Controllable fibroin extracellular scaffold gel material for injection and method for preparing same

Abstract

Disclosed in the present invention is a method for preparing a controllable fibroin extracellular scaffold gel material for injection. A composite filler consists of the following raw materials in mass ratio: 5-50 parts of fibroin, 0.01-5 parts of cellulose, and 0.7-10 parts of a hyaluronate. The method comprises: (1) preparation of a fibroin microfiber gel: adding cellulose to an aqueous fibroin solution to prepare a gel, and crushing and sieving to give the fibroin microfiber gel; (2) preparation of an extracellular scaffold gel material: mixing a hyaluronate with the fibroin microfiber gel to physically crosslink same, so as to form the fibroin extracellular scaffold gel material. The modified fibroin hydrogel prepared by the method can promote in-situ tissue regeneration after injection and features a controllable molecular weight and a controllable crosslinking degree. The modified fibroin hydrogel possesses a structure similar to that of the extracellular matrix (ECM), a degradation process matching the in-situ tissue regeneration process, and improved mechanical properties, degradation properties, biocompatibility, etc., without the use of chemical crosslinking agents. The modified fibroin hydrogel is injectable and prospective in the field of medical beauty injection.

Description

Controllable injection silk fibroin extracellular matrix gel material and preparation method thereof TECHNICAL FIELD

[0001] The present application relates to medical cosmetic filling materials, in particular to a controllable injection extracellular matrix gel material and a preparation method thereof. BACKGROUND

[0002] There are various ways to regenerate tissues, such as intracellular / extracellular polymerization, in situ synthesis of functional polymers on living cells, or in vitro preparation of scaffolds and implantation of cells after culture for regeneration. However, these methods have great limitations. Intracellular / extracellular polymerization is a new and immature field, and the in situ synthesis of functional polymers by cells requires complex means to achieve; and in vitro tissue engineering requires complex cell culture conditions to obtain functional tissues in vitro, and autocrine and paracrine signaling effects are difficult to reproduce in vitro culture.

[0003] In situ tissue engineering is also an effective means of tissue engineering regeneration by combining the original regeneration ability of tissues with engineered biomaterials. In this process, the biomaterials provide a structural framework to promote the attachment and migration of host stem cells and progenitor cells, and to promote the differentiation of these cells into specific cell types to achieve repair or regeneration.

[0004] The ultimate goal of tissue regeneration is to use certain means to achieve cell migration and proliferation, to remodel the extracellular matrix (ECM) to affect cell function, such as reorganization of the cytoskeleton, activation of integrins, and normal expression of genes. ECM has a unique spatial organization, and in directional tissues such as muscle and nerve, the unique arrangement of cells and ECM can be observed. In situ tissue engineering achieves guided cell regeneration by designing scaffolds with near-ECM structures to create artificial ECM morphologies, thereby achieving guided differentiation, cell encapsulation, in situ regeneration and protection, ECM remodeling, and tissue repair.

[0005] In the process of in situ tissue regeneration, the scaffold provided by the biomaterials should facilitate the penetration of cells and tissues after implantation, and then the scaffold should be decomposed and completely resorbed. The key point in this process is that the scaffold should be controlled to degrade to guide in situ tissue regeneration, and this process requires balancing the degradation rate of the scaffold and the regeneration rate of the tissue to complete the structural and functional regeneration.

[0006] The raw materials for in situ tissue engineering biomaterials are widely available, and the materials need to respond to biological signals and interact with the immune system and endogenous cells to stimulate regeneration. At the same time, the structure and biological properties need to be precisely controlled during the design process to guide endogenous cells to the injury site. This technology opens up unprecedented ways to customize biomacromolecular properties, achieve precise spatial control, and seamlessly integrate the material into the biological entity.

[0007] Regenerated silk fibroin is widely used in the fields of biological medicine and tissue engineering due to its excellent mechanical properties, good biocompatibility, biodegradability, and multifunctional structure. It has a unique protein spatial structure, and the formation of certain β-sheet structure in regenerated silk fibroin can form nanoscale microfibers, which overlap and aggregate to form a flocculent network structure. In the process of tissue regeneration, this property can be used to prepare scaffolds to simulate the structure of ECM, so as to achieve cell proliferation and regeneration and remodeling of ECM.

[0008] In injectable tissue filling, implanting tissue engineering scaffolds to promote in situ tissue regeneration is an effective repair method. There are few products on the market that use this method for repair. The common products are represented by hyaluronic acid, which is a pure physical filler without regenerative effect and easy to swell due to water absorption. Another type is represented by poly-L-lactic acid and polycaprolactone microspheres, but their structure is loose and easily decomposed by the tissue environment and proteases, making it difficult to maintain long-term filling effect. These products are very different from the microstructure of human skin itself. In contrast, regenerated silk fibroin microfibers have a relatively dense and stable molecular structure, with a suitable degradation period, making them good tissue regeneration filling materials. If a scaffold is prepared using this material as a substrate and the scaffold is designed to simulate ECM, it can more effectively stimulate tissue regeneration.

[0009] Among the existing inventions, silk fibroin and hyaluronic acid are cross-linked using chemical cross-linking agents such as BDDE to form a silk fibroin hyaluronic acid three-dimensional interpenetrating network composite gel material, but the cross-linking degree is uncontrollable, and the use of chemical cross-linking agents has certain cytotoxicity. After accumulation of residues in the body, it is easy to cause redness, allergy, carcinogenesis, and other risks.

[0010] Therefore, it is a key research and development direction of the silk fibroin tissue engineering material to find a silk fibroin extracellular matrix gel material which is controllable in crosslinking degree and molecular weight, matches the in-situ tissue regeneration process, is similar to the microstructure of human skin, and has good biocompatibility, water absorption, good mechanical properties and injectability. Different skin tissues have different tensions, and the rheological properties of the filling materials required are also different. The injection silk fibroin extracellular matrix gel material disclosed by the application is matched with different skin microstructures and different skin regeneration processes through different process adjustments, and realizes controllable in-situ tissue regeneration.

[0011] The injection silk fibroin extracellular matrix gel material disclosed by the application realizes the wrinkle removal and lifting effect through physical occupation in the initial stage of injection into the skin, and has no phenomenon of water absorption volume expansion; in the later stage, the gel material can promote in-situ tissue regeneration under the extracellular matrix structure of the gel, and the area proportion of collagen in pathological sections is increased, and the in-vivo degradation rate of the gel material is controllable by adjusting the molecular weight and the ratio of components, and the retention rate of the gel in the body is reflected. SUMMARY

[0012] In view of the above problems, the application provides a silk fibroin extracellular matrix gel material which is controllable in crosslinking degree and molecular weight, matches the in-situ tissue regeneration process, is similar to the microstructure of human skin, has good biocompatibility, water absorption, good mechanical properties and injectability, and does not use a chemical crosslinking agent to reduce the corresponding residues.

[0013] The technical scheme of the application is as follows:

[0014] The injection silk fibroin extracellular matrix gel is controllable in crosslinking degree and molecular weight, and matches the in-situ tissue regeneration process, and can be a controllable injection extracellular matrix gel material.

[0015] 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 according to the following steps:

[0016] (1) Preparation of silk fibroin microfiber gel: silk fibroin aqueous solution is added to cellulose to prepare a gel, the gel is crushed and sieved to obtain a silk fibroin microfiber gel;

[0017] (2) Preparation of extracellular matrix gel material: hyaluronate is mixed with the silk fibroin microfiber gel to physically crosslink the silk fibroin microfiber gel, so as to form a silk fibroin extracellular matrix gel material.

[0018] The gel material does not include any chemical crosslinking agent.

[0019] The cellulose is one or more than two of hydroxypropyl methyl cellulose, hydroxypropyl cellulose, carboxymethyl cellulose, hydroxyethyl cellulose, preferably hydroxypropyl methyl cellulose. The hyaluronate is one or more than two of sodium hyaluronate, potassium hyaluronate, magnesium hyaluronate in any proportion, preferably sodium hyaluronate.

[0020] Further, the preparation method of the silk fibroin composite filler of the present application is as follows: (1) silk fibroin microfiber gel: put the mulberry silk into 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, heat and boil for 10-120 minutes, dry at 25-100℃, dissolve in a 9.3 mol / L lithium bromide aqueous solution, and water bath at 25-100℃ for 10-120 minutes until the silk protein is fully dissolved, filter out insoluble particulate impurities with a 50-200 mesh filter bag, and after desalination and concentration, obtain a silk fibroin aqueous solution with a specific molecular weight. Add a cellulose solution to a silk fibroin solution with a mass concentration of 20%, and then treat with an ultrasonic wave crusher. After gelation, homogenize at 1500-24000 rpm / min for 5 minutes, sieve, and collect the silk fibroin microfiber gel passing through a 200-325 mesh sieve; (2) preparation of extracellular matrix gel: mix the hyaluronic acid particles with the silk fibroin microfiber, and physically cross-link at 25-60℃ for 2-8 hours, dialyze with a dialysis solution, fill, sterilize, and form a silk fibroin extracellular matrix gel material.

[0021] Further, by means of the standing process + temperature control, the degree of β-sheet and the number of hydrogen bonds can be controlled to control the cross-linking degree of the gel. The alkyl group promotes intermolecular hydrophobic interaction, while the hydrogen bonds between the hydroxypropyl group in the hydroxypropyl methyl cellulose and water molecules delay gel formation. Under the hydrophobic interaction of the cellulose, the β-sheet structure is uniformly distributed, and the cross-linking density is higher, and its cross-linking form is similar to that of the extracellular matrix.

[0022] Further, the extracellular matrix gel is composed of raw materials in the following mass ratio: silk fibroin 5-50 parts, cellulose 0.01-5 parts, and hyaluronate 0.7-10 parts. Preferably, silk fibroin 10-50 parts, cellulose 0.01-5 parts, and hyaluronate 0.7-5 parts.

[0023] Further preferably, the ratio of silk fibroin to hyaluronate is greater than or equal to 2:1, 3:1, or 4:1 or more.

[0024] In addition, the ratio of silk fibroin to hyaluronate is preferably greater than or equal to 1.5:1, or less than or equal to 1.618:1, or 1.5≤ silk fibroin / hyaluronate≤1.618.

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

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

[0027] Further, after the silk fibroin microfiber gel and the hyaluronic acid particles are mixed in step (2), it is preferable to stand at 40-55 DEG C.

[0028] Further, after the silk fibroin microfiber gel and the hyaluronic acid particles are mixed in step (2), it is preferable to stand for 4-6 h.

[0029] The present application provides the silk fibroin extracellular matrix gel material, which has the advantages of controllable size, high molecular weight, controllable cross-linking degree, easy filling, high skin affinity and the like, and is more suitable as a soft tissue filling material than conventional silk fibroin fillers. Different skin parts have different tension and strength, and the corresponding materials have different rheological properties, i.e. elastic modulus and viscous modulus. The alkyl group promotes the intermolecular hydrophobic interaction, induces the phase separation of the blending system (mixed solution) by the nucleation-growth mechanism. The phase separation leads to the generation of SF-rich phase, increases the local concentration of SF, and promotes the transition of SF molecules from random coil / helix structure to β-sheet structure. This process is somewhat similar to the "macromolecular crowding effect" in protein folding, and the β-sheet structure formed between SF molecules acts as a physical crosslinking point in the gel network. With the progress of phase separation, the phase structure is coarsened. At the same time, the β-sheet structure formed between SF molecular chains gradually increases until the gel network throughout the system is formed. The β-sheet structure formed between SF molecular chains is further increased and improved, providing more crosslinking points, so that the gel network can develop, and the molecular chain movement in the dispersed phase is limited, and part of the molecular chain is even severely limited, and the formed phase structure is "frozen" down, then the gel is formed.

[0030] In addition, the hydrogen bond between the hydroxypropyl in cellulose, such as hydroxypropyl methyl cellulose, and water molecules can delay the gel formation, so that the internal structure of the gel is formed slowly and uniformly. Therefore, the macroscopic mechanical properties of the gel are stronger. By adjusting the β-sheet speed and proportion of silk fibroin at different temperatures, the physical crosslinking degree of the gel material of the present application can be adjusted.

[0031] The soft tissue filling material refers to a microplastic filling material for facial wrinkle removal.

[0032] Advantages of the present application

[0033] (1) The raw material of the hydrogel is silk fibroin which is low in cost and certified by the Food and Drug Administration (FDA), so the obtained hydrogel has good biocompatibility and biodegradability.

[0034] (2) The mechanical strength of the hydrogel is controllable, and a series of hydrogels with different mechanical strengths can be prepared by changing the molecular weight and the ratio, so as to meet the use requirements of different facial skin.

[0035] (3) The hydrogel is similar to the extracellular skeleton in structure, size and function; the composition is fibrous protein with a diameter of 5-30 nm, which plays an important role in maintaining the normal morphology of cells, bearing certain external force, and maintaining the normal operation of various functions of cells, and can be comparable to the mechanical properties of facial skin tissue; the extracellular skeleton also plays an important role in the transportation and transportation of substances, and the hydrogel can promote the transfer and differentiation of surrounding fibroblasts (see Figure 3), realizing tissue regeneration.

[0036] (4) The preparation method of the hydrogel is green, low in cost and easy to realize, and is expected to be used for commercial large-scale production. The silk fibroin nanofiber hydrogel can be widely used in biomedical materials and nanofunctional materials. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 is a SEM, TEM and AFM diagram of the silk fibroin extracellular skeleton gel disclosed in Example 1 of the present application, and the AFM diagrams of four different molecular weight silk fibroin microfibers

[0038] Figure 2 is a schematic diagram of the cross-linking form of the silk fibroin microfiber gel and cellulose

[0039] Figure 3 is the in vivo retention rate of the sample and the comparative example implanted subcutaneously for different times in Example 3-2

[0040] Figure 4 is a comparison of the inflammatory reactions of mice in Example 3-2 and the comparative example implanted in vivo for 2 weeks;

[0041] Figure 5 is the collagen production rate of the sample and the comparative example implanted subcutaneously for different times in Example 3-2; the greater the collagen area ratio, the stronger the function of the product in stimulating skin tissue regeneration.

[0042] Figure 6 is a schematic diagram of the comparison of the collagen regeneration promoting ability of the sample and the comparative example in Example 3-2 implanted in the experiment DETAILED DESCRIPTION

[0043] The present disclosure relates generally to soft tissue fillers, such as dermal fillers and subdermal fillers, based on silk fibroin, cellulose, hyaluronic acid (HA) and pharmaceutically acceptable salts of HA, such as sodium hyaluronate. The silk fibroin-based compositions of the present disclosure have enhanced stability, the extracellular matrix gel material is similar to the extracellular matrix (ECM) structure in the human body, cells can synthesize and deposit new proteins on the surface of the biomaterial after implantation, remodel the part of ECM, and the degradation process matches the tissue regeneration process, thereby achieving in situ regeneration of the tissue.

[0044] The stable compositions maintain at least one or all of the following aspects: translucent to opalescent gel, suitable pH for use in patients, squeeze force and / or rheological characteristics, silk fibroin concentration, HA concentration, sterility, and osmolality after effective autoclaving and / or long-term storage. Methods or processes for making such silk fibroin-based compositions and products made by these methods or processes are also provided.

[0045] The present disclosure will be further described in conjunction with specific examples, but the scope of protection of the present disclosure is not limited to this:

[0046] Example 1: Controllable silk fibroin extracellular matrix gel with different molecular weight of silk fibroin

[0047] Silk fibroin microfiber gel: degumming, dissolving, desalination and concentration of mulberry silkworm silk, respectively selecting 50 kDa-100 kDa, 100 kDa-150 kDa, 150 kDa-200 kDa, 200 kDa-350 kDa pore size system, preparing regenerated silk fibroin solution with molecular weight of 50 kDa-100 kDa, 100 kDa-150 kDa, 150 kDa-200 kDa, 200 kDa-350 kDa, and mass concentration of 20%.

[0048] Prepare a 5% hydroxypropyl methyl cellulose solution, take 1 ml, and add it to 125 ml of the above silk fibroin aqueous solution, so that the silk fibroin is 5 parts and the cellulose is 0.01 parts, and mix under the condition of vortex mixer 3000 rpm / min for 1 min. Ultrasonic treatment is performed by ultrasonic crusher, amplitude 75%, intermittent ultrasonic (ultrasonic 50 s, stop 10 s), total ultrasonic 5 min.

[0049] Then, gel at 90°C, homogenize at 24000 rpm / min for 5 min, sieve, and collect the silk fibroin microfiber gel passing through the 200 mesh sieve.

[0050] Preparation of extracellular matrix gel material: 2.5 parts of sodium hyaluronate particles with a molecular weight of 1000-1500 kDa were prepared in three parts, and mixed with the silk fibroin microfiber gel in step 1 respectively. Then, crosslinking at 55°C for 6 hours.

[0051] Dialysis was carried out under magnetic stirring with PBS buffer at pH 7.4 as dialysate, and the dialysate was replaced every 2 hours or so. Dialysis was carried out for about 72 hours.

[0052] The dialyzed gel was filled and autoclaved. The obtained silk fibroin extracellular matrix gel was recorded as sample 1-1, 1-2, 1-3, 1-4 respectively.

[0053] Table 1 is the rheological data of the controllable silk fibroin extracellular matrix gel prepared by different molecular weight silk fibroin.

[0054] Table 1 is the rheological data of the controllable silk fibroin extracellular matrix gel prepared by different molecular weight silk fibroin.

[0055]

[0056] With the increase of molecular weight, the elastic modulus and viscous modulus of the formed gel increase significantly, and different molecular weight of silk fibroin microfiber gel can be selected according to the use. With the increase of molecular weight, the elastic modulus and viscous modulus of the formed gel increase significantly, and different molecular weight of silk fibroin microfiber gel can be selected according to the use. As shown in Figure 1, the SEM, TEM and AFM images of the silk fibroin extracellular matrix gel prepared in the example, and the AFM images of four different molecular weight silk fibroin microfiber. The results show that the SF gel is composed of a network of nanofibers, the length of the nanofiber is about several hundred nanometers, and the width is about 5 nm. Because there is obvious entanglement and bifurcation between the microfibers, a visible gel body can be formed. As shown in Figure 2, according to the different molecular weight of silk fibroin, the crosslinking degree of the gel can be effectively controlled.

[0057] According to the difference of the crosslinking degree of the gel, the degradation effect in the tissue is different, so the controllability of gel degradation can be realized.

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

[0059] Silk fibroin microfiber gel: degumming and dissolving of mulberry silk, desalination and concentration, selecting a system with a pore size of 50-150 kDa, preparing a regenerated silk fibroin solution with a molecular weight of 50-150 kDa, and a mass concentration of 20%.

[0060] The hydroxypropyl methyl cellulose solution was prepared and added to the above-mentioned aqueous silk fibroin solution, and mixed in a vortex mixer at 3000 rpm / min for 1 min. Ultrasonic treatment was performed using an ultrasonic disruptor at an amplitude of 80% for a total of 5 min, with intermittent ultrasonic treatment (ultrasonic treatment for 50 s and pause for 10 s).

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

[0062] Preparation of extracellular matrix gel material: The sodium hyaluronate particles with a molecular weight of 500 kDa-1000 kD; 1000 kDa-1500 kD; 1500 kDa-2000 kD; 2000 kDa-2400 kDa were mixed with the silk fibroin microfibril gel in step 1, respectively. The ratio of silk fibroin: cellulose: hyaluronic acid was 15:2:5. Subsequently, crosslinking was performed at 25°C for 12 hours.

[0063] Dialysis was performed using a phosphate buffer solution with a pH value of 7.4 as the dialysis solution under magnetic stirring, and the dialysis solution was replaced every 2 hours or so. Dialysis was performed for about 72 hours.

[0064] The dialyzed gel was filled and autoclaved, and the samples were labeled as 2-1, 2-2, 2-3, and 2-4, respectively.

[0065] In Example 2, the rheological data of the controllable silk fibroin extracellular matrix gel prepared using hyaluronic acid with different molecular weights are shown in Table 2 below. The elastic modulus of 2-4 is slightly higher, so the molecular weight of hyaluronic acid is preferably 1500 kDa-2400 kDa.

[0066] Table 2 Rheological data of controllable silk fibroin extracellular matrix gel prepared using hyaluronic acid with different molecular weights

[0067]

[0068] Example 3: Controllable silk fibroin extracellular matrix gel using different types of cellulose

[0069] Silk fibroin microfibrils: 40 g of degummed, dissolved, and desalted regenerated silk fibroin solution was prepared by concentrating the solution to a mass concentration of 20% using a system with a pore size cutoff of 50 kDa-150 kDa.

[0070] Prepare 5% hydroxypropyl cellulose solution, add to the above-mentioned aqueous silk fibroin solution, mix in a vortex mixer at 3000 rpm / min for 1 min. Perform ultrasonic treatment with an ultrasonic cell disruptor, amplitude 70%, intermittent ultrasonic (ultrasonic 50 s, stop 10 s), total ultrasonic 5 min.

[0071] Prepare 5% hydroxypropyl methyl cellulose solution, add to the above-mentioned aqueous silk fibroin solution, mix in a vortex mixer at 3000 rpm / min for 1 min. Perform ultrasonic treatment with an ultrasonic cell disruptor, amplitude 70%, intermittent ultrasonic (ultrasonic 50 s, stop 10 s), total ultrasonic 5 min.

[0072] Prepare 5% carboxymethyl cellulose solution, add to the above-mentioned aqueous silk fibroin solution, mix in a vortex mixer at 3000 rpm / min for 1 min. Perform ultrasonic treatment with an ultrasonic cell disruptor, amplitude 70%, intermittent ultrasonic (ultrasonic 50 s, stop 10 s), total ultrasonic 5 min.

[0073] Then, stand at -20℃ to form a gel, homogenize at 28000 rpm / min for 5 min, sieve, and collect the silk fibroin microfiber gel that passes through a 300-mesh sieve.

[0074] Preparation of extracellular scaffold gel material: prepare sodium hyaluronate particles with a molecular weight of 1500-2000 kDa, and mix with the silk fibroin microfiber gel in step 1. The ratio of silk fibroin: cellulose: hyaluronic acid is 4:1:1. Then, crosslink at 47℃ for 2 hours.

[0075] Use PBS buffer with pH value of 7.4 as dialysis liquid, and replace the dialysis liquid every 2 hours or so under magnetic stirring. Dialyze for about 72 hours.

[0076] Fill the gel after dialysis treatment, autoclave, and record as samples 3-1, 3-2, 3-3.

[0077] Table 3 Test data of silk fibroin extracellular scaffold gel material with different types of cellulose

[0078]

[0079] At the same concentration, the gel uniformity of hydroxypropyl cellulose is very poor, with large blocks of gel and a small amount of water mixed in the system, the gel uniformity of hydroxypropyl methyl cellulose is very good, and the elastic modulus is suitable for soft tissue filling, the gel formed by carboxymethyl cellulose has a lot of precipitate, and is not completely gelled, with a large amount of precipitate.

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

[0081] Under the conditions of Example 3, samples with different proportions of components were prepared respectively, and their rheological properties were tested;

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

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

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

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

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

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

[0088] Rheological data test method: using a rheometer at (25±0.2)℃, using a 25mm rotor, a plate gap of 1mm, a deformation of 0.5%, scanning at a shear rate from 0.01 Hz to 100 Hz,

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

[0090] Table 4 Rheological data of controllable silk fibroin extracellular matrix gel prepared by different component proportions

[0091]

[0092] By controlling the component proportion variables, the influence degree of each component on the mechanical properties of the material was analyzed. The increase of the concentration of each component will increase the elastic modulus of the material, and the concentration of silk fibroin has the greatest influence on the mechanical properties of the material and is decisive. In the gelation mechanism we proposed, the β-sheet structure formed between silk fibroin molecules plays a key role in the formation of the gel network. This explains the positive correlation between the gel modulus and the content of silk fibroin found in the rheological experiment. Simply put, the β-sheet structure formed between the silk fibroin molecular chains gradually increases the microfibrils that constitute the gel, which is the gel network throughout the system. The higher the concentration of silk fibroin, the more fibers, the denser the gel network, and the higher the elastic modulus.

[0093] Example 5:

[0094] Preparation of silk fibroin microfiber: choose a system with a pore size of 50 kDa-150 kDa, prepare a regenerated silk fibroin solution with a molecular weight of 50-150 kDa, and ultrasonically treat to obtain sample 5-1. Prepare a 5% hydroxypropyl methylcellulose solution 5ml, add to 12.5ml of the above silk fibroin aqueous solution, and mix in a vortex mixer at 3000rpm / min for 1min. Ultrasonic treatment is carried out by ultrasonic crusher, amplitude 70%, intermittent ultrasonic (ultrasonic 50s, stop 10s), ultrasonic for a total of 5min to obtain sample 5-2. Sample 5-3 is obtained in the manner of example 3. On the basis of sample 5-1, 0.60 g of sodium hyaluronate particles with a molecular weight of 1500 kDa-2000 kDa are added, and mixed to form sample 5-4.

[0095] Table 5 Modulus test results of four different component samples

[0096]

[0097] The modulus of the four samples was tested, and the results are shown in Table 4 below. The test results show that in sample 5-4, when only hyaluronic acid is added to the silk fibroin microfiber, the modulus is only slightly improved. In macroscopic observation, it can also be found that sample 5-4 cannot form a gel scaffold under naked eye. The modulus results of sample 5-2 prove that the intermolecular hydrophobic interaction of cellulose in silk fibroin microfiber makes the modulus greatly improved. In sample 5-3, it can be found that after the physical crosslinking process of the addition of the three components is completed, the sample obtains high modulus, proving that it obtains a stable gel structure.

[0098] Comparative example (CN102836465B):

[0099] (1) Preparation of silk fibroin microspheres: degumming and dissolving of mulberry silk, desalination and concentration, and preparation of a silk fibroin solution with a mass concentration of 5% using purified water.

[0100] At 25°C, the above-mentioned 5% silk fibroin solution was allowed to stand to form a white gel material 5g, which was homogenized by a homogenizer at 24000rpm / min for 5min to obtain silk fibroin gel particles with different diameters 5g, which were filtered and the silk fibroin particles passing through a 200 mesh sieve were collected 3.5g. A small amount of collected silk fibroin particles was dried to constant weight to determine the water content of the silk fibroin particles, and the mass concentration of the silk fibroin particles was determined to be 3.23%.

[0101] (2) Silk fibroin-hyaluronic acid composite gel: 0.7 g of sodium hyaluronate was dissolved in 7 ml of 1% sodium hydroxide solution to prepare a 0.1 g / ml sodium hyaluronate solution, and 0.712 g of silk fibroin particles obtained in step (1) (0.023 / 0.0323 = 0.712 g, the dry weight of the silk fibroin particles was 0.023 g) was added to the sodium hyaluronate solution, and 56 μl (0.056 g) of BDDE (1,4-butanediol diglycidyl ether) was added thereto, and the mixture was mixed uniformly and then was left to stand at 40°C for 4 hours for crosslinking, thereby forming a silk fibroin-HA composite gel material.

[0102] The silk fibroin-HA composite gel material was added to a dialysis bag with MW 8,000-14,000, and PBS buffer with pH 7.4 and temperature 37°C was used as the dialysis solution, and the dialysis treatment was performed under magnetic stirring. The composite gel material after the dialysis treatment was homogenized at 24000 rpm / min for 10 min using a homogenizer, and then was extruded through a 60-mesh sieve using a syringe, and the composite gel particles were collected. After sterilization at 120°C for 15 min using high-temperature high-pressure steam, the sterile packaging was performed, and the product was filled into a disposable syringe. Thus, 35 g of the silk fibroin-hyaluronic acid composite gel for injection was obtained, which can be used as a subcutaneous injection product.

[0103] The test method for the crosslinking agent was as follows: the sample of Example 3-2 and the sample of the comparative example were taken in the same amount and were immersed in PBS, and the solution outside the dialysis bag was taken as the test solution at 2w, 4w and 8w, respectively. The residual amount of BDDE was tested by gas chromatography, and the specific test method was as follows:

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

[0105] 2) Chromatographic conditions

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

[0107] Column temperature: initial temperature 150°C, increased to 260°C at 30°C / min, and maintained for 10 min.

[0108] Detector: FID.

[0109] Carrier gas: N2.

[0110] Injection port temperature: 260°C.

[0111] Detector temperature: 300°C.

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

[0113] 3) Sample pretreatment: The solution to be tested was precisely weighed and placed in a 10 mL volumetric flask. Appropriate amount of acetone was added, shaken and diluted to the mark. The solution was filtered and the filtrate was obtained.

[0114] 4) Sample test: 1 μL of the sample solution and the control solution were precisely measured and injected into the gas chromatograph. The chromatogram was recorded and the peak area was calculated by external standard method.

[0115] Table 6 Crosslinking agent residual data of Example 3-2 and the comparative example

[0116]

[0117] From the crosslinking degree residual data, it can be seen that, since the present application is physical crosslinking and does not contain any crosslinking agent, there is no crosslinking agent residue at each time. In the comparative example, since the crosslinking agent is added, the chemical crosslinking agent is gradually precipitated with the degradation of hyaluronic acid, which may cause inflammatory reaction of the tissue and is harmful to the tissue. It can also be found in Figure 3 that the inflammatory reaction of the comparative example is higher than that of the example.

[0118] Application example:

[0119] The gels obtained from Example 3-2 and the comparative example were implanted subcutaneously in mice, respectively. After a certain period of time, the samples were taken to make pathological sections. HE staining was used to observe the inflammatory reaction of the materials implanted in the body, and immunohistochemical staining was used to observe the promotion of collagen regeneration.

[0120] Table 7 Histopathological evaluation standard for characterizing inflammatory reaction after material implantation

[0121]

[0122] After the materials were implanted in the body, the inflammatory reaction was most severe and obvious around 2 weeks. Figure 5 shows the comparison of inflammatory reactions of mice implanted with Example 3-2 and the comparative example for 2 weeks. The dark dots in the figure are lymphocyte nuclei. It can be seen that there are a large number of lymphocytes infiltrating around the implant of the comparative example, and the inflammatory score is high. There are only a small amount of lymphocytes around Example 3-2, and the inflammatory reaction is small. The biocompatibility of the gel described in the present application is better than that of the comparative example.

[0123] The inflammatory reaction of the implantation experiment of Example 3-2 and the comparative example is shown in Figure 4, the collagen generation promotion rate of the sample in Example 3-2 and the comparative example implanted subcutaneously for different time is shown in Figure 5, and the collagen regeneration promotion ability of the implantation experiment of the sample in Example 3-2 and the comparative example is shown in Figure 6. As shown in the following figure, after the sample in Example 3-2 is implanted in mice, the inflammatory reaction is lower than that of the comparative example, the collagen generation ability is higher than that of the comparative example, and the generation ability evidence can be seen from the immunohistochemical comparison diagram. Therefore, the silk fibroin extracellular skeleton gel material claimed in the application has higher in-situ tissue growth ability than the comparative example due to the similar structure to ECM, and does not produce higher inflammatory reaction, and has good biocompatibility.

[0124] Specifically, Figure 3 is the in-vivo retention rate of the sample in Example 3-2 and the comparative example implanted subcutaneously for different time

[0125] In the early stage of implantation, the hyaluronic acid in the product of the comparative example swells by absorbing water, while the hydrogel described in the application does not swell by absorbing water, and maintains the initial volume, which clinically does not make the facial injection site “steamed”. With the extension of the implantation time, the product of the comparative example degrades quickly, while the hydrogel described in the application has a stable degradation rate.

[0126] Figure 4 is the inflammatory reaction comparison of Example 3-2 and the comparative example mice after being implanted in vivo for 2 weeks. After the sample in Example 3-2 is implanted in mice, the inflammatory reaction is lower than that of the comparative example, which proves that the silk fibroin extracellular skeleton gel material has good biocompatibility due to the similar structure to ECM, and does not produce higher inflammatory reaction.

[0127] Figure 5 is the collagen generation promotion rate of the sample in Example 3-2 and the comparative example implanted subcutaneously for different time. The greater the collagen area ratio, the stronger the function of the product in stimulating skin tissue regeneration.

[0128] Figure 6 is a schematic diagram of the collagen regeneration promotion ability comparison of the sample in Example 3-2 and the comparative example implanted for 14 weeks.

[0129] The pathological section is subjected to immunohistochemical staining to analyze the collagen regeneration promotion ability of the material. The deeper the color and the larger the area, the more collagen, and the better the regeneration effect of the material. Before the material is completely degraded, the more collagen is secreted with the extension of the implantation time. Figure 6 shows the section immunohistochemical staining condition when the sample in Example 3-2 and the comparative example are implanted in mice for 14 weeks. The tissue color of Example 3-2 is obviously darker and the area is larger, so the sample in Example 3-2 has stronger regeneration ability than the comparative example.

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

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

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

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

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

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

[0136] The three-dimensional structure inside the gel, including pore size and porosity, can regulate cell behavior, and the adhesion and proliferation of cells require appropriate porosity. When the product ratio changes, since microfibrils are the key to forming the gel, when the silk fibroin content increases, samples 6-2 and 6-3 have greater porosity. The proliferation of cells on the material can also prove that samples 6-2 and 6-3 have higher cell proliferation rates than 6-1 and 6-4, providing more adhesion sites and a suitable microenvironment for survival, that is, the preparation of silk fibroin extracellular matrix gel is better when 1.5 ≤ silk fibroin / hyaluronate ≤ 1.618 is met.

[0137] Porosity data test method: After freeze-drying, the gel scaffold is placed in a n-hexane solvent with a volume of V1, and the n-hexane is fully filled into the pore structure of the scaffold under a certain negative pressure. At this time, the solvent volume is recorded as V2. Then the scaffold filled with n-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: 96-well plate culture of L929 cells, after 3-5 days of culture, MTT solution was added to each well for treatment. The enzyme-linked immunoassay instrument was set at 490 nm wavelength to determine the absorption value. Cell proliferation rate = (experimental group absorption value - blank control absorption value) / (control group absorption value - blank control absorption value) × 100%.

[0139] Table 8 Porosity and cell proliferation rate data of controllable silk fibroin extracellular matrix gel prepared by different component ratios

[0140]

[0141] By controlling the variable of the proportion of silk fibroin and hyaluronate, the influence on the internal pores of the gel is analyzed. Suitable pore size is essential for tissue formation to facilitate the diffusion of nutrients, oxygen and waste between cells. The hydrogel network with appropriate proportion is stable, has suitable pore size and porosity, and is conducive to the adhesion and growth of cells on the surface. Through the experiment in Example 6, it is found that when 1.5≤ silk fibroin / hyaluronate≤1.618 is met, the silk fibroin extracellular scaffold gel with good porosity and cell proliferation rate can be obtained.

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; The gel material does not include any chemical crosslinking agent.

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; and the hyaluronate solution is prepared by using a 0.1%-1% sodium hydroxide aqueous solution to prepare a hyaluronate solution.

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

5. The material of claim 1, wherein: after the gel material is prepared, it is purified by dialysis, and the dialysis liquid is an isotonic phosphate buffer solution. The ratio of silk fibroin to hyaluronate is greater than or equal to 2:1, 3:1 or 4:1 or more.

6. Material according to one of claims 1 to 5, characterized in that: The gel comprises 5-50 parts of silk fibroin, 0.01-5 parts of cellulose and 0.7-10 parts of hyaluronate.

7. Material according to one of claims 1 to 5, characterized in that: The gel comprises 10-50 parts of silk fibroin, 0.01-5 parts of cellulose and 0.7-5 parts of hyaluronate.

8. Material according to one of claims 1 to 5, characterized in that: 1.5≤silk fibroin / hyaluronate≤1.

618.

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

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

11. The preparation method of claim 10, wherein: (1) the silk fibroin aqueous solution is prepared by placing mulberry silkworm silk in a boiling 0.1-5% sodium carbonate aqueous solution or a mixed solution of sodium carbonate and sodium bicarbonate, drying at 25-100°C, dissolving in a 9.3 mol / L lithium bromide aqueous solution, and water-bathing at 25-100°C for 10-120 minutes until the silk fibroin is fully dissolved, filtering off insoluble particulate impurities with a 50-200 mesh filter bag, desalting and concentrating to obtain a silk fibroin aqueous solution with a specific molecular weight; after a 20% silk fibroin solution is added to a cellulose solution, the same is treated by an ultrasonic crusher; after the gel is formed, it is homogenized at 1500-24000 rpm / min 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.

12. Use of the controllable silk fibroin extracellular matrix gel material according to one of claims 1 to 9, 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.

13. Use according to claim 12, 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.

14. Use according to claim 12, characterized in that: The medical and aesthetic filler or the medical filler is used for face, trunk or limbs.

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