signals to respond and interact with the immune system and endogenous cells to stimulate regeneration. Simultaneously, the design process necessitates precise control over the structure and biological properties to guide endogenous cells to the site of injury. This technology opens unprecedented avenues for customizing the properties of biomacromolecules, achieving precise spatial control, and seamlessly integrating materials into biological entities.

[0007] Regenerated silk fibroin is highly valued for its superior mechanical properties, excellent biocompatibility, biodegradability, and multifunctional structure, and is widely used in the fields of biomedicine and tissue engineering. It possesses a unique protein spatial structure; the formation of specific β-sheet structures within regenerated silk fibroin can create nanoscale microfibers that overlap and aggregate to form a flocculent network structure. In the process of tissue regeneration, this property can be utilized to prepare scaffolds that mimic the structure of the endocrine microstructure (ECM), thereby achieving cell proliferation, regeneration, and ECM remodeling.

[0008] In injectable tissue fillers, implanting tissue-engineered scaffolds to promote in-situ tissue regeneration is an effective repair method. Products using this method are rare on the market. Common products are purely physical fillers, such as hyaluronic acid, which have no regenerative effect and are prone to water absorption and swelling. Another type is microsphere products, such as poly-L-lactic acid and polycaprolactone, but their loose structure makes them easily decomposed by the tissue environment and proteases, making it difficult to maintain long-term filling effects. These products differ greatly from the microstructure of human skin itself. In contrast, regenerated silk fibroin microfibers, due to their relatively dense and stable molecular structure and suitable degradation cycle, are excellent tissue regeneration filler materials. If scaffolds are prepared using this material as a base, and the scaffolds are structurally designed to simulate the ECM (extracellular matrix), tissue regeneration can be stimulated more effectively.

[0009] In existing inventions, silk fibroin and hyaluronic acid are cross-linked using chemical cross-linking agents such as BDDE to form a composite gel material with a three-dimensional interpenetrating network of silk fibroin and hyaluronic acid. However, the degree of cross-linking is uncontrollable, and the use of chemical cross-linking agents has certain cytotoxicity. After residual accumulation in the body, it can easily cause risks such as redness, swelling, allergies, and cancer.

[0010] Therefore, finding a silk fibroin extracellular matrix gel material with controllable cross-linking degree and molecular weight, whose degradation process matches the in-situ tissue regeneration process, and which is similar to the microstructure of human skin, while also possessing good biocompatibility, water absorption, good mechanical properties, and injectability, is a key research direction for silk fibroin tissue engineering materials. Different skin tissues have different tensions, requiring different rheological properties of the filler material. The injectable silk fibroin extracellular matrix gel material described in this invention, through different process adjustments, matches different skin microstructures and the regeneration process at different skin locations, achieving controllable in-situ tissue regeneration.

[0011] The injectable silk fibroin extracellular matrix gel material of this invention achieves wrinkle removal and lifting effects through physical occupancy in the early stage after injection into the skin, without the phenomenon of water absorption and volume expansion. In the later stage, under the extracellular matrix structure of the gel, the gel material can promote in-situ tissue regeneration, which is manifested by the increase in the area ratio of collagen in pathological sections. Furthermore, by adjusting the molecular weight and ratio of the components, the degradation rate of the gel material in vivo can be controlled, which is reflected in the retention rate of the gel in vivo. Summary of the Invention

[0012] To address the aforementioned issues, this invention aims to provide a silk fibroin extracellular skeleton gel material with controllable crosslinking degree and molecular weight, whose degradation process matches the in-situ tissue regeneration process, and which is similar to the microstructure of human skin. It also possesses good biocompatibility, water absorption, good mechanical properties, and injectability, and requires the absence of chemical crosslinking agents to reduce corresponding residues.

[0013] The technical solution of this invention is as follows:

[0014] An injectable silk fibroin extracellular matrix gel with controllable crosslinking degree and molecular weight, and a degradation process that matches the in-situ tissue regeneration process, can be a controllable injectable extracellular matrix gel material.

[0015] The gel comprises 5-50 parts silk fibroin, 0.01-5 parts cellulose, and 0.7-10 parts hyaluronic acid. The gel is prepared according to the following steps:

[0016] (1) Preparation of silk fibroin microfiber gel: A gel was prepared by adding cellulose to an aqueous solution of silk fibroin, crushing and sieving to obtain silk fibroin microfiber gel;

[0017] (2) Preparation of extracellular matrix gel material: Hyaluronic acid salts are mixed with silk fibroin microfiber gel to physically cross-link them and form silk fibroin extracellular matrix gel material.

[0018] The gel material does not contain any chemical cross-linking agents.

[0019] The cellulose is one or more of hydroxypropyl methylcellulose, hydroxypropyl cellulose, carboxymethyl cellulose, and hydroxyethyl cellulose, preferably hydroxypropyl methylcellulose. The hyaluronic acid salt is one or more of sodium hyaluronate, potassium hyaluronate, and magnesium hyaluronate in any proportion, preferably sodium hyaluronate.

[0020] Furthermore, the preparation method of the silk fibroin composite filler of the present invention is as follows: (1) Silk fibroin microfiber gel: Silkworm silk is placed in a boiling sodium carbonate aqueous solution or a mixed solution of sodium carbonate and sodium bicarbonate with a mass fraction of 0.1-5%, heated to boiling for 10-120 minutes, dried at 25-100℃, dissolved in a 9.3mol / L lithium bromide aqueous solution, and bathed in a water bath at 25-100℃ for 10-120 minutes until the silk fibroin is fully dissolved. Insoluble particulate impurities are filtered out using a 50-200 mesh filter bag, and after desalting and concentration, a silk fibroin aqueous solution with a specific molecular weight is obtained. A 20% silk fibroin solution is added to a cellulose solution and then ultrasonically treated with an ultrasonic crusher. After gelation, homogenize for 5 min at 1500-24000 rpm / min, sieve, and collect silk fibroin microfiber gel that passes through 200-325 mesh sieve; (2) Preparation of extracellular skeleton gel: Hyaluronic acid particles are mixed with silk fibroin microfiber, and allowed to stand at 25-60℃ for 2-8 h to allow physical cross-linking. Dialyze with dialysate, fill, sterilize, and form silk fibroin extracellular skeleton gel material.

[0021] Furthermore, by controlling the static setting process and temperature, the degree of β-sheet and the number of hydrogen bonds can be controlled to regulate the degree of gel crosslinking. Alkoxy groups promote intermolecular hydrophobic interactions, while the hydrogen bonds between the hydroxypropyl groups in hydroxypropyl methylcellulose and water molecules delay gel formation. Under the hydrophobic interactions of cellulose, the β-sheet structure is evenly distributed, and the crosslinking density is higher, with a crosslinking pattern similar to that of the extracellular skeleton.

[0022] Furthermore, the extracellular scaffold gel is composed of the following raw materials in the following mass ratio: 5-50 parts silk fibroin, 0.01-5 parts cellulose, and 0.7-10 parts hyaluronic acid. Preferably, it consists of 10-50 parts silk fibroin, 0.01-5 parts cellulose, and 0.7-5 parts hyaluronic acid.

[0023] Further optimization of silk fibroin: hyaluronic acid ratio greater than or equal to 2:1, 3:1 or 4:1 or higher.

[0024] In addition, the preferred ratio of silk fibroin to hyaluronic acid is ≥1.5:1, or ≤1.618:1, or 1.5≤silk fibroin / hyaluronic acid≤1.618.

[0025] Furthermore, in step (1), the molecular weight of the silk fibroin is between 50 kDa and 350 kDa.

[0026] Furthermore, the molecular weight of the cross-linked hyaluronic acid particles in step (2) is between 500 kDa and 2400 kDa, preferably between 1500 kDa and 2400 kDa.

[0027] Furthermore, after mixing the silk fibroin microfiber gel and hyaluronic acid particles in step (2), it is preferable to let it stand at 40-55°C.

[0028] Further, after mixing the silk fibroin microfiber gel and hyaluronic acid particles in step (2), it is preferably left to stand for 4-6 hours.

[0029] This invention provides a silk fibroin extracellular matrix gel material, which has advantages such as controllable size, high molecular weight, controllable crosslinking degree, easy filling, and high skin affinity, making it more suitable as a soft tissue filler compared to conventional silk fibroin fillers. Different skin sites have different tension and strength, corresponding to different rheological properties of the material, namely elastic modulus and viscous modulus. Alkoxy groups promote hydrophobic interactions between molecules, inducing phase separation in the blended system (mixed solution) via a nucleation-growth mechanism. Phase separation leads to the formation of an SF-enriched phase, increasing the local concentration of SF and promoting the transformation of SF molecules from random coil / helical structures to β-sheet structures. This process is somewhat similar to the "macromolecular crowding effect" in protein folding. The β-sheet structures formed between SF molecules act as physical crosslinking points in the gel network, and the phase structure coarsens as phase separation proceeds. At the same time, the β-sheet structures formed between SF molecular chains gradually increase until a gel network is formed throughout the system. The β-sheet structure formed between SF molecular chains is further increased and improved, providing more cross-linking points, which enables the gel network to develop. Meanwhile, the movement of molecular chains in the dispersed phase is restricted, and some molecular chains are even severely restricted. The already formed phase structure is "frozen", and then a gel is formed.

[0030] Furthermore, the hydrogen bonds between hydroxypropyl groups and water molecules in cellulose, such as hydroxypropyl methylcellulose, delay gel formation, resulting in a slower and more uniform formation process of the gel's internal structure. Therefore, the gel exhibits stronger macroscopic mechanical properties. The degree of physical cross-linking of the gel material described in this invention can be adjusted by regulating the β-sheet rate and ratio of silk fibroin at different temperatures.

[0031] The soft tissue filler material refers to filler materials used in minimally invasive cosmetic procedures such as facial wrinkle removal.

[0032] Beneficial effects of the present invention

[0033] (1) The raw material of the hydrogel described in this invention is silk fibroin, which is inexpensive and certified by the U.S. Food and Drug Administration (FDA), and therefore the hydrogel obtained has good biocompatibility and biodegradability.

[0034] (2) The mechanical strength of the hydrogel described in this invention is controllable. As can be seen from the performance of elastic modulus and viscosity modulus, a series of hydrogels with different mechanical strengths can be prepared by simply changing the molecular weight and ratio, thereby meeting the usage requirements of different facial skin.

[0035] (3) The hydrogel described in this invention is similar to the extracellular skeleton in terms of structure, size, and function; its components are all fibrous proteins with a diameter of 5-30 nm, which play an important role in maintaining the normal morphology of cells, withstanding certain external forces, and maintaining the normal operation of various cell functions, and can be compared with the mechanical properties of natural facial skin tissue; the extracellular skeleton also plays an important role in the transport and translocation of substances, and the hydrogel described in this invention can also promote the transfer and differentiation of surrounding fibroblasts (see Figure 3 ), to achieve tissue regeneration.

[0036] (4) The preparation method of the hydrogel described in this invention is green, environmentally friendly, low-cost, and easy to implement, and is expected to be used for large-scale commercial production. The silk protein nanofiber hydrogel of this invention can be widely used in biomedical materials and nanomaterials. Attached Figure Description

[0037] Figure 1 SEM, TEM and AFM images of the silk fibroin extracellular skeleton gel disclosed in Example 1 of this application, and AFM images of four different molecular weight silk fibroin microfibers.

[0038] Figure 2 Schematic diagram of the cross-linking mechanism between silk fibroin microfiber gel and cellulose;

[0039] Figure 3 The in vivo retention rates of the samples and comparative examples implanted subcutaneously at different times in Examples 3-2;

[0040] Figure 4 To compare the inflammatory responses of mice in Example 3-2 and the control group 2 weeks after implantation;

[0041] Figure 5 The collagen production rate of the samples and comparative examples implanted under the skin at different times in Examples 3-2 is shown; the larger the collagen area ratio, the stronger the product's function of stimulating skin tissue regeneration.

[0042] Figure 6 This is a schematic diagram comparing the collagen regeneration promotion capabilities of the samples and comparative examples in Example 3-2 during the implantation experiment. Detailed Implementation

[0043] This invention generally relates to soft tissue fillers, such as dermal fillers and subcutaneous fillers, based on silk fibroin, cellulose, hyaluronic acid (HA), and pharmaceutically acceptable salts of HA (e.g., sodium hyaluronate). The silk fibroin-based compositions of this invention exhibit enhanced stability. This extracellular matrix gel material is structurally similar to the extracellular matrix (ECM) in the human body. After implantation, cells can synthesize and deposit new proteins on the surface of the biomaterial, remodeling this portion of the ECM. Its degradation process matches the tissue regeneration process, thereby achieving in-situ tissue regeneration.

[0044] The stable composition retains at least one or all of the following properties after effective autoclaving and / or prolonged storage: a translucent to milky white gel, a suitable pH for patient use, extrusion pressure and / or rheological characteristics, silk fibroin concentration, HA concentration, sterility, and molar osmotic pressure concentration. Methods or processes for preparing such silk fibroin-based compositions and products prepared by these methods or processes are also provided.

[0045] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:

[0046] Example 1: Controllable silk fibroin extracellular skeleton gel with selected silk fibroin molecules of different molecular weights

[0047] Silk fibroin microfiber gel: Silkworm silk was degummed, dissolved, desalted, and concentrated. Systems with pore sizes of 50kDa-100kDa, 100kDa-150kDa, 150kDa-200kDa, and 200kDa-350kDa were selected to prepare regenerated silk fibroin solutions with molecular weights of 50kDa-100kDa, 100kDa-150kDa, 150kDa-200kDa, and 200kDa-350kDa, respectively, all with a mass concentration of 20%.

[0048] Prepare a 5% hydroxypropyl methylcellulose solution. Take 1 ml of the solution and add it to 125 ml of the aforementioned silk fibroin aqueous solution, so that the silk fibroin is 5 parts and the cellulose is 0.01 parts. Mix the mixture in a vortex mixer at 3000 rpm / min for 1 min. Then, ultrasonically treat the mixture with an ultrasonic disruptor at 75% amplitude, using intermittent ultrasonication (50 s for ultrasonication, 10 s for pause), for a total of 5 min.

[0049] Then, the gel was allowed to stand at 90°C, homogenized at 24000 rpm / min for 5 min, sieved, and the silk fibroin microfiber gel that passed through a 200-mesh sieve was collected.

[0050] Preparation of extracellular matrix gel material: Take 2.5 parts of sodium hyaluronate particles with a molecular weight of 1000kDa-1500kDa and prepare three portions, which are then mixed with the silk fibroin microfiber gel in step 1. Subsequently, crosslinking is carried out at 55℃ for 6 hours.

[0051] Using PBS buffer (pH 7.4) as the dialysate, dialysis was performed with magnetic stirring, and the dialysate was changed approximately every 2 hours. Dialysis lasted approximately 72 hours.

[0052] The dialysis gels were filled into containers and autoclaved. The obtained silk fibroin extracellular skeleton gels were designated as samples 1-1, 1-2, 1-3, and 1-4, respectively.

[0053] Table 1 shows the rheological data of controllable silk fibroin extracellular skeleton gels prepared from silk fibroin of different molecular weights.

[0054] Table 1. Gel rheological data of extracellular skeletons of silk fibroin with different molecular weights

[0055]

[0056] As the molecular weight increases, the elastic modulus and viscous modulus of the formed gel increase significantly. Silk fibroin microfiber gels of different molecular weights can be selected depending on the application. For example... Figure 1 The images shown are SEM, TEM, and AFM images of the silk fibroin extracellular matrix gel prepared in the examples, as well as AFM images of four different molecular weight silk fibroin microfibers. The results show that the SF gel is composed of a nanofiber network, with the nanofibers being approximately several hundred nanometers in length and about 5 nm in width. Due to the obvious entanglement and branching between the microfibers, a gel visible to the naked eye can be formed. The AFM images of microfibers with different molecular weights show that the degree of cross-linking of the gel can be effectively controlled according to the different molecular weights of the silk fibroin. Figure 2 As shown.

[0057] Depending on the degree of cross-linking of the gel, its degradation effect in tissues varies, thus enabling controllable gel degradation.

[0058] Example 2: Controllable silk fibroin extracellular skeleton gel with different molecular weights of sodium hyaluronate

[0059] Silk fibroin microfiber gel: Silkworm silk is degummed, dissolved, desalted and concentrated. A system with a pore size of 50-150kDa is selected to prepare a regenerated silk fibroin solution with a molecular weight of 50-150kDa and a mass concentration of 20%.

[0060] Prepare a hydroxypropyl methylcellulose solution and add it to the aforementioned silk fibroin aqueous solution. Mix the mixture in a vortex mixer at 3000 rpm for 1 min. Then, perform ultrasonic treatment using an ultrasonic homogenizer with an amplitude of 80% and intermittent ultrasonication (50 s for ultrasonication, 10 s for pause) for a total of 5 min.

[0061] Then, the mixture was allowed to stand at -20℃ to form a gel, homogenized at 20000 rpm / min for 5 min, sieved, and the silk fibroin microfiber gel that passed through a 325-mesh sieve was collected to prepare 4 samples.

[0062] Preparation of extracellular matrix gel materials: Sodium hyaluronate particles with molecular weights ranging from 500kDa to 1000kDa, 1000kDa to 1500kDa, 1500kDa to 2000kDa, and 2000kDa to 2400kDa were mixed with the silk fibroin microfiber gel from step 1, respectively, to achieve a silk fibroin:cellulose:hyaluronic acid ratio of 15:2:5. Subsequently, crosslinking was carried out at 25°C for 12 hours.

[0063] Using phosphate buffer solution with a pH of 7.4 as the dialysate, dialysis was performed with magnetic stirring, and the dialysate was changed approximately every 2 hours. Dialysis lasted approximately 72 hours.

[0064] The dialysis gels were filled into containers, autoclaved, and the samples were labeled as 2-1, 2-2, 2-3, and 2-4.

[0065] In Example 2, the rheological data of controllable silk fibroin extracellular skeleton gels prepared with hyaluronic acid of different molecular weights are shown in Table 2 below. Among them, 2-4 have slightly higher elastic modulus, so the preferred molecular weight of hyaluronic acid is 1500 kDa-2400 kDa.

[0066] Table 2. Rheological data of controllable silk fibroin extracellular skeleton gels prepared from hyaluronic acid of different molecular weights.

[0067]

[0068] Example 3: Controllable silk fibroin extracellular skeleton gels of different types of cellulose

[0069] Silk fibroin microfiber: 40g of silkworm silk was degummed, dissolved, desalted and concentrated. A system with a pore size of 50kDa-150kDa was selected to prepare a regenerated silk fibroin solution with a molecular weight of 50-150kDa and a mass concentration of 20%.

[0070] Prepare a 5% hydroxypropyl cellulose solution and add it to the aforementioned silk fibroin aqueous solution. Mix the mixture for 1 min using a vortex mixer at 3000 rpm / min. Then, perform ultrasonic treatment using an ultrasonic disruptor with an amplitude of 70% and intermittent ultrasonication (50 s for ultrasonication followed by a 10 s pause) for a total of 5 min.

[0071] Prepare a 5% hydroxypropyl methylcellulose solution and add it to the aforementioned silk fibroin aqueous solution. Mix the mixture in a vortex mixer at 3000 rpm for 1 min. Then, perform ultrasonic treatment using an ultrasonic disruptor with an amplitude of 70% and intermittent ultrasonication (50 s for ultrasonication, 10 s for pause) for a total of 5 min.

[0072] Prepare a 5% carboxymethyl cellulose solution and add it to the aforementioned silk fibroin aqueous solution. Mix the mixture in a vortex mixer at 3000 rpm for 1 min. Then, perform ultrasonic treatment using an ultrasonic disruptor with an amplitude of 70% and intermittent ultrasonication (50 s for ultrasonication, 10 s for pause) for a total of 5 min.

[0073] Then, the mixture was allowed to stand at -20℃ to form a gel, homogenized at 28000 rpm / min for 5 min, sieved, and the silk fibroin microfiber gel that passed through a 300-mesh sieve was collected.

[0074] Preparation of extracellular matrix gel material: Sodium hyaluronate particles with a molecular weight of 1500kDa-2000kDa were prepared and mixed with the silk fibroin microfiber gel from step 1, resulting in a silk fibroin:cellulose:hyaluronic acid ratio of 4:1:1. Subsequently, crosslinking was performed at 47°C for 2 hours.

[0075] Using PBS buffer (pH 7.4) as the dialysate, dialysis was performed with magnetic stirring, and the dialysate was changed approximately every 2 hours. Dialysis lasted approximately 72 hours.

[0076] The dialysis-treated gels were filled into containers, autoclaved, and labeled as samples 3-1, 3-2, and 3-3.

[0077] Table 3. Test data of silk fibroin extracellular skeleton gel materials with different types of cellulose added.

[0078]

[0079] At the same concentration, hydroxypropyl cellulose has very poor gel uniformity, with large clumps of gel and a small amount of water mixed in the system. Hydroxypropyl methylcellulose has very good gel uniformity and an elastic modulus suitable for soft tissue filling. Carboxymethyl cellulose forms gels with a lot of sediment and is not completely gelled, resulting in a large amount of sediment.

[0080] Example 4: Controllable silk fibroin extracellular cytoskeleton gel

[0081] Under the conditions of Example 3, samples with different part ratios were prepared and their rheological properties were tested.

[0082] Sample 4-1: 10 parts silk fibroin, 1.25 parts cellulose, and 5 parts sodium hyaluronate;

[0083] Sample 4-2: 10 parts silk fibroin, 1.88 parts cellulose, and 5 parts sodium hyaluronate;

[0084] Sample 4-3: 20 parts silk fibroin, 2.5 parts cellulose, and 4 parts sodium hyaluronate;

[0085] Sample 4-4: 15 parts silk fibroin, 1.25 parts cellulose, and 5 parts sodium hyaluronate;

[0086] Samples 4-5: 15 parts silk fibroin, 1.88 parts cellulose, and 5 parts sodium hyaluronate;

[0087] Samples 4-6: 20 parts silk fibroin, 1.25 parts cellulose, and 5 parts sodium hyaluronate;

[0088] Rheological data testing method: A rheometer was used at (25±0.2)℃ with a 25mm rotor, a plate gap of 1mm, and a fixed deformation of 0.5%. The data was scanned at shear rates from 0.01 Hz to 100Hz.

[0089] The elastic modulus and viscous modulus of the material were obtained at 1 Hz (simulating skin tissue tension).

[0090] Table 4. Rheological data of controllable silk fibroin extracellular skeleton gels prepared with different group ratios.

[0091]

[0092] By controlling the proportions of each component, the influence of each component on the mechanical properties of the material was analyzed. Increased concentration of each component led to an increase in the elastic modulus of the material, with the concentration of silk fibroin having the greatest, decisive, impact on the mechanical properties. In our proposed gelation mechanism, the β-sheet structure formed between silk fibroin molecules plays a crucial role in the formation of the gel network. This explains the positive correlation between gel modulus and silk fibroin content observed in rheological experiments. Simply put, the microfibers formed by the gradually increasing β-sheet structure between silk fibroin molecular chains are key to gel composition and constitute the gel network that runs through the system. Higher silk fibroin concentration results in more fibers, a denser gel network, and a higher elastic modulus.

[0093] Example 5:

[0094] Preparation of silk fibroin microfibers: A system with a pore size cutoff of 50kDa-150kDa was selected to prepare a regenerated silk fibroin solution with a molecular weight of 50-150kDa. The solution was ultrasonically treated to obtain sample 5-1. 5 ml of a 5% hydroxypropyl methylcellulose solution was prepared and added to 12.5 ml of the aforementioned silk fibroin aqueous solution. The mixture was vortexed at 3000 rpm / min for 1 min. The mixture was then ultrasonically treated with an ultrasonic disruptor at 70% amplitude, intermittently ultrasonicated (50 s for sonication, 10 s for pause), for a total of 5 min to obtain sample 5-2. Sample 5-3 was obtained following the procedure in Example 3. Based on sample 5-1, 0.60 g of sodium hyaluronate particles with a molecular weight of 1500kDa-2000kDa were added and mixed to form sample 5-4.

[0095] Table 5. Sample modulus test results for four different components

[0096]

[0097] Modulus tests were performed on four samples, and the results are shown in Table 4 below. The results show that in samples 5-4, the modulus increase was small when only hyaluronic acid was added to the silk fibroin microfibers. Macroscopic observation also revealed that sample 5-4 did not form a gel scaffold under naked-eye observation. The modulus results of sample 5-2, however, demonstrate that the hydrophobic interactions between cellulose molecules in the silk fibroin microfibers resulted in a significant increase in modulus. In sample 5-3, it was observed that after the physical cross-linking process following the addition of the three components, the sample achieved a high modulus, indicating the formation of a stable gel structure.

[0098] Comparative Example (CN102836465B):

[0099] (1) Preparation of silk fibroin microspheres: silkworm silk was degummed, dissolved, desalted and concentrated, and then prepared into a silk fibroin solution with a mass concentration of 5% using purified water.

[0100] At 25°C, the above 5% silk fibroin solution was allowed to stand to form 5g of white gel material. The gel material was homogenized at 24000rpm / min for 5min to obtain 5g of silk fibroin gel particles of different diameters. After filtration, 3.5g of silk fibroin particles that passed through a 200-mesh sieve were collected. A small amount of the collected silk fibroin particles were weighed and dried to constant weight to determine the water content of the silk fibroin particles and to determine the mass concentration of the silk fibroin particles as 3.23%.

[0101] (2) Silk fibroin-hyaluronic acid composite gel: Dissolve 0.7g of sodium hyaluronate in 7ml of 1% sodium hydroxide solution to prepare a 0.1g / ml sodium hyaluronate solution, and add 0.712g of silk fibroin particles obtained in step (1) (0.023 / 0.0323=0.712g, the dry weight of silk fibroin particles is 0.023g) to the sodium hyaluronate solution. After thorough mixing, add 56μl (0.056g) of BDDE (1,4-butanediol diglycidyl ether), mix evenly, and place at 40℃ for 4 hours to crosslink, thus forming silk fibroin-HA composite gel material.

[0102] The above-mentioned silk fibroin-HA composite gel material was added to a dialysis bag with a MW 8,000-14,000 capacity. Using PBS buffer (pH 7.4, temperature 37℃) as the dialysis solution, dialysis was performed under magnetic stirring. The dialyzed composite gel material was homogenized at 24,000 rpm / min for 10 min, then squeezed through a 60-mesh sieve in a syringe. The composite gel particles were collected, sterilized by high-temperature autoclaving at 120℃ for 15 min, and then aseptically dispensed into disposable syringes. This yields 35g of injectable silk fibroin-hyaluronic acid composite gel, suitable for subcutaneous injection.

[0103] The test method for crosslinking agents is as follows: Take the same amount of the sample from Example 3-2 and the comparative sample, place them in a dialysis bag, and then immerse them in PBS. Collect the solution outside the dialysis bag at 2 weeks, 4 weeks, and 8 weeks respectively as the test solution. The residual amount of BDDE is tested using gas chromatography. The specific test method is as follows:

[0104] 1) Instruments and reagents: gas chromatograph, FID detector, DB-17 column (30 m x 0.32 mm, film thickness 0.50 μm), 1,4-butanediol diglycidyl ether standard, acetone.

[0105] 2) Chromatographic conditions

[0106] Column: DB-17 (0.32 mm x 30 m, film thickness 0.50 μm)

[0107] Column temperature: Initial temperature 150℃, increased to 260℃ at 30℃ / min, and held for 10 min.

[0108] Detector: FID.

[0109] Carrier gas: N2.

[0110] Inlet temperature: 260℃.

[0111] Detector temperature: 300℃.

[0112] Carrier gas flow rate: 5 mL / min.

[0113] 3) Sample pretreatment: Accurately weigh the solution to be tested, place it in a 10 mL volumetric flask, add an appropriate amount of acetone, shake and dilute to the mark, shake well, filter, and take the filtrate.

[0114] 4) Sample loading test: Accurately measure 1 μL of each of the test solution and the reference solution, inject them into the gas chromatograph, record the chromatogram, and calculate the peak area according to the external standard method.

[0115] Table 6. Crosslinking agent residue data for Examples 3-2 and Comparative Examples.

[0116]

[0117] The data on residual crosslinking shows that, since this invention uses physical crosslinking and contains no crosslinking agent, no crosslinking agent residue was observed at any point in time. In contrast, in the comparative example, due to the addition of a crosslinking agent, the chemical crosslinking agent gradually precipitated out as the hyaluronic acid degraded, potentially causing inflammatory reactions in the tissue and being harmful to the tissue. Figure 3 It was also found that the inflammatory response was higher in the control group than in the control group.

[0118] Application example:

[0119] The gels obtained in Examples 3-2 and the comparative example were implanted subcutaneously into mice. After a certain period of time, the samples were taken to make pathological sections. HE staining was used to observe the inflammatory response of the implanted materials in vivo, and immunohistochemical staining was used to observe the effect on collagen regeneration.

[0120] Table 7 shows the histopathological evaluation criteria for characterizing the inflammatory response after material implantation.

[0121]

[0122] After the material is implanted, the inflammatory response is most intense and pronounced approximately two weeks later. Figure 5 This image shows a comparison of the inflammatory responses in mice of Example 3-2 and the control group two weeks after implantation. The dark dots in the image represent lymphocyte nuclei. It can be seen that the control group implanted the same way as the control group had a large number of lymphocytes infiltrating the surrounding tissue, resulting in a high inflammation score. In contrast, Example 3-2 showed only a very small number of lymphocytes around the implant, indicating a low inflammatory response. Therefore, the gel described in this invention exhibits superior biocompatibility compared to the control group.

[0123] A comparison of inflammatory responses in Example 3-2 and the comparative mouse implantation experiment is shown in the figure. Figure 4 The collagen production promotion rates of the samples and comparative examples implanted subcutaneously for different times in Examples 3-2 are shown in the figure. Figure 5 The schematic diagram comparing the collagen regeneration promotion capacity of the samples and comparative examples in Example 3-2 is shown below. Figure 6As shown in the figure below, after implantation of Example 3-2 in mice, the inflammatory response was lower than that of the control group, while the collagen production capacity was higher than that of the control group. Evidence of its production capacity can be seen in the immunohistochemical comparison diagram. It can be seen that the silk fibroin extracellular matrix gel material proposed in this invention has a structure similar to ECM, its in situ tissue growth capacity is higher than that of the control group, and it does not produce a high inflammatory response, thus having good biocompatibility.

[0124] Specifically: Figure 3 The in vivo retention rates of the samples and comparative examples implanted subcutaneously at different times in Examples 3-2 are shown.

[0125] In the initial implantation stage, the hyaluronic acid in the comparative product absorbs water and swells, while the hydrogel described in this invention does not absorb water and swells, maintaining its initial volume. Clinically, this means that the facial injection site does not "bread out". As the implantation time increases, the comparative product degrades rapidly, while the hydrogel described in this invention exhibits a stable degradation rate.

[0126] Figure 4 The study compared the inflammatory responses of mice in Example 3-2 and the control group two weeks after implantation. After implantation of Example 3-2 in mice, the inflammatory response was lower than that in the control group, demonstrating that the silk fibroin extracellular matrix gel material has good biocompatibility, a structure similar to ECM, and does not produce a high inflammatory response.

[0127] Figure 5 The collagen production rate of the samples and comparative examples implanted under the skin at different times in Examples 3-2 is shown; the larger the collagen area ratio, the stronger the product's function of stimulating skin tissue regeneration.

[0128] Figure 6 This is a schematic diagram comparing the collagen regeneration promotion capacity of the samples in Example 3-2 and the comparative example in the 14-week implantation experiment.

[0129] Immunohistochemical staining was performed on pathological sections to analyze the material's ability to promote collagen regeneration. Deeper staining and larger staining areas indicated a higher proportion of collagen and better regeneration. Before complete material degradation, the longer the implantation time, the more collagen secretion was promoted. Figure 6 The immunohistochemical staining of the tissue sections from the samples in Example 3-2 and the comparative example after 14 weeks of implantation into mice was selected. The tissues in Example 3-2 showed significantly deeper staining and a larger area, indicating that the regeneration ability of the sample in Example 3-2 was stronger than that of the comparative example.

[0130] Example 6: Controllable silk fibroin extracellular matrix gel

[0131] Under the conditions of Example 3, samples with different part ratios were prepared, the porosity of each sample was determined by liquid displacement method, and cell co-culture was performed to observe cell adhesion and proliferation on the material.

[0132] Sample 6-1: 13 parts silk fibroin, 2.5 parts cellulose, and 10 parts sodium hyaluronate;

[0133] Sample 6-2: 15 parts silk fibroin, 2.5 parts cellulose, and 10 parts sodium hyaluronate;

[0134] Sample 6-3: 16.18 parts silk fibroin, 2.5 parts cellulose, and 10 parts sodium hyaluronate;

[0135] Sample 6-4: 18 parts silk fibroin, 2.5 parts cellulose, and 10 parts sodium hyaluronate.

[0136] The three-dimensional structure of the gel, including pore size and porosity, can regulate cell behavior; cell adhesion and proliferation require suitable porosity. When the product ratio changes, samples 6-2 and 6-3 exhibit greater porosity when the silk fibroin content increases, as microfibers are key components of the gel. Cell proliferation on the material also demonstrates this; samples 6-2 and 6-3 show higher cell proliferation rates compared to 6-1 and 6-4, providing more adhesion sites and a suitable microenvironment for cell survival. In other words, preparing silk fibroin extracellular scaffold gels is more effective when the ratio of 1.5 ≤ silk fibroin / hyaluronic acid ≤ 1.618 is met.

[0137] Porosity data testing method: The lyophilized gel scaffold is placed in a hexane solvent of volume V1. Under a certain negative pressure, the hexane is used to fully fill the pore structure of the scaffold. The solvent volume at this time is recorded as V2. Then the scaffold filled with hexane is taken out, and the solvent volume at this time is recorded as V3. The porosity of the porous scaffold is calculated by the formula: Porosity (%) = [(V1 − V3) ⁄ (V2 − V3)] × 100%.

[0138] Cell proliferation rate: L929 cells were cultured in 96-well plates for 3-5 days, and then MTT solution was added to each well for treatment. The absorbance was measured at 490 nm using an ELISA reader. Cell proliferation rate = (Experimental group absorbance - Blank control absorbance) / (Control group absorbance - Blank control absorbance) × 100%.

[0139] Table 8. Data on porosity and cell proliferation rate of controllable silk fibroin extracellular skeleton gels prepared with different component ratios.

[0140]

[0141] The effects of controlling the proportions of silk fibroin and hyaluronic acid on the internal pore size of the gel were analyzed. Appropriate pore size is essential for tissue formation to facilitate the diffusion of nutrients, oxygen, and waste between cells. A properly proportioned hydrogel network is stable, possessing suitable pore size and porosity, which is beneficial for cell adhesion and growth on the surface. Experiments in Example 6 showed that a silk fibroin extracellular scaffold gel with good porosity and cell proliferation rate can be obtained when 1.5 ≤ silk fibroin / hyaluronic acid ≤ 1.618.

Claims

1. A controllable injectable silk fibroin extracellular matrix gel material, characterized by: The gel comprises 5-50 parts of silk fibroin, 0.01-5 parts of cellulose and 0.7-10 parts of hyaluronate, and is prepared by the following steps: (1) Preparation of silk fibroin microfiber gel: silk fibroin aqueous solution is added to cellulose to prepare a gel, which is crushed and sieved to obtain a silk fibroin microfiber gel; the cellulose is hydroxypropyl methyl cellulose; (2) Preparation of extracellular matrix gel material: hyaluronate is mixed with the silk fibroin microfiber gel to physically crosslink the same to form a silk fibroin extracellular matrix gel material; wherein, according to the weight ratio, 1.5≤silk fibroin / hyaluronate≤1.618, and no chemical crosslinking agent is included in the gel material.

2. The material of claim 1, wherein: the molecular weight of the silk fibroin is 50 kDa to 350 kDa; and the silk fibroin aqueous solution is prepared by degumming silk of mulberry silkworm or tussah silkworm through dissolution and purification.

3. The material of claim 1, wherein: the molecular weight of the hyaluronate is 500 kDa to 2400 kDa.

4. The material of claim 1, wherein: the molecular weight of the hyaluronate is in the range of 1500 kDa-2400 kDa.

5. The material of claim 1, wherein: after preparation of the gel material, dialysis purification is performed, and the dialysis liquid is an isotonic phosphate buffer solution. The gel comprises 5-50 parts of silk fibroin, 0.01-5 parts of cellulose and 0.7-10 parts of hyaluronate. The gel comprises 10-50 parts of silk fibroin, 0.01-5 parts of cellulose and 0.7-5 parts of hyaluronate. Silk fibroin / hyaluronate = 1.

618.

9. A preparation method of the material according to any one of claims 1-8, wherein:

6. Material according to one of claims 1 to 5, characterized in that: (1) Preparation of silk fibroin microfiber gel: silk fibroin aqueous solution is added to cellulose to prepare a gel, which is crushed and sieved to obtain a silk fibroin microfiber gel; 7. Material according to one of claims 1 to 5, characterized in that: (2) Preparation of extracellular matrix gel material: hyaluronate is mixed with the silk fibroin microfiber gel to physically crosslink the same to form a silk fibroin extracellular matrix gel material.

8. The material of any of claims 1-5, wherein:

10. The preparation method of claim 9, wherein: (1) mulberry silkworm silk is placed in a boiling aqueous solution of sodium carbonate with a mass fraction of 0.1-5% or a mixed solution of sodium carbonate and sodium bicarbonate, heated and boiled for 10-120 minutes, dried at 25-100℃, dissolved in a 9.3 mol / L lithium bromide aqueous solution, and water-bathed at 25-100℃ for 10-120 minutes until the silk fibroin is fully dissolved, filtered with a 50-200 mesh filter bag to remove insoluble particulate impurities, desalted and concentrated to obtain a silk fibroin aqueous solution with a specific molecular weight; after the 20% silk fibroin solution is added to a cellulose solution, it is treated by an ultrasonic crusher; after the gel is formed, it is homogenized at 1500-24000 rpm for 5 minutes, sieved, and the silk fibroin microfiber gel passing through a 200-325 mesh sieve is collected. ​ ​ ​ ​ (2) Preparation of extracellular matrix gel: prepare hyaluronic acid particles, mix with silk fibroin microfiber gel, stand at 25-60 DEG C for 2-12h, make it physical crosslinking, dialysis, filling, sterilization, form silk fibroin extracellular matrix gel material.

11. Use of the controllable silk fibroin extracellular matrix gel material according to one of claims 1 to 8, characterized in that: The medical and aesthetic filler or the medical filler is used for preparing a medical and aesthetic filler or a medical filler.

12. The use according to claim 11, characterized in that: The medical and aesthetic filler or the medical filler is used for skin tissue or soft tissue filling repair, and can promote in-situ tissue regeneration.

13. The use according to claim 11, characterized in that: The medical and aesthetic filler or the medical filler is used for face, trunk or limbs.

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