A regenerated silk fibroin solid microsphere scaffold and an injectable soft tissue regeneration filler

Abstract

The present invention provides a regenerated silk fibroin solid microsphere scaffold and an injectable soft tissue regeneration filler thereof, wherein the injectable soft tissue filler is prepared by compounding a double-crosslinked regenerated silk fibroin solid microsphere scaffold and a crosslinked sodium hyaluronate gel microsphere. When used as a filling material, it has the immediate filling effect of the crosslinked sodium hyaluronate gel and good plasticity. At the same time, the regenerated silk fibroin solid microsphere scaffold in the filler can also stimulate the regeneration of tissue fibroblasts and collagen, prolonging the effect maintenance time of the filler, and is an innovative tissue regeneration medical cosmetic filling material.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical preparations, and in particular to a regenerated silk fibroin solid microsphere scaffold and an injectable soft tissue regeneration filler thereof. Background Art

[0002] With the rapid development of the medical beauty industry in recent years, injectable soft tissue filling materials have received more and more attention. By injecting soft tissue filling materials, facial beauty effects such as cheek augmentation, improvement of nasolabial folds, full jaw, tight chin contour, and rhinoplasty can be achieved. At present, the most commonly used soft tissue filling material in the medical beauty industry, cross-linked sodium hyaluronate gel, has good immediate filling and shaping effects. However, due to the rapid degradation rate of sodium hyaluronate in the tissue environment, and the low hardness of the single-property gel material itself, it does not have the tissue regeneration function of stimulating the growth of tissue cells and the production of collagen. As a result, patients usually need repeated injections every 6 months or so, which greatly increases the treatment costs and adverse feelings of patients.

[0003] Since regenerated silk fibroin was approved as a biomaterial by the U.S. Food and Drug Administration (FDA) in 1993, it has been widely used in the fields of biomedicine and tissue engineering due to its excellent mechanical properties, good biocompatibility, biodegradability and versatility of structural adjustment. Regenerated silk fibroin can be processed into materials with different structures such as solutions, silk films, microspheres / microparticles, hydrogels, scaffolds and electrospun membranes, and used in the fields of wound repair, drug carriers, tissue filling and artificial organs. In recent years, regenerated silk fibroin products that have been launched on the market include the SERI surgical stent developed by Allergan for abdominal wall reconstruction and plastic surgery. Sofregen, an American company, has developed a SilkVoice natural silk protein injection for the treatment of vocal cord medialization and vocal cord insufficiency. Suzhou Soho Biomaterials Co., Ltd. has developed a wound dressing made of regenerated silk fibroin for the healing and repair of scald and burn wounds. In addition to the products that have been launched on the market, some companies have also conducted clinical trials on their products, including regenerated silk fibroin bandages for ligament reconstruction, regenerated silk fibroin tissue meshes for femoral groove hernia, regenerated silk fibroin sutures for oral surgery, regenerated silk fibroin porous tissue regeneration scaffolds for meniscus cartilage repair, etc. At present, there is still a blank in the preparation of regenerated silk fibroin for facial plastic surgery and filling products.

[0004] When regenerated silk fibroin is used as an injectable tissue filling material, it needs to be prepared into hydrogel microspheres or solid microsphere scaffolds that can be injected by a syringe, and then injected into the tissue to achieve the effect of tissue filling. Since the internal molecular structure distribution of regenerated silk fibroin hydrogel microspheres is relatively loose, it is very easy to be quickly decomposed by the tissue microenvironment and various proteases after being implanted into the body tissue, thus failing to achieve a long-term tissue filling effect. According to relevant research reports, the gel volume of regenerated silk fibroin gel microspheres decreased by about 50% after being implanted subcutaneously in rats for 3 months. Compared with regenerated silk fibroin hydrogel microspheres, regenerated silk fibroin solid microsphere scaffolds have a more dense and stable molecular structure, and their in vivo degradation cycle is as long as more than 1 year. In addition, the regenerated silk fibroin solid microsphere scaffold has a higher hardness, which can effectively stimulate the proliferation of tissue fibroblasts and collagen secretion in the tissue, and stimulate autologous tissue regeneration. Therefore, the regenerated silk fibroin solid microsphere scaffold is more suitable as a tissue regeneration filling material for applications in the field of facial plastic surgery.

[0005] At present, the preparation method of regenerated silk fibroin solid microsphere scaffold is mainly to prepare regenerated silk fibroin into hydrogel microspheres by emulsion method, self-assembly method, ion coagulation method, phase separation method and micro-preparation method, and then dry the hydrogel microspheres and prepare them into solid microsphere scaffolds. The common characteristics of the above methods are complicated preparation process, low preparation efficiency, and not suitable for industrialized scale-up production. In particular, in the preparation method of the above regenerated silk fibroin solid microsphere scaffold, after dissolving the degummed silk in an alkaline inorganic salt solution, it is first necessary to remove the inorganic salt in the solution through a long and multiple dialysis purification process, and then obtain the regenerated silk fibroin solution for preparing the solid microsphere scaffold. As is known to all, dialysis purification takes a long time, consumes a large amount of water, is prone to secondary pollution, and has a small sample processing volume, which is not suitable for large-scale, industrialized product mass production processes. In addition, the internal molecules of the regenerated silk fibroin solid microsphere scaffold prepared by the above method are mostly single β-folded physical cross-linked structures, and their structural stability and degradation cycle still need to be improved. Summary of the invention

[0006] In view of the deficiencies of the prior art, the present invention provides a regenerated silk fibroin solid microsphere scaffold and an injectable soft tissue regeneration filler thereof.

[0007] The technical scheme of the present invention is as follows: an injectable soft tissue regeneration filler, comprising a double-cross-linked regenerated silk fibroin solid microsphere scaffold, cross-linked sodium hyaluronate gel microspheres and mannitol.

[0008] Preferably, the injectable soft tissue regeneration filler comprises, by weight, 5-30 parts of double-crosslinked regenerated silk fibroin solid microsphere scaffold, 100 parts of crosslinked sodium hyaluronate gel microspheres and 5.25-6.5 parts of mannitol.

[0009] Preferably, the injectable soft tissue regeneration filler is composed of 5 parts of double-crosslinked regenerated silk fibroin solid microsphere scaffold, 100 parts of cross-linked sodium hyaluronate gel microspheres and 5.25 parts of mannitol by weight.

[0010] Preferably, the injectable soft tissue regeneration filler is composed of 10 parts by weight of double-crosslinked regenerated silk fibroin solid microsphere scaffold, 100 parts of cross-linked sodium hyaluronate gel microspheres and 5.5 parts of mannitol.

[0011] Preferably, the injectable soft tissue regeneration filler is composed of 30 parts by weight of double-crosslinked regenerated silk fibroin solid microsphere scaffold, 100 parts of cross-linked sodium hyaluronate gel microspheres and 6.5 parts of mannitol.

[0012] In some embodiments, the present invention provides a method for preparing the injectable soft tissue regeneration filler, comprising the following steps: adding a double-crosslinked regenerated silk fibroin solid microsphere scaffold to sodium hyaluronate gel microspheres, and stirring and mixing them evenly to obtain a composite gel of the two; adding mannitol to the composite gel of the double-crosslinked regenerated silk fibroin solid microsphere scaffold and the sodium hyaluronate gel microspheres, and stirring until completely dissolved; and then sterilizing the composite gel with wet heat to obtain an injectable soft tissue regeneration filler.

[0013] In some embodiments, the double-crosslinked regenerated silk fibroin solid microsphere scaffold is prepared by the following method:

[0014] 1) Cut the silk cocoons into pieces and add them into a solution of alkali A, boil them, wash them with clean water and dry them to obtain degummed silk;

[0015] 2) Weighing a certain amount of degummed silk and dissolving it in a salt A solution to obtain a regenerated silk fibroin solution;

[0016] 3) adding a crosslinking agent to the regenerated silk fibroin solution, stirring and dispersing the solution uniformly, and then crosslinking the solution at room temperature to obtain a regenerated silk fibroin gel having chemical crosslinking;

[0017] 4) adding the chemically cross-linked regenerated silk fibroin gel to solution A, stirring and incubating, to obtain a regenerated silk fibroin gel having a chemically and physically double cross-linked structure;

[0018] 5) adding the double cross-linked regenerated silk fibroin gel into ultrapure water, stirring, washing and purifying, to obtain a purified double cross-linked regenerated silk fibroin gel;

[0019] 6) drying the double-crosslinked regenerated silk fibroin gel to obtain regenerated silk fibroin powder;

[0020] 7) crushing the regenerated silk fibroin powder to obtain a regenerated silk fibroin solid microsphere scaffold with a chemical and physical double cross-linking structure.

[0021] Preferably, in step 1), the base A is selected from sodium carbonate or potassium carbonate; the concentration of the base A solution is 0.2 M; and the boiling time is 6-8 h.

[0022] Preferably, in step 2), the salt A is LiBr; the concentration of the salt A solution is 9.3M; the mass concentration of the degummed silk after dissolution is 4-20%, preferably 7-10%.

[0023] Preferably, in step 3), the crosslinking agent is BDDE; the mass ratio of the crosslinking agent BDDE to the regenerated silk fibroin is 1:5-20, preferably 1:5-10; and the crosslinking time is 12-48h, preferably 36-48h.

[0024] Preferably, in step 4), solution A is an ethanol solution, a methanol solution or a propanol solution, preferably a 75-99% ethanol solution; the mass ratio of the regenerated silk fibroin hydrogel to solution A is 1:4-40, preferably 1:10-20; and the incubation time is 0.1-48h, preferably 0.5-2h.

[0025] Preferably, the washing times in step 5) are 3-5 times, preferably 5 times.

[0026] Preferably, the drying method in step 6) is freeze drying or vacuum drying, preferably freeze drying.

[0027] Preferably, the pulverizing method in step 7) is one or a combination of mechanical pulverizing, air flow pulverizing and ball milling pulverizing, preferably a combination of mechanical pulverizing and ball milling pulverizing.

[0028] Preferably, the pulverizing method is mechanical pulverization with a stirring speed of 5000-25000 rpm, preferably 15000-20000 rpm.

[0029] Preferably, the pulverizing method is air flow pulverizing with a pulverizing pressure of 0.2-0.7 MPa, preferably 0.3-0.4 MPa; and a feeding pressure of 0.3-0.8 MPa, preferably 0.4-0.5 MPa.

[0030] Preferably, the pulverizing method is ball milling at a speed of 300-800 rpm, preferably 600 rpm.

[0031] Preferably, the average particle size Dv(50) of the double-crosslinked regenerated silk fibroin solid microsphere scaffold obtained in step 7) is 6.76-61.6 μm, preferably 6-40 μm.

[0032] In some embodiments, the cross-linked sodium hyaluronate gel microspheres are prepared by the following method:

[0033] S1 Dissolve NaOH in ultrapure water, then add sodium hyaluronate and stir until completely dissolved;

[0034] S2: adding BDDE crosslinking agent to the sodium hyaluronate solution obtained in S1, stirring and mixing evenly, and keeping it at room temperature for crosslinking;

[0035] S3: adding ultrapure water and concentrated hydrochloric acid to the cross-linked sodium hyaluronate gel and stirring;

[0036] S4 collects the cross-linked sodium hyaluronate gel by filtering, and adds the cross-linked sodium hyaluronate gel to a grinder for grinding and granulation to obtain cross-linked sodium hyaluronate gel microspheres;

[0037] S5 collects cross-linked sodium hyaluronate gel microspheres, adds them into a centrifuge, and centrifuges and washes the gel microspheres to obtain purified cross-linked sodium hyaluronate gel microspheres.

[0038] The invention provides an injectable soft tissue filler for use in preparing tissue filling materials.

[0039] The invention discloses an injectable soft tissue filler for use in preparing a tissue regeneration medical cosmetic filling material.

[0040] Beneficial effects of the present invention: The injectable soft tissue filler provided by the present invention is prepared by compounding a double-crosslinked regenerated silk fibroin solid microsphere scaffold with a crosslinked sodium hyaluronate gel microsphere. When used as a filling material, it has the immediate filling effect of the crosslinked sodium hyaluronate gel and good plasticity. At the same time, the regenerated silk fibroin solid microsphere scaffold in the filler can also stimulate the regeneration of tissue fibroblasts and collagen, prolonging the effect duration of the filler, and is an innovative tissue regeneration medical cosmetic filling material. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 Flow chart of the preparation method of double cross-linked regenerated silk fibroin solid microsphere scaffold;

[0042] Figure 2 A schematic flow chart of a method for preparing an injectable soft tissue regeneration filler;

[0043] Figure 3 Photograph of the double cross-linked regenerated silk fibroin gel obtained according to the preparation method of Example 1 of the present invention;

[0044] Figure 4 Scanning electron microscope image (a) and aerodynamic particle size distribution diagram (b) of the double-crosslinked regenerated silk fibroin solid microsphere scaffold obtained according to the preparation method of Example 1 of the present invention;

[0045] Figure 5 Scanning electron microscope image (a) and aerodynamic particle size distribution diagram (b) of the double-crosslinked regenerated silk fibroin solid microsphere scaffold obtained according to the preparation method of Example 7 of the present invention;

[0046] Figure 6 Scanning electron microscope image (a) and aerodynamic particle size distribution diagram (b) of the double-crosslinked regenerated silk fibroin solid microsphere scaffold obtained according to the preparation method of Example 8 of the present invention;

[0047] Figure 7 The amino acid content detection data of the double-crosslinked regenerated silk fibroin solid microsphere scaffold obtained by the preparation method of Example 8 of the present invention;

[0048] Figure 8 Infrared spectrum of the double-crosslinked regenerated silk fibroin solid microsphere scaffold obtained according to the preparation method of Example 8 of the present invention;

[0049] Fig. 9 Electron microscopic photograph (a) and particle size distribution diagram (b) of cross-linked sodium hyaluronate gel microspheres obtained according to the preparation method of Example 9 of the present invention;

[0050] Fig.10 A photo of the injectable soft tissue regeneration filler obtained according to the preparation method of Example 10 of the present invention;

[0051] Fig.11 Cytotoxicity data of the injectable soft tissue regeneration filler obtained by the preparation method of Example 10 of the present invention; Fig.11 (a) is a graph showing the cell survival rate after the extract of the injectable soft tissue filler was co-cultured with fibroblasts; Fig.11 (b) is a fluorescent photograph of live and dead staining of fibroblasts after co-culture on the injectable soft tissue regeneration filler;

[0052] Fig.12 Anatomical photographs of the injectable soft tissue regeneration filler obtained by the preparation method of Example 10 of the present invention injected into the subcutaneous tissue of rats for different time periods;

[0053] Fig.13 H&E staining photos of tissue sections of rats injected with the injectable soft tissue regeneration filler obtained by the preparation method of Example 10 of the present invention at different time periods;

[0054] Fig.14 Masson staining photos of tissue sections of rats injected with the injectable soft tissue regeneration filler obtained by the preparation method of Example 10 of the present invention at different time periods; DETAILED DESCRIPTION

[0055] 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.

[0056] Example 1

[0057] The embodiment of the present invention provides a method for preparing a double-crosslinked regenerated silk fibroin solid microsphere scaffold, and the preparation method comprises the following steps:

[0058] 1) Take 100g of silk cocoons, add 0.2M Na2CO3 and boil for 6h, then wash with clean water and dry to obtain degummed silk;

[0059] 2) 50 g of degummed silk was dissolved in 450 mL of LiBr solution (9.3 M) to obtain a regenerated silk fibroin solution with a mass concentration of 10%;

[0060] 3) adding 10 g of BDDE crosslinking agent to the regenerated silk fibroin solution, stirring for 15 min and then crosslinking at room temperature for 36 h to obtain a chemically crosslinked regenerated silk fibroin gel;

[0061] 4) adding 500 g of chemically cross-linked regenerated silk fibroin gel into 5 L of 99% ethanol solution and incubating at room temperature for 0.5 h to perform secondary physical cross-linking on the regenerated silk fibroin gel;

[0062] 5) adding the double-crosslinked regenerated silk fibroin gel into ultrapure water, stirring and washing for 5 times to purify the gel;

[0063] 6) placing the double-crosslinked regenerated silk fibroin hydrogel into a freeze drying box, and freeze drying to obtain regenerated silk fibroin powder;

[0064] 7) The regenerated silk fibroin powder is placed in a mechanical stirring pulverizer, and high-speed mechanical pulverization is performed at a speed of 15000 rpm to obtain a double-crosslinked regenerated silk fibroin solid microsphere scaffold.

[0065] Figure 3 A photograph showing regenerated silk fibroin gel after chemical cross-linking. Figure 4 (a) shows a scanning electron microscope image of a double-crosslinked regenerated silk fibroin solid microsphere scaffold obtained according to the preparation method of an embodiment of the present invention. Figure 4 (b) shows the particle size distribution of the double cross-linked regenerated silk fibroin solid microsphere scaffold obtained according to the preparation method of the embodiment of the present invention. Figure 4 (b) It can be seen that the particle size Dv(50) of the double-crosslinked regenerated silk fibroin solid microsphere scaffold is 61.6 μm.

[0066] Example 2

[0067] The embodiment of the present invention provides a method for preparing a double-crosslinked regenerated silk fibroin solid microsphere scaffold, and the preparation method comprises the following steps:

[0068] 1) Take 100g of silk cocoons, add 0.2M Na2CO3 and boil for 8h, then wash with clean water and dry to obtain degummed silk;

[0069] 2) 35 g of degummed silk was dissolved in 465 mL of LiBr solution (9.3 M) to obtain a 7% regenerated silk fibroin solution;

[0070] 3) adding 7 g of BDDE crosslinking agent to the regenerated silk fibroin solution, stirring for 15 min and then crosslinking at room temperature for 48 h to obtain a chemically crosslinked regenerated silk fibroin gel;

[0071] 4) adding 500 g of chemically cross-linked regenerated silk fibroin gel into 10 L of 75% ethanol solution and incubating at room temperature for 2 h to perform secondary physical cross-linking on the regenerated silk fibroin gel;

[0072] 5) adding the double-crosslinked regenerated silk fibroin gel into ultrapure water, stirring and washing for 5 times to purify the gel;

[0073] 6) placing the double-crosslinked regenerated silk fibroin hydrogel into a freeze drying box, and freeze drying to obtain regenerated silk fibroin powder;

[0074] 7) The regenerated silk fibroin powder is placed in a mechanical stirring pulverizer, and the powder is stirred and pulverized at a high speed of 20,000 rpm to obtain a double-crosslinked regenerated silk fibroin solid microsphere scaffold.

[0075] Example 3

[0076] The embodiment of the present invention provides a method for preparing a double-crosslinked silk fibroin solid microsphere scaffold, the preparation method comprising the following steps:

[0077] 1) Take 100g of silk cocoons, add 0.2M Na2CO3 and boil for 8h, then wash with clean water and dry to obtain degummed silk;

[0078] 2) Take 20 g of degummed silk and dissolve it in 480 mL of LiBr solution (9.3 M) to obtain a 4% regenerated silk fibroin solution;

[0079] 3) adding 4 g of BDDE crosslinking agent to the regenerated silk fibroin solution, stirring for 15 min and then crosslinking at room temperature for 48 h to obtain a chemically crosslinked regenerated silk fibroin gel;

[0080] 4) adding the chemically cross-linked regenerated silk fibroin gel into 5 L of 75% ethanol solution and incubating at room temperature for 24 h to perform secondary physical cross-linking on the regenerated silk fibroin gel;

[0081] 5) adding the double-crosslinked regenerated silk fibroin gel into ultrapure water, stirring and washing for 5 times to purify the gel;

[0082] 6) placing the double-crosslinked regenerated silk fibroin hydrogel into a freeze drying oven, and freeze drying to obtain regenerated silk fibroin powder;

[0083] 7) The regenerated silk fibroin powder is placed in a mechanical stirring pulverizer, and the powder is stirred and pulverized at a high speed of 10,000 rpm to obtain a double-crosslinked regenerated silk fibroin solid microsphere scaffold.

[0084] Example 4

[0085] The embodiment of the present invention provides a method for preparing a double-crosslinked regenerated silk fibroin solid microsphere scaffold, and the preparation method comprises the following steps:

[0086] 1) Take 100g of silk cocoons, add 0.2M Na2CO3 and boil for 8h, then wash with clean water and dry to obtain degummed silk;

[0087] 2) Take 20 g of degummed silk and dissolve it in 480 mL of LiBr solution (9.3 M) to obtain a 4% regenerated silk fibroin solution;

[0088] 3) adding 4 g of BDDE crosslinking agent to the regenerated silk fibroin solution, stirring for 15 min and then crosslinking at room temperature for 48 h to obtain a chemically crosslinked regenerated silk fibroin gel;

[0089] 4) adding the chemically cross-linked regenerated silk fibroin gel into 5 L of 75% ethanol solution and incubating at room temperature for 24 h to perform secondary physical cross-linking on the regenerated silk fibroin gel;

[0090] 5) adding the double-crosslinked regenerated silk fibroin gel into ultrapure water, stirring and washing for 5 times to purify the gel;

[0091] 6) placing the double-crosslinked regenerated silk fibroin hydrogel into a freeze drying oven, and freeze drying to obtain regenerated silk fibroin powder;

[0092] 7) The regenerated silk fibroin powder is placed in a mechanical stirring pulverizer, and the powder is stirred and pulverized at a high speed of 5000 rpm to obtain a double-crosslinked regenerated silk fibroin solid microsphere scaffold.

[0093] Example 5

[0094] The embodiment of the present invention provides a method for preparing a double-crosslinked silk fibroin solid microsphere scaffold, the preparation method comprising the following steps:

[0095] 1) Take 100g of silk cocoons, add 0.2M Na2CO3 and boil for 8h, then wash with clean water and dry to obtain degummed silk;

[0096] 2) 50 g of degummed silk was dissolved in 450 mL of LiBr solution (9.3 M) to obtain a 10% regenerated silk fibroin solution;

[0097] 3) adding 5 g of BDDE crosslinking agent to the regenerated silk fibroin solution, stirring for 15 min, and then crosslinking at room temperature for 48 h to obtain a chemically crosslinked regenerated silk fibroin gel;

[0098] 4) adding the chemically cross-linked regenerated silk fibroin gel into 5 L of 75% ethanol solution and incubating at room temperature for 0.5 h to perform secondary physical cross-linking on the regenerated silk fibroin gel;

[0099] 5) adding the double-crosslinked regenerated silk fibroin gel into ultrapure water, stirring and washing for 5 times to purify the gel;

[0100] 6) placing the double-crosslinked regenerated silk fibroin hydrogel into a freeze drying oven, and freeze drying to obtain regenerated silk fibroin powder;

[0101] 7) The regenerated silk fibroin powder is placed in a mechanical stirring pulverizer, and the powder is stirred and pulverized at a high speed of 15000 rpm to obtain a double-crosslinked regenerated silk fibroin solid microsphere scaffold.

[0102] Example 6

[0103] The embodiment of the present invention provides a method for preparing a double-crosslinked regenerated silk fibroin solid microsphere scaffold, and the preparation method comprises the following steps:

[0104] 1) Take 100g of silk cocoons, add 0.2M Na2CO3 and boil for 8h, then wash with clean water and dry to obtain degummed silk;

[0105] 2) 50 g of degummed silk was dissolved in 450 mL of LiBr solution (9.3 M) to obtain a 10% regenerated silk fibroin solution;

[0106] 3) adding 2 g of BDDE crosslinking agent to the regenerated silk fibroin solution, stirring for 15 min and then crosslinking at room temperature for 48 h to obtain a chemically crosslinked regenerated silk fibroin gel;

[0107] 4) adding the chemically cross-linked regenerated silk fibroin gel into 5 L of 75% ethanol solution and incubating at room temperature for 24 h to perform secondary physical cross-linking on the regenerated silk fibroin gel;

[0108] 5) adding the double-crosslinked regenerated silk fibroin gel into ultrapure water, stirring and washing for 5 times to purify the gel;

[0109] 6) placing the double-crosslinked regenerated silk fibroin hydrogel into a freeze drying oven, and freeze drying to obtain regenerated silk fibroin powder;

[0110] 7) The regenerated silk fibroin powder is placed in a mechanical stirring pulverizer, and the powder is stirred and pulverized at a high speed of 5000 rpm to obtain a double-crosslinked regenerated silk fibroin solid microsphere scaffold.

[0111] Example 7

[0112] The regenerated silk fibroin solid microsphere scaffold prepared by mechanical stirring and crushing in Example 1 was added to the air flow mill, and the regenerated silk fibroin solid microsphere scaffold was secondary crushed under the conditions of a crushing pressure of 0.3 MPa and a feed pressure of 0.4 MPa to prepare a small-sized regenerated silk fibroin solid microsphere scaffold.

[0113] Figure 5 (a) shows a scanning electron microscope image of a small-sized double-cross-linked regenerated silk fibroin solid microsphere scaffold obtained according to the preparation method of an embodiment of the present invention. Figure 5 (b) shows the particle size distribution of the double cross-linked regenerated silk fibroin solid microsphere scaffold obtained according to the preparation method of the embodiment of the present invention. Figure 5 (b) It can be seen that the particle size Dv(50) of the double-cross-linked regenerated silk fibroin solid microsphere scaffold is 33.1 μm.

[0114] Example 8

[0115] The embodiment of the present invention provides a method for preparing a double-crosslinked regenerated silk fibroin solid microsphere scaffold, and the preparation method comprises the following steps:

[0116] 1) Take 100g of silk cocoons, add 0.2M Na2CO3 and boil for 8h, then wash with clean water and dry to obtain degummed silk;

[0117] 1) Take 50 g of degummed silk and dissolve it in 450 mL of LiBr solution (9.3 M) to obtain a 10% regenerated silk fibroin solution;

[0118] 2) adding 10 g of BDDE crosslinking agent to the regenerated silk fibroin solution, stirring for 15 min and then crosslinking at room temperature for 48 h to obtain a chemically crosslinked regenerated silk fibroin gel;

[0119] 3) adding the chemically cross-linked regenerated silk fibroin gel into 5 L of 75% ethanol solution and incubating at room temperature for 2 h to perform secondary physical cross-linking on the regenerated silk fibroin gel;

[0120] 4) adding the double-crosslinked regenerated silk fibroin gel into ultrapure water, stirring and washing for 5 times to purify the gel;

[0121] 5) placing the double-crosslinked regenerated silk fibroin hydrogel into a freeze drying box, and freeze drying to obtain regenerated silk fibroin powder;

[0122] 6) Add the regenerated silk fibroin powder into a ball mill, and mill the regenerated silk fibroin powder for 36 minutes at a rotation speed of 600 rpm to prepare another small-sized regenerated silk fibroin solid microsphere scaffold.

[0123] Figure 6 (a) shows a scanning electron microscope image of a small-sized double-cross-linked regenerated silk fibroin solid microsphere scaffold obtained according to the preparation method of an embodiment of the present invention. Figure 6 (b) shows the particle size distribution of the double cross-linked regenerated silk fibroin solid microsphere scaffold obtained according to the preparation method of the embodiment of the present invention. Figure 6 (b) It can be seen that the particle size Dv(50) of the double-crosslinked regenerated silk fibroin solid microsphere scaffold is 6.76 μm.

[0124] Figure 7 The amino acid content detection data of the small-sized double-crosslinked regenerated silk fibroin solid microsphere scaffold obtained by the preparation method of the embodiment of the present invention are shown. Figure 7 It can be seen that the double-crosslinked regenerated silk fibroin solid microsphere scaffold contains 18 characteristic amino acids of regenerated silk fibroin.

[0125] Figure 8 The infrared spectrum data of the small-sized double-crosslinked regenerated silk fibroin solid microsphere scaffold obtained by the preparation method of the embodiment of the present invention are shown. Figure 8 It can be seen that the double cross-linked regenerated silk protein solid microsphere scaffold is at 1700cm -1 ~1600cm -1 , 1590cm -1 ~1460cm -1 , and 1280cm -1 ~1190cm -1 There are characteristic peaks at the bottom, corresponding to the amide I band, amide II band and amide III band of silk fibroin.

[0126] Example 9

[0127] The embodiment of the present invention provides a method for preparing cross-linked sodium hyaluronate gel microspheres, the preparation method comprising the following steps:

[0128] 1) Dissolve 5 g NaOH in 495 mL ultrapure water, then add 100 g sodium hyaluronate (injection grade, molecular weight 500-1000 KDa) and stir until completely dissolved;

[0129] 2) Add 2 g of BDDE crosslinker to the sodium hyaluronate solution, stir and mix evenly, and keep it at room temperature for crosslinking for 24 hours;

[0130] 3) Add 5 L of ultrapure water and 5 mL of concentrated hydrochloric acid to the cross-linked sodium hyaluronate gel and stir for 2 h;

[0131] 4) filtering and collecting the cross-linked sodium hyaluronate gel, adding the cross-linked sodium hyaluronate gel into a grinder, grinding and granulating at a gear speed of 10,000 rpm to obtain cross-linked sodium hyaluronate gel microspheres;

[0132] 5) Collect the cross-linked sodium hyaluronate gel microspheres, add them into a centrifuge, and centrifuge and wash the gel microspheres (centrifugal speed is 2000 rpm, washing time is 2 h) to obtain purified cross-linked sodium hyaluronate gel microspheres.

[0133] Fig. 9 (a) shows an electron microscope photograph of cross-linked sodium hyaluronate gel microspheres obtained according to the preparation method of an embodiment of the present invention. Fig. 9 (b) shows the particle size distribution of cross-linked sodium hyaluronate gel microspheres obtained according to the preparation method of the embodiment of the present invention. Fig. 9 (b) It can be seen that the particle size Dv(50) of the cross-linked sodium hyaluronate gel microspheres is 401.6 μm.

[0134] Example 10

[0135] The present invention provides a method for preparing an injectable soft tissue regeneration filler, the method comprising the following steps:

[0136] 1) Accurately weigh 5 g of the double-crosslinked regenerated silk fibroin solid microsphere scaffold prepared in Example 1 and 100 g of the cross-linked sodium hyaluronate gel microsphere prepared in Example 9, and stir and mix them evenly;

[0137] 2) Add 5.25 g of mannitol to the mixed gel and continue stirring until the mannitol is completely dissolved;

[0138] 3) Place the mixed gel in a moist heat sterilizer and sterilize at 121°C for 8 minutes;

[0139] 4) Filling the sterilized mixed gel into a Luer lock sterile syringe to obtain an injectable cross-linked sodium hyaluronate soft tissue filler containing a double-cross-linked regenerated silk fibroin solid microsphere scaffold.

[0140] Fig.10 The photo of the injectable soft tissue filler obtained according to the preparation method of Example 10 of the present invention is shown. Fig.10 It can be seen that the double-cross-linked regenerated silk fibroin solid microsphere scaffold is uniformly and stably dispersed in the cross-linked sodium hyaluronate gel microspheres.

[0141] Fig.11 The cytotoxicity data of the injectable soft tissue regeneration filler obtained by the preparation method of Example 10 of the present invention are shown. Fig.11As shown in (a), after the extract of the injectable soft tissue filler was co-cultured with fibroblasts, the cell survival rate reached more than 94%. Fig.11 (b) shows a fluorescent photograph of live and dead cells after co-culture of fibroblasts on the injectable soft tissue regeneration filler. From the photograph, it can be seen that the cells grow well on the filler and the cell growth density is high, indicating that the injectable soft tissue filler has excellent biocompatibility.

[0142] Fig.12 The anatomical pictures of the injectable soft tissue regeneration filler obtained by the preparation method of Example 10 of the present invention injected into the subcutaneous tissue of rats at the 4th, 12th and 24th weeks are shown. Fig.12 As shown, the filler had not completely degraded 24 weeks after implantation in the tissue, a certain volume of filler still existed at the injection site, and obvious angiogenesis occurred inside the filler, proving that injectable soft tissue fillers not only have a long-term tissue filling effect, but also have excellent tissue compatibility and the function of promoting tissue regeneration.

[0143] Fig.13 The injectable soft tissue regeneration filler obtained by the preparation method of Example 10 of the present invention was injected into the subcutaneous tissue of rats, and the hematoxylin-eosin (H&E) staining photos of tissue sections at 4 weeks, 12 weeks and 24 weeks were shown. From the figure, it can be seen that the subcutaneous injected tissue of the rat and its surrounding cells are in good condition, and there is no obvious cell atrophy in the tissue site after 24 weeks of injection.

[0144] Fig.14 The injectable soft tissue regeneration filler obtained by the preparation method of Example 10 of the present invention was injected into the subcutaneous tissue of rats, and the collagen fiber Masson staining photos of the tissue sections at the 4th week, the 12th week and the 24th week were shown. It can be seen from the figure that as time goes by, the collagen proliferation effect of the subcutaneous injection tissue and its surrounding areas of the rats is significant, and the tissue sections are stained dark blue (because the attached figure is a grayscale image, the dark blue color cannot be seen, but in the actual effect figure, the dark blue color is distributed in other areas except the circular cells in the figure).

[0145] Embodiment 11

[0146] The present invention provides a method for preparing an injectable soft tissue regeneration filler, the method comprising the following steps:

[0147] 1) Accurately weigh 10 g of the double-crosslinked regenerated silk fibroin microspheres prepared in Example 1 and 100 g of the crosslinked sodium hyaluronate gel microspheres prepared in Example 9, and stir and mix them evenly;

[0148] 2) Add 5.5 g of mannitol to the mixed gel and continue stirring until the mannitol is completely dissolved;

[0149] 3) Place the mixed gel in a moist heat sterilizer and sterilize at 121°C for 15 minutes;

[0150] 4) Filling the sterilized mixed gel into a Luer lock sterile syringe to obtain an injectable cross-linked sodium hyaluronate soft tissue filler containing a double-cross-linked regenerated silk fibroin solid microsphere scaffold.

[0151] Example 12

[0152] An embodiment of the present invention provides a method for preparing an injectable soft tissue filler, the method comprising the following steps:

[0153] 1) Accurately weigh 30 g of the double-crosslinked regenerated silk fibroin microspheres prepared in Example 1 and 100 g of the crosslinked sodium hyaluronate gel microspheres prepared in Example 9, and stir and mix them evenly;

[0154] 2) Add 6.5 g of mannitol to the mixed gel and continue stirring until the mannitol is completely dissolved;

[0155] 3) Place the mixed gel in a moist heat sterilizer and sterilize at 121°C for 15 minutes;

[0156] 4) Filling the sterilized mixed gel into a Luer lock sterile syringe to obtain an injectable cross-linked sodium hyaluronate soft tissue filler containing a double-cross-linked regenerated silk fibroin solid microsphere scaffold.

[0157] Those skilled in the art will recognize that, although multiple exemplary embodiments of the present invention have been shown and described in detail herein, many other variations or modifications that conform to the principles of the present invention can still be directly determined or derived from the contents disclosed in the present invention without departing from the spirit and scope of the present invention. Therefore, the scope of the present invention should be understood and identified as covering all such other variations or modifications.

Claims

1. An injectable soft tissue regeneration filler, characterized in that: The double cross-linked regenerated silk fibroin solid microsphere scaffold comprises 5-30 parts by weight, 100 parts by weight of cross-linked sodium hyaluronate gel microspheres and 5.25-6.5 parts by weight of mannitol; the double cross-linked regenerated silk fibroin solid microsphere scaffold is prepared according to the following method: 1) Cut the silk cocoons into pieces and add them into a solution of alkali A, boil them, wash them with water and dry them to obtain degummed silk; 2) Weigh a certain amount of degummed silk and dissolve it in salt A solution to obtain a regenerated silk fibroin solution; 3) adding a cross-linking agent to the regenerated silk fibroin solution, stirring and dispersing the solution uniformly, and then cross-linking the solution at room temperature to obtain a regenerated silk fibroin gel having chemical cross-linking; 4) adding the chemically cross-linked regenerated silk fibroin gel to solution A, stirring and incubating, and obtaining a regenerated silk fibroin gel having a chemically and physically double cross-linked structure; 5) adding the double cross-linked regenerated silk fibroin gel into ultrapure water, stirring, washing and purifying, to obtain a purified double cross-linked regenerated silk fibroin gel; 6) Drying the double-crosslinked regenerated silk fibroin gel to obtain regenerated silk fibroin powder; 7) crushing the regenerated silk fibroin powder to obtain a regenerated silk fibroin solid microsphere scaffold with a chemical and physical double cross-linking structure; In step 3), the crosslinking agent is BDDE; the mass ratio of the crosslinking agent BDDE to the regenerated silk fibroin is 1:5-10; and the crosslinking time is 36-48h; In the step 4), solution A is a 75-99% ethanol solution; the mass ratio of the regenerated silk fibroin hydrogel to solution A is 1:10-20; and the incubation time is 0.5-2 h.

2. The injectable soft tissue regeneration filler according to claim 1, characterized in that: The injectable soft tissue regeneration filler is composed of 5 parts of double-crosslinked regenerated silk fibroin solid microsphere scaffold, 100 parts of crosslinked sodium hyaluronate gel microspheres and 5.25 parts of mannitol by weight.

3. The injectable soft tissue regeneration filler according to claim 1, characterized in that: The injectable soft tissue regeneration filler consists of 10 parts of double-crosslinked regenerated silk fibroin solid microsphere scaffolds, 100 parts of crosslinked sodium hyaluronate gel microspheres and 5.5 parts of mannitol by weight.

4. The injectable soft tissue regeneration filler according to claim 1, characterized in that: The injectable soft tissue regeneration filler is composed of 30 parts of double-crosslinked regenerated silk fibroin solid microsphere scaffolds, 100 parts of cross-linked sodium hyaluronate gel microspheres and 6.5 parts of mannitol by weight.

5. The injectable soft tissue regeneration filler according to claim 1, characterized in that: In step 1), the base A is selected from sodium carbonate or potassium carbonate; the concentration of the base A solution is 0.2 M; and the boiling time is 6-8 h; In the step 2), the salt A is LiBr; the concentration of the salt A solution is 9.3 M; the mass concentration of the degummed silk after dissolution is 7-10%; The washing times in step 5) are 5 times; The drying method in step 6) is freeze drying; The pulverizing method in step 7) is one or a combination of mechanical stirring pulverizing, air flow pulverizing and ball milling pulverizing; the stirring speed of the mechanical stirring pulverizing is 15000-20000 rpm; the pulverizing pressure of the air flow pulverizing is 0.3-0.4 MPa; the feeding pressure is 0.4-0.5 MPa; the speed of the ball milling pulverizing is 600 rpm; The average particle size Dv 50 of the double-crosslinked regenerated silk fibroin solid microsphere scaffold obtained in step 7) is 6-40 µm.

6. The injectable soft tissue regeneration filler according to claim 1, characterized in that: The cross-linked sodium hyaluronate gel microspheres are prepared according to the following method: S1 Dissolve NaOH in ultrapure water, then add sodium hyaluronate and stir until completely dissolved; S2 Add BDDE crosslinking agent to the sodium hyaluronate solution obtained in S1, stir and mix evenly, and keep it at room temperature for crosslinking; S3: Add ultrapure water and concentrated hydrochloric acid to the cross-linked sodium hyaluronate gel and stir; S4: collecting the cross-linked sodium hyaluronate gel by filtration, adding the cross-linked sodium hyaluronate gel to a grinder for grinding and granulation to obtain cross-linked sodium hyaluronate gel microspheres; S5 collects the cross-linked sodium hyaluronate gel microspheres, adds them into a centrifuge, and centrifuges and washes the gel microspheres to obtain purified cross-linked sodium hyaluronate gel microspheres.

7. The method for preparing the injectable soft tissue regeneration filler according to claim 1, comprising the following steps: Adding the double-crosslinked regenerated silk fibroin solid microsphere scaffold into the sodium hyaluronate gel microsphere, and stirring and mixing them evenly to obtain a composite gel of the two; Mannitol is added to the composite gel of the double-crosslinked regenerated silk fibroin solid microsphere scaffold and the sodium hyaluronate gel microsphere, and stirred until completely dissolved; the composite gel is then sterilized by wet heat to obtain an injectable soft tissue regeneration filler.

8. Use of the injectable soft tissue filler according to any one of claims 1 to 4 in the preparation of tissue filling materials.

9. Use of the injectable soft tissue filler according to any one of claims 1 to 4 in the preparation of tissue regeneration medical cosmetic filling materials.

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