Regenerated silk fibroin-collagen gel injectable soft tissue regeneration filler as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a regenerated silk fibroin-collagen gel injectable soft tissue regeneration filler as well as a preparation method and application thereof. According to the preparation method disclosed by the invention, regenerated silk fibroin-collagen (mRSF-COL) gel particles are prepared by cross-linking SS-PEG-SS and combining sieving and crushing; after the mRSF-COL gel particles are compounded with auxiliary materials such as sodium hyaluronate, the injectable soft tissue regeneration filling agent is prepared. The mRSF has excellent plasticity, provides a controllable and stable structure for the filler, and has excellent biological characteristics, such as good cell compatibility, blood compatibility, biodegradability, non-toxicity and low immunogenicity; the introduction of COL further enhances the stability of the filler structure, and improves the repair and tissue regeneration capacities at the same time, so that the soft tissue filler prepared by the invention has a wide application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to a regenerated silk fibroin-collagen gel injectable soft tissue regeneration filler, and a preparation method and application thereof. Background Art

[0002] As we age, the elasticity and smoothness of the skin will gradually weaken, and wrinkles will form, and the surface gloss will gradually disappear. At the same time, subcutaneous fat on the face will gradually be lost, the thickness of the dermis will decrease, and the soft tissue of the face will be easily loosened and drooped due to the influence of gravity and muscle atrophy. In order to improve this situation, facial soft tissue filling has emerged as a minimally invasive treatment method, which is specifically used to solve problems such as facial volume tissue loss, contour deformation and static wrinkles. Facial soft tissue filling can not only effectively improve the beauty of the face, but also restore the firmness and elasticity of the skin, making people rejuvenated. At present, facial soft tissue filling materials are mainly non-autologous tissue injections, which are easy to operate and have significant effects. The ideal filling material must have excellent biocompatibility, significant effectiveness and high safety to ensure that the filling effect is natural and lasting without causing any harm to the human body.

[0003] Silk fibroin (SF) is a natural protein derived from the endothelial cells of the silk gland in the silkworm. Due to its excellent biocompatibility, processability and chemical modifiability, it has attracted extensive attention in various fields such as biomedicine, tissue engineering, food processing, photochemistry and biosensing. SF is one of the main components of silk, accounting for about 70%-80% of the total weight of silk. Mulberry silk is currently the main source of SF. Regenerated silk fibroin (mRSF) is a protein material obtained by extracting, dissolving and reprocessing SF from natural silk. Different processing processes can obtain different properties and structures. mRSF exhibits excellent biocompatibility, that is, it does not produce obvious immune response when interacting with biological tissues. In addition, the biodegradation rate of mRSF can be adjusted within a controllable range, which means that its decomposition rate can be adjusted as needed. It also has strong cell adhesion ability, which helps cells grow and attach to its surface. Under mild processing conditions, mRSF can be made into various forms of biomaterials. This means it can be molded into a variety of desired shapes and forms, making it ideal for use in medical aesthetics such as facial soft tissue augmentation.

[0004] Collagen (COL) is one of the most abundant proteins in the human body and is the main component of connective tissue. At present, collagen products on the domestic and foreign markets are widely used in the field of clinical medicine, covering the repair and reconstruction of skin, bones, cartilage, cardiovascular system, oral cavity and luminal tissue, as well as medical cosmetic surgery and other fields. Among them, the repeating units in the amino acid sequence of recombinant human collagen III (rhCOL-III) are highly consistent with the specific functional regions in the amino acid sequence of human collagen, which makes it have good biocompatibility and can be seamlessly connected with human tissue, showing great application potential in the field of facial soft tissue filling. Animal-derived collagen is widely used in anti-aging, promoting skin elasticity, improving joint function, promoting bone density, etc. It is also an important material for facial filling, especially for filling nasolabial folds, nasolabial folds, temples and other parts. Animal-derived type I collagen (COL-I) has better biocompatibility, biodegradability and weak antigenicity. It can not only activate coagulation factors in animals and promote blood coagulation, but also serve as a growth matrix for fibroblasts and vascular endothelial cells, which is beneficial to cell proliferation and accelerates the healing of damaged tissues. COL can help restore the three-dimensional sense and elasticity of the face and make the facial lines softer through injection. It has a broad application prospect in the field of soft tissue injection filling.

[0005] The commonly used commercial cross-linking method, glutaraldehyde, is highly toxic. BDDE needs to be cross-linked under alkaline conditions, but alkaline conditions will make COL unstable and decompose. Polyethylene glycol disuccinimidyl succinate (SS-PEG-SS) is a polymer derivative with hydrophilicity, low toxicity and biocompatibility. It is widely used in the fields of biomedicine, pharmaceuticals and materials science, especially in drug delivery and biomarkers. It has important application potential. The molecular structure of SS-PEG-SS carries multiple reactive groups (succinate and imine groups), which can react chemically with functional groups in other molecules to achieve cross-linking. Therefore, it is possible to consider introducing SS-PEG-SS into the gel structure to promote cross-linking.

[0006] In summary, this study combined the advantages of mRSF and COL, cross-linked them through SS-PEG-SS, and prepared them into regenerated silk fibroin-collagen (mRSF-COL) gel injectable soft tissue regeneration filler. This composite gel makes up for the defects of traditional soft tissue fillers, with stable structure, moderate swelling degree, high elastic modulus, good needle permeability, significant repair effect, and many other advantages, including a wide range of clinical applications, diversified production process selection, and controllable production costs. Summary of the invention

[0007] In view of the deficiencies of the prior art, the present invention provides a regenerated silk fibroin-collagen gel injectable soft tissue regeneration filler and a preparation method and application thereof.

[0008] The technical scheme of the present invention is as follows: a regenerated silk fibroin-collagen gel injectable soft tissue regeneration filler, which is composed of 4 parts of mRSF-COL gel particles and 10 parts of HA solution by weight.

[0009] In some embodiments, the mass concentration of the HA solution is 0.02-0.2 g / mL.

[0010] In some embodiments, mRSF-COL gel particles are prepared by the following method:

[0011] 1) Add mRSF to PBS and stir to fully dissolve to obtain an mRSF solution; add COL to PBS and stir to fully dissolve the COL solution; add SS-PEG-SS to PBS and ultrasonically dissolve to fully dissolve to obtain an SS-PEG-SS solution;

[0012] 2) stirring and mixing the mRSF solution and the COL solution to obtain a mRSF-COL mixed solution; adding the SS-PEG-SS solution to the mRSF-COL mixed solution, stirring and mixing to obtain a mRSF-COL gel;

[0013] 3) The mRSF-COL gel is crushed into small pieces, and then the gel is stirred and washed with a PBS solution, the washed gel is collected by filtration, and then sieved and crushed for the first time; the gel is continued to be stirred and washed with a PBS solution; the washed gel is collected by filtration, and then sieved and crushed for the second time to obtain mRSF-COL gel particles.

[0014] Preferably, the mass concentration of the mRSF solution in step 1) is 150-350 mg / mL, preferably 200 mg / mL.

[0015] Preferably, in step 1), COL is selected from COL-I or rhCOL-III; the mass concentration of the COL solution is 50-200 mg / mL, preferably 200 mg / mL.

[0016] Preferably, in step 1), the mass concentration of SS-PEG-SS is 100-700 mg / mL, preferably 500 mg / mL.

[0017] Preferably, in step 2), the mixing volume ratio of the mRSF solution to the COL solution is 2:1 to 1:2, preferably 1:1.

[0018] Preferably, in step 2), the volume ratio of the SS-PEG-SS solution to the mRSF-COL mixed solution is 1:1 to 1:9, preferably 1:9.

[0019] Preferably, in step 2), the cross-linking reaction temperature is 25 to 37° C., preferably 37° C.; the cross-linking reaction time is 1 to 6 hours, preferably 4 hours.

[0020] Preferably, in step 3), the aperture of the sieve used in the first screening and crushing is 3.2 mm; and the aperture of the sieve used in the second screening and crushing is 200 μm.

[0021] In some embodiments, the preparation method of the regenerated silk fibroin-collagen gel injectable soft tissue regeneration filler is: adding HA to PBS, stirring and dissolving evenly to obtain a HA solution; adding mRSF-COL gel particles to the HA solution, stirring and dissolving evenly; after compounding, filling with a prefilled syringe, and sterilizing with wet heat to obtain the result.

[0022] Preferably, the mass concentration of the mRSF-COL gel particles in the HA solution is 10% to 50%, preferably 40%.

[0023] Preferably, the moist heat sterilization temperature is 121° C. and the sterilization time is 12 min.

[0024] The present invention provides use of the regenerated silk fibroin-collagen gel injectable soft tissue regeneration filler in preparing tissue filling materials.

[0025] The present invention provides use of the regenerated silk fibroin-collagen gel injectable soft tissue regeneration filler in preparing tissue regeneration medical cosmetic filling materials.

[0026] Beneficial effects of the present invention: According to the scheme of the embodiment of the present invention, regenerated silk fibroin-collagen (mRSF-COL) gel particles are prepared by cross-linking technology combined with screening and crushing. Further, the composite microgel is compounded with auxiliary materials such as sodium hyaluronate to prepare an injectable soft tissue regeneration filler. mRSF has excellent plasticity, provides a controllable and stable structure for the filler, and has excellent biological properties, such as good cell compatibility, blood compatibility, biodegradability, non-toxicity and low immunogenicity. The introduction of COL further enhances the stability of the filler structure, while improving the repair and tissue regeneration capabilities, and has broad application prospects in the direction of soft tissue fillers.

[0027] The regenerated silk fibroin-collagen (mRSF-COL) gel injectable soft tissue regeneration filler prepared in the present invention has the advantages of excellent biocompatibility, accelerated cell proliferation, and promoted angiogenesis. In addition, the injectable soft tissue regeneration filler prepared by compounding the composite microgel with the sodium hyaluronate auxiliary material not only has the advantages of outstanding filling effect and good plasticity when used as a tissue engineering and regenerative medicine material, but also the composite microgel in the filler can induce the regeneration of tissue fibroblasts and collagen, prolonging the effect maintenance time of the filler, and is an innovative tissue engineering and regenerative medicine material. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 A schematic flow chart showing a method for preparing an mRSF-COL gel injectable soft tissue regeneration filler according to one embodiment of the present invention;

[0029] Figure 2 The scanning electron microscope image of the freeze-dried mRSF-COL gel prepared according to Example 8 of the present invention is shown;

[0030] Figure 3 The scanning electron microscope image of the freeze-dried mRSF-COL gel particles prepared according to Example 8 of the present invention is shown;

[0031] Figure 4 The particle size distribution diagram of mRSF-COL gel particles prepared according to Example 8 of the present invention is shown;

[0032] Figure 5 The pushing force data of the mRSF-COL gel injectable soft tissue regeneration filler prepared according to Example 8 of the present invention is shown;

[0033] Figure 6 The cytotoxicity data of the mRSF-COL injectable gels prepared according to Examples 7-8 of the present invention and Comparative Examples 1-2 are shown;

[0034] Figure 7 The cell live-death staining of the mRSF-COL gel injectable soft tissue regeneration filler prepared according to Examples 7 and 8 of the present invention is shown;

[0035] Figure 8 The cell proliferation data of the mRSF-COL gel injectable soft tissue regeneration filler prepared according to Examples 7 and 8 of the present invention are shown;

[0036] Fig. 9 The anatomical picture of the mRSF-COL gel injectable soft tissue regeneration filler prepared according to Example 8 of the present invention injected into the subcutaneous tissue of rats for 3 months is shown;

[0037] Fig.10 The H&E staining picture of tissue sections of rats injected with the mRSF-COL gel injectable soft tissue regeneration filler prepared according to Example 8 of the present invention for 3 months is shown;

[0038] Fig.11 The Masson staining picture of the tissue section of the rat injected with the mRSF-COL gel injectable soft tissue regeneration filler prepared according to Example 8 of the present invention for 3 months;

[0039] Fig.12 The CD31 immunofluorescence staining picture of tissue sections of rats injected with the mRSF-COL gel injectable soft tissue regeneration filler prepared according to Example 8 of the present invention for 3 months is shown;

[0040] Related Notes

[0041] mRSF: regenerated silk fibroin; COL: collagen; COL-I: type I collagen; rhCOL-III: recombinant humanized type III collagen; SS-PEG-SS: polyethylene glycol disuccinimidyl succinate; PBS: phosphate buffered solution. DETAILED DESCRIPTION

[0042] The following embodiments can enable those skilled in the art to more fully understand the present invention, but the present invention is not limited to the scope of the embodiments.

[0043] Example 1

[0044] A method for preparing an mRSF-COL gel injectable soft tissue regeneration filler, the preparation method comprising the following steps:

[0045] Step S1: Add 0.3g mRSF to 0.9mL PBS and stir to fully dissolve; add 0.06g rhCOL-III to 0.9mL PBS and stir to fully dissolve; add 0.1g SS-PEG-SS to 0.2Ml PBS and ultrasonically dissolve; mix the above 0.9mL mRSF solution and 0.9mL rhCOL-III solution and stir to mix evenly; add the above 0.2mL SS-PEG-SS solution thereto, stir to mix evenly, and stand at 37°C for cross-linking for 4h to obtain mRSF-COL gel with poor mechanical properties.

[0046] Example 2

[0047] A method for preparing an injectable soft tissue regeneration filler of mRSF-COL gel, the method comprising the following steps: step S1: adding 0.288g of mRSF to 0.9mL of PBS, stirring to fully dissolve; adding 0.072g of rhCOL-III to 0.9mL of PBS, stirring to fully dissolve; adding 0.1g of SS-PEG-SS to 0.2mL of PBS, ultrasonically fully dissolving; mixing the above 0.9mL of mRSF solution and 0.9mL of rhCOL-III solution, stirring to mix evenly; adding the above 0.2mL of SS-PEG-SS solution thereto, stirring to mix evenly, and standing at 37°C for cross-linking for 4h to obtain mRSF-COL gel with poor mechanical properties.

[0048] Example 3

[0049] A method for preparing an injectable soft tissue regeneration filler of mRSF-COL gel, the preparation method comprising the following steps: step S1: adding 0.27g mRSF to 0.9mL PBS, stirring to fully dissolve; adding 0.09g rhCOL-III to 0.9mL PBS, stirring to fully dissolve; adding 0.1g SS-PEG-SS to 0.2mL PBS, ultrasonically fully dissolving; mixing the above 0.9mL mRSF solution and 0.9mL rhCOL-III solution, stirring to mix evenly; adding the above 0.2mL SS-PEG-SS solution thereto, stirring to mix evenly, and standing at 37°C for crosslinking for 2h to obtain mRSF-COL gel with poor mechanical properties.

[0050] Example 4

[0051] A method for preparing an mRSF-COL gel injectable soft tissue regeneration filler, the preparation method comprising the following steps: step S1: adding 0.24g mRSF to 0.9mL PBS, stirring to fully dissolve; adding 0.12g rhCOL-III to 0.9mL PBS, stirring to fully dissolve; adding 0.1g SS-PEG-SS to 0.2mL PBS, ultrasonically fully dissolving; mixing the above 0.9mL mRSF solution and 0.9mL rhCOL-III solution, stirring to mix evenly; adding the above 0.2mL SS-PEG-SS solution thereto, stirring to mix evenly, and standing at 37°C for crosslinking for 2h to obtain mRSF-COL gel, which still has poor mechanical properties.

[0052] Example 5

[0053] A method for preparing an injectable soft tissue regeneration filler of mRSF-COL gel, the method comprising the following steps: step S1: adding 0.18g mRSF to 0.9mL PBS, stirring to fully dissolve; adding 0.18g rhCOL-III to 0.9mL PBS, stirring to fully dissolve; adding 0.1g SS-PEG-SS to 0.2mL PBS, ultrasonically fully dissolving; mixing the above 0.9mL mRSF solution and 0.9mL rhCOL-III solution, stirring to mix evenly; adding the above 0.2mL SS-PEG-SS solution thereto, stirring to mix evenly, and standing at 37°C for cross-linking for 1h to obtain mRSF-COL gel.

[0054] Example 6

[0055] A method for preparing an mRSF-COL gel injectable soft tissue regeneration filler, the preparation method comprising the following steps: step S1: adding 0.18g mRSF to 0.9mL PBS, stirring to fully dissolve; adding 0.18g COL-I to 0.9mL PBS, stirring to fully dissolve; adding 0.1g SS-PEG-SS to 0.2mL PBS, ultrasonically fully dissolving; mixing the above 0.9mL mRSF solution and 0.9mL COL-I solution, stirring to mix evenly; adding the above 0.2mL SS-PEG-SS solution thereto, stirring to mix evenly, and immediately obtaining mRSF-COL gel.

[0056] Example 7

[0057] A method for preparing an mRSF-COL gel injectable soft tissue regeneration filler, the preparation method comprising the following steps:

[0058] Step S1: Add 0.18 g mRSF to 0.9 mL PBS and stir to fully dissolve; add 0.18 g rhCOL-III to 0.9 mL PBS and stir to fully dissolve; add 0.1 g SS-PEG-SS to 0.2 mL PBS and ultrasonically dissolve; mix the above 0.9 mL mRSF solution and 0.9 mL rhCOL-III solution and stir to mix evenly; add the above 0.2 mL SS-PEG-SS solution thereto, stir to mix evenly, and stand at 37°C for cross-linking for 1 hour to obtain mRSF-COL gel.

[0059] Step S2: The cross-linked gel is crushed into small pieces, and then the gel is stirred and washed with PBS solution for 2 times, each time for 1 hour, to remove the residual catalyst and cross-linking agent in the gel; the washed gel is collected by filtration and passed through a 50-mesh stainless steel sieve; the gel is continued to be stirred and washed with PBS solution for 3 times, each time for 1 hour; the washed gel is collected by filtration and passed through an 80-mesh stainless steel sieve to obtain mRSF-COL gel particles.

[0060] Step S3: Add 0.2 g HA to 10 mL PBS and stir to dissolve evenly; add 4 g mRSF-COL gel particles prepared in step S2 to the above 10 mL HA solution and stir to dissolve evenly; after compounding, fill with a 1 mL prefilled syringe and sterilize with wet heat at 121°C for 12 min to obtain the mRSF-COL gel injectable soft tissue regeneration filler.

[0061] Example 8

[0062] A method for preparing an mRSF-COL gel injectable soft tissue regeneration filler, the preparation method comprising the following steps:

[0063] Step S1: Add 0.18 g mRSF to 0.9 mL PBS and stir to fully dissolve; add 0.18 g COL-I to 0.9 mL PBS and stir to fully dissolve; add 0.1 g SS-PEG-SS to 0.2 mL PBS and ultrasonically dissolve; mix the above 0.9 mL mRSF solution and 0.9 mL COL-I solution and stir to mix evenly; add the above 0.2 mL SS-PEG-SS solution thereto and stir to mix evenly to immediately obtain mRSF-COL gel.

[0064] Step S2: The cross-linked gel is crushed into small pieces, and then the gel is stirred and washed with PBS solution for 2 times, each time for 1 hour, to remove the residual catalyst and cross-linking agent in the gel; the washed gel is collected by filtration and passed through a 50-mesh stainless steel sieve; the gel is continued to be stirred and washed with PBS solution for 3 times, each time for 1 hour; the washed gel is collected by filtration and passed through an 80-mesh stainless steel sieve to obtain mRSF-COL gel particles.

[0065] Step S3: Add 0.2 g HA to 10 mL PBS and stir to dissolve evenly; add 4 g mRSF-COL gel particles prepared in step S2 to the above 10 mL HA solution and stir to dissolve evenly; after compounding, fill with a 1 ml prefilled syringe and sterilize with wet heat at 121°C for 12 min to obtain the mRSF-COL gel injectable soft tissue regeneration filler.

[0066] Comparative Example 1

[0067] Step S1: add 0.18g rhCOL-III to 1mL PBS and stir to fully dissolve; add 0.18g mRSF to 1mL PBS and stir to fully dissolve; add 125mg EDC to the mRSF solution and stir for 30min, then add 85mg NHS to the mRSF solution and stir for 20min to obtain a mRSF mixed solution; add the rhCOL-III solution dropwise to the mRSF mixed solution, stir at a high speed of 1500r to gel the solution, then stop stirring and let stand for 48h to obtain a mRSF-COL-NHS / EDC gel.

[0068] Step S2: the cross-linked gel is crushed into small pieces, and then the gel is stirred and washed with PBS solution for 2 times, each time for 1 hour, to remove the residual catalyst and cross-linking agent in the gel; the washed gel is collected by filtration and passed through a 50-mesh stainless steel sieve; the gel is continued to be stirred and washed with PBS solution for 3 times, each time for 1 hour; the washed gel is collected by filtration and passed through an 80-mesh stainless steel sieve to obtain mRSF-COL-NHS / EDC gel particles.

[0069] Step S3: Add 0.2 g HA to 10 mL PBS and stir to dissolve evenly; add 4 g mRSF-COL-NHS / EDC gel particles prepared in step S2 to the above 10 mL HA solution and stir to dissolve evenly; after compounding, fill with a 1 ml prefilled syringe and sterilize with wet heat at 121°C for 12 min to obtain mRSF-COL-NHS / EDC injectable gel.

[0070] Comparative Example 2

[0071] Step S1: Add 0.18g COL-I to 1mL PBS and stir to fully dissolve; add 0.18g mRSF to 1ml PBS and stir to fully dissolve; add 125mg EDC to the mRSF solution and stir for 30min, then add 85mg NHS to the mRSF solution and stir for 20min to obtain a mRSF mixed solution; add the COL-I solution dropwise to the mRSF mixed solution, stir at a high speed of 1500r to gel the solution, then stop stirring and let stand for 48h to obtain a mRSF-COL-NHS / EDC gel.

[0072] Step S2: the cross-linked gel is crushed into small pieces, and then the gel is stirred and washed with PBS solution for 2 times, each time for 1 hour, to remove the residual catalyst and cross-linking agent in the gel; the washed gel is collected by filtration and passed through a 50-mesh stainless steel sieve; the gel is continued to be stirred and washed with PBS solution for 3 times, each time for 1 hour; the washed gel is collected by filtration and passed through an 80-mesh stainless steel sieve to obtain mRSF-COL-NHS / EDC gel particles.

[0073] Step S3: Add 0.2g HA to 10mL PBS and stir to dissolve evenly; add 4g mRSF-COL-NHS / EDC gel particles prepared in step S2 to the above 10mL HA solution and stir to dissolve evenly; after compounding, fill with a 1ml prefilled syringe and sterilize with wet heat at 121℃ for 12min to obtain mRSF-COL-NHS / EDC injectable gel. Performance analysis of mRSF-COL gel injectable soft tissue regeneration filler:

[0074] Figure 2 The scanning electron microscope image of the freeze-dried mRSF-COL gel prepared in Example 8 is shown. It can be seen from the image that the mRSF-COL gel has a porous network structure with a pore size of about 20-50 μm.

[0075] Figure 3 The scanning electron microscope image of the mRSF-COL gel particles prepared in Example 8 after freeze-drying is shown.

[0076] Figure 4 The particle size distribution diagram of the mRSF-COL gel prepared in Example 8 is shown. From the diagram, it can be observed that the particle size distribution of the mRSF-COL gel is around 100 μm.

[0077] Figure 5 The pushing force data of the mRSF-COL gel injectable soft tissue regeneration filler prepared in Example 8 are shown. It can be seen from the figure that the average pushing force of the filler is 9.44N, the maximum pushing force is 18.74N, and the minimum pushing force is 6.19N. The pushing force value of the filler meets the national standard for soft tissue fillers. In contrast, the maximum pushing forces that the mRSF-COL-NHS / EDC injectable gel prepared in Comparative Examples 1-2 can withstand are 156.98N and 198.21N, respectively, and cannot be successfully pushed out.

[0078] Test Example 1

[0079] Fibroblasts were plated in 96-well cell culture plates and cell culture dishes, with approximately 4,000 cells per well. After 24 hours, the cells were completely attached to the wall and incubated with the extract of mRSF-COL gel injectable soft tissue regeneration filler. After 24 hours, CCK-8 detection reagent was added to the 96-well plate, and the cell absorbance was detected by an ELISA instrument to calculate the cell survival rate.

[0080] Figure 6The cytotoxicity data of the mRSF-COL injectable gel prepared in Examples 7-8 and Comparative Examples 1-2 are shown. It can be seen from the figure that the cell survival rate of the mRSF-COL injectable gel prepared in Comparative Examples 1 and 2 is above 75%, and has good cell compatibility. The cell survival rate of the mRSF-COL gel injectable soft tissue regeneration filler group prepared in Examples 7 and 8 is above 120%, showing the ability to promote cell proliferation, which is more conducive to tissue regeneration and repair.

[0081] Test Example 2

[0082] Fibroblasts were plated in a cell culture dish, with about 100,000 cells per well. After 24 hours, the cells were completely attached to the wall and incubated with the extract of mRSF-COL gel injectable soft tissue regeneration filler. After 24 hours, the cells in the cell culture dish were stained with a live / dead cell kit, with live cells stained green and dead cells stained red to detect cell viability.

[0083] Figure 7 The live / dead cell staining pictures of the mRSF-COL gel injectable soft tissue regeneration filler prepared in Example 7 and Example 8 are shown. It can be seen from the picture that the number of live cells in the mRSF-COL gel injectable soft tissue regeneration filler group prepared in Example 7 and Example 8 is consistent with that in the control group, and almost no dead cells are observed, showing good cell compatibility.

[0084] Test Example 3

[0085] Fibroblasts were plated in a 24-well cell culture plate, with approximately 20,000 cells per well. After 24 hours, the cells were completely attached to the wall, and mRSF-COL gel injectable soft tissue regeneration filler was added to co-culture with the cells. After 1, 2, and 3 days of co-culture, CCK-8 detection reagent was added to the 24-well plate, and the cell absorbance was detected by an ELISA instrument to calculate the cell survival rate.

[0086] Figure 8 The cell proliferation data of the mRSF-COL gel injectable soft tissue regeneration filler prepared in Example 7 and Example 8 are shown. It can be seen from the figure that the mRSF-COL gel injectable soft tissue regeneration filler prepared in Example 7 and Example 8 can effectively promote the proliferation of fibroblasts in vitro, among which the mRSF-COL gel injectable soft tissue regeneration filler prepared in Example 8 has a more significant effect in promoting cell proliferation.

[0087] Test Example 4

[0088] The mRSF-COL gel injectable soft tissue regeneration filler prepared in Example 8 was implanted subcutaneously into Sprague Dawley rats. The rats were killed at the first and third months, and the skin tissue implanted with the hydrogel was removed and fixed for hematoxylin-eosin (H&E), Masson's trichrome, and immunofluorescence (IF) staining.

[0089] Fig. 9 The diagram shows the tissue extraction pictures of the mRSF-COL gel injectable soft tissue regeneration filler prepared in Example 8 after being implanted subcutaneously in rats at the 1st and 3rd months. It can be seen from the diagram that the implant has not been completely degraded 3 months after being implanted in the tissue.

[0090] Fig.10 H&E staining images of tissue sections extracted from rats at 1 month and 3 months after the mRSF-COL gel injectable soft tissue regeneration filler prepared in Example 8 was implanted subcutaneously. From the images, it can be seen that a small amount of inflammatory cells and fibroblasts exist around the implant, indicating that the mRSF-COL gel injectable soft tissue regeneration filler has good biocompatibility and will not cause severe local tissue inflammation.

[0091] Fig.11 The Masson staining images of the extracted tissue sections of the mRSF-COL gel injectable soft tissue regeneration filler prepared in Example 8 implanted subcutaneously in rats at 1 month and 3 months are shown. From the images, it can be seen that a large amount of collagen fiber regeneration is newly generated in the tissue surrounding the mRSF-COL gel injectable soft tissue regeneration filler.

[0092] Fig.12 The CD31 immunofluorescence staining images of the extracted tissue sections of the rats at 1 month and 3 months after the mRSF-COL gel injectable soft tissue regeneration filler prepared in Example 8 was implanted subcutaneously are shown. From the images, it can be seen that there are obvious CD31 vascular endothelial cell membrane proteins distributed around and inside the mRSF-COL gel injectable soft tissue regeneration filler, indicating that the mRSF-COL gel injectable soft tissue regeneration filler can induce the growth of autologous vascular tissue.

Claims

1. A regenerated silk fibroin-collagen gel injectable soft tissue regeneration filler, characterized in that: The composition is composed of 4 parts of mRSF-COL gel particles and 10 parts of HA solution by weight, wherein the mass concentration of the HA solution is 0.02-0.2 g / mL.

2. The injectable soft tissue regeneration filler according to claim 1, characterized in that: mRSF-COL gel particles were prepared by the following method: 1) Add mRSF to PBS and stir to fully dissolve to obtain mRSF solution; add COL to PBS and stir to fully dissolve COL solution; add SS-PEG-SS to PBS and ultrasonically dissolve to fully dissolve to obtain SS-PEG-SS solution; 2) stirring and mixing the mRSF solution and the COL solution to obtain a mRSF-COL mixed solution; adding the SS-PEG-SS solution to the mRSF-COL mixed solution, stirring and mixing to obtain a mRSF-COL gel; 3) The mRSF-COL gel was crushed into small pieces, and then the gel was washed with PBS solution, and the washed gel was collected by filtration and crushed by the first sieving; Continue to wash the gel with PBS solution; The washed gel was collected by filtration and sieved for a second time to obtain mRSF-COL gel particles.

3. The injectable soft tissue regeneration filler according to claim 2, characterized in that: The mass concentration of the mRSF solution in step 1) is 150-350 mg / mL, preferably 200 mg / mL; the COL in step 1) is selected from COL-I or rhCOL-III; the mass concentration of the COL solution is 50-200 mg / mL, preferably 200 mg / mL; the mass concentration of the SS-PEG-SS in step 1) is 100-700 mg / mL, preferably 500 mg / mL.

4. The injectable soft tissue regeneration filler according to claim 2, characterized in that: In step 2), the mixing volume ratio of the mRSF solution to the COL solution is 2:1 to 1:2, preferably 1:

1.

5. The injectable soft tissue regeneration filler according to claim 2, characterized in that: In step 2), the volume ratio of SS-PEG-SS solution to mRSF-COL mixed solution is 1:1 to 1:9, preferably 1:9; the cross-linking reaction temperature in step 2) is 25 to 37° C., preferably 37° C.; the cross-linking reaction time is 1 to 6 hours, preferably 4 hours.

6. The injectable soft tissue regeneration filler according to claim 2, characterized in that: In step 3), the aperture of the sieve used in the first screening and crushing is 3.2 mm; the aperture of the sieve used in the second screening and crushing is 200 μm.

7. The preparation method of the regenerated silk fibroin-collagen gel injectable soft tissue regeneration filler according to claim 1 is as follows: adding HA to PBS, stirring and dissolving uniformly to obtain a HA solution; adding mRSF-COL gel particles to the HA solution, stirring and dissolving uniformly; and filling with a prefilled syringe after compounding, and sterilizing with wet heat to obtain the product.

8. The preparation method according to claim 7, characterized in that: The mass concentration of mRSF-COL gel particles in the HA solution is 10% to 50%, preferably 40%; the wet heat sterilization temperature is 121° C.; and the sterilization time is 12 minutes.

9. Use of the regenerated silk fibroin-collagen gel injectable soft tissue regeneration filler according to claim 1 in preparing tissue filling materials.

10. Use of the regenerated silk fibroin-collagen gel injectable soft tissue regeneration filler according to claim 1 in the preparation of tissue regeneration medical cosmetic filling materials.

Images