A preparation method of textured bacterial cellulose microgel and skin filling composition

Abstract

The present invention relates to a preparation method of textured bacterial cellulose microgel and a skin filling composition. The preparation method of the textured bacterial cellulose microgel comprises treating a bacterial cellulose gel sheet with hydrogen peroxide, alkali and acid, and further comprises a step of treating with alkaline earth metal salt and / or transition metal salt.

Description

Technical Field

[0001] The present invention relates to the field of medical cosmetology, and in particular to an injectable dermal filler. Background Art

[0002] Medical cosmetology is to improve the external shape of the human body through medical means, namely drugs, surgery and other technologies to make up for the original relative defects, including the loss of collagen due to aging, which makes the skin loose and wrinkled, and the need to repair the external shape due to some congenital defects, burns and mechanical injuries. The injection of skin fillers is the most convenient and effective way to solve the above problems. This is because the injection of skin fillers is fast-acting and non-surgical, and can achieve significant and lasting effects without damaging other parts of the human body. At present, the commonly used skin filler materials in clinical practice include: natural degradable polymer materials such as hyaluronic acid and collagen, and synthetic degradable polymer materials such as poly-L-lactic acid and polycaprolactone. The former mainly plays a physical filling role, while the latter has a certain biological stimulation effect. After injection, it causes a slight inflammatory reaction, stimulates the human body to produce new collagen, and achieves the effect of improving wrinkles, lifting and tightening, and increasing skin elasticity; but the above materials still have many shortcomings, such as a short effective time after implantation in the body, weak tissue regeneration activity, and adverse inflammatory reactions.

[0003] Bacterial cellulose (BC) is synthesized by microorganisms such as Acetobacter, Agrobacterium, Rhizobium and Sarcina. It is a linear polysaccharide composed of D-pyranoglucoside connected by β-1,4-glucoside bonds, forming parallel, non-helical conformations and non-branched structures.

[0004] Bacterial cellulose gel has good biocompatibility, water retention and mechanical properties, especially the unique extracellular matrix-like three-dimensional nanofiber network structure. It can be used as a tissue engineering scaffold such as bone / cartilage, as well as medical devices such as artificial blood vessels and artificial skin. However, the direct use of paste-like bacterial cellulose gel for injection filling has limited ability to promote tissue regeneration, and the microstructure of paste-like bacterial cellulose gel is not conducive to providing three-dimensional mechanical support for regenerated tissue and space for cell proliferation. Summary of the invention

[0005] One of the present inventions provides a method for preparing a textured bacterial cellulose microgel, which comprises treating a bacterial cellulose gel sheet with hydrogen peroxide, alkali and acid.

[0006] In one embodiment, the method further comprises the step of treating with an alkaline earth metal salt and / or a transition metal salt.

[0007] In a specific embodiment, the alkaline earth metal salt and / or transition metal salt is at least one of magnesium chloride, calcium chloride, strontium chloride, zinc chloride, magnesium nitrate, calcium nitrate, strontium nitrate and zinc nitrate.

[0008] In a specific embodiment, the method further comprises the step of subjecting the bacterial cellulose gel sheet to freeze-thaw treatment.

[0009] In a specific embodiment, the method further comprises the step of stretching the bacterial cellulose gel sheet.

[0010] In a specific embodiment, the method comprises the following steps:

[0011] 1) treating the bacterial cellulose gel sheet with an aqueous hydrogen peroxide solution to obtain a hydrogen peroxide-treated bacterial cellulose gel sheet;

[0012] 2) treating the hydrogen peroxide-treated bacterial cellulose gel sheet with an aqueous solution of an alkaline earth metal and / or transition metal salt to obtain a salt-treated bacterial cellulose gel sheet;

[0013] 3) subjecting the salt-treated bacterial cellulose gel sheet to freeze-thaw treatment to obtain a freeze-thaw-treated bacterial cellulose gel sheet;

[0014] 4) treating the freeze-thaw treated bacterial cellulose gel sheet with alkali to obtain an alkali treated bacterial cellulose gel sheet;

[0015] 5) stretching the alkali-treated bacterial cellulose gel sheet to obtain a stretched bacterial cellulose gel sheet;

[0016] 6) treating the stretched bacterial cellulose gel sheet with acid to obtain a textured bacterial cellulose gel sheet.

[0017] In a specific embodiment, the method further comprises step 7), washing the textured bacterial cellulose gel sheet obtained in step 6) to obtain a washed textured bacterial cellulose gel sheet.

[0018] In a specific embodiment, the method further comprises step 8), homogenizing and crushing the cleaned textured bacterial cellulose gel sheet to obtain a homogenized textured bacterial cellulose microgel.

[0019] In a specific embodiment, in step 1), the mass / volume ratio of the bacterial cellulose gel sheet to the aqueous hydrogen peroxide solution is 1 g:(1-10 mL).

[0020] In a specific embodiment, the volume concentration of the aqueous solution of hydrogen peroxide is 2% to 10%.

[0021] In a specific embodiment, the bacterial cellulose gel sheet is immersed in the aqueous hydrogen peroxide solution for 1 to 2 hours, wherein ultrasonication is performed for 10 to 30 minutes.

[0022] In a specific embodiment, in step 2), the mass / volume ratio of the bacterial cellulose gel sheet to the aqueous salt solution is 1 g:(1-10 mL), for example 1 g:(2-10 mL).

[0023] In a specific embodiment, the molar concentration of the aqueous salt solution is 0.01 to 1 mol / L.

[0024] In a specific embodiment, the bacterial cellulose gel sheet treated with hydrogen peroxide is immersed in the aqueous salt solution for 2 to 24 hours, wherein ultrasonication is performed for 0.5 to 1 hour.

[0025] In one specific embodiment, in step 4), the base is sodium hydroxide.

[0026] In a specific embodiment, the mass / volume ratio of the bacterial cellulose gel sheet to the aqueous alkali solution is 1 g:(1-10 mL), for example 1 g:(5-10 mL); and the alkali concentration is 0.1 mol / L.

[0027] In a specific embodiment, the freeze-thaw treated bacterial cellulose gel sheet is immersed in the alkaline aqueous solution for 6 to 24 hours.

[0028] In one embodiment, in step 6), the acid is hydrochloric acid.

[0029] In a specific embodiment, the mass / volume ratio of the bacterial cellulose gel sheet to the acid aqueous solution is 1 g:(1-20 mL), for example 1 g:(5-20 mL); and the acid concentration is 0.1 mol / L.

[0030] In a specific embodiment, the alkali-treated bacterial cellulose gel sheet is soaked in the acid aqueous solution for 1 to 2 hours.

[0031] In a specific embodiment, in step 3), freeze-thaw is performed 1 to 5 times.

[0032] In one embodiment, in step 5), the alkali-treated bacterial cellulose gel sheet is stretched to an elongation of 10% to 60%.

[0033] In one embodiment, in step 7), the textured bacterial cellulose gel sheet is washed with water (eg, deionized water).

[0034] In one embodiment, in step 8), the washed textured bacterial cellulose gel sheet is homogenized and crushed in physiological saline or water for injection to obtain a homogenized textured bacterial cellulose microgel dispersion.

[0035] In a specific embodiment, in the textured bacterial cellulose microgel dispersion, the mass content of the physiological saline or water for injection is 10% to 99%.

[0036] In one embodiment, the textured bacterial cellulose microgel is capable of passing through a 22G to 29G syringe needle.

[0037] The second aspect of the present invention provides an injectable skin filler, which comprises a textured bacterial cellulose microgel dispersion prepared by the method described in any one of the present inventions, and optionally other fillers.

[0038] In a specific embodiment, the skin filling composition further comprises a growth factor that promotes skin growth.

[0039] In a specific embodiment, the growth factors include epidermal growth factor (EGF) and / or fibroblast growth factors (FGFs) and the like.

[0040] In a specific embodiment, the other fillers include at least one of sodium carboxymethylcellulose, sodium hyaluronate and sodium carboxymethyl chitosan.

[0041] In a specific embodiment, based on the mass of the textured bacterial cellulose microgel dispersion as 100%, the mass fraction of the other fillers is 0.2% to 20%, preferably, the mass fraction of the other fillers is 0.2% to 1%.

[0042] Beneficial effects of the present invention:

[0043] The preparation method of the textured bacterial cellulose microgel provided by the present invention is simple and mild. The microstructure and aggregation state of the bacterial cellulose microgel can be regulated by a removable in-situ generated micro-template and a mechanical orientation effect. The prepared textured bacterial cellulose microgel has a good three-dimensional porous scaffold structure, provides a three-dimensional mechanical support structure and a space for cell proliferation for tissue regeneration, and can also better promote the synthesis of type I collagen, so it can be used as a skin filler, etc. It can be directly used for injection filling after further homogenization. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 The dermal filler of Example 9 is shown packaged into a syringe.

[0045] Figure 2The SEM image of the textured bacterial cellulose microgel prepared in Example 1 is shown.

[0046] Figure 3 The SEM image of the textured bacterial cellulose microgel prepared in Example 8 is shown.

[0047] Figure 4 The SEM image of the bacterial cellulose microgel prepared in Comparative Example 1 is shown. DETAILED DESCRIPTION

[0048] The above contents of the present invention are further described in detail below in the form of preferred implementation cases, but they do not constitute a limitation to the present invention.

[0049] Unless otherwise specified, the reagents in the embodiments of the present invention can be purchased through commercial channels.

[0050] Bacterial cellulose gel in sheet or paste form was purchased from Shanghai Puzhen Biotechnology Co., Ltd.

[0051] Preparation of textured bacterial cellulose microgels

[0052] Example 1

[0053] 1) taking 5 g of bacterial cellulose gel sheet, immersing it in 50 mL of 5% hydrogen peroxide aqueous solution for 1 hour, wherein ultrasonic treatment is performed at 50 W for 30 minutes to obtain a hydrogen peroxide-treated bacterial cellulose gel sheet;

[0054] 2) taking out the bacterial cellulose gel sheet treated with hydrogen peroxide in step 1), immersing it in 10 mL of 1 mol / L magnesium chloride solution for 2 h, and performing ultrasonic treatment at 50 W for 0.5 h to obtain a salt-treated bacterial cellulose gel sheet;

[0055] 3) taking out the salt-treated bacterial cellulose gel sheet from step 2), and freezing and thawing at -40°C / room temperature for 5 times to obtain a freeze-thaw-treated bacterial cellulose gel sheet;

[0056] 4) immersing the freeze-thaw treated bacterial cellulose gel sheet in 50 mL of 0.1 mol / L sodium hydroxide solution for 6 hours to obtain an alkali-treated bacterial cellulose gel sheet;

[0057] 5) taking out the alkali-treated bacterial cellulose gel sheet from step 4) and stretching it to an elongation of 60% to obtain a stretched bacterial cellulose gel sheet;

[0058] 6) immersing the stretched bacterial cellulose gel sheet in 100 mL of 0.1 mol / L hydrochloric acid for 2 hours to obtain a textured bacterial cellulose gel sheet;

[0059] 7) taking out the textured bacterial cellulose gel sheet from step 6) and washing it thoroughly with deionized water to obtain a washed textured bacterial cellulose gel sheet;

[0060] 8) Then, 1 gram of the washed bacterial cellulose gel sheet was added into 99 mL of normal saline, and the sheet was homogenized and broken into pieces that could pass through a 29G syringe needle to obtain a textured bacterial cellulose microgel dispersion.

[0061] Example 2

[0062] 1) taking 10 g of bacterial cellulose gel sheet, immersing it in 100 mL of 2% hydrogen peroxide aqueous solution for 2 h, wherein ultrasonic treatment was performed at 100 W for 10 min to obtain a hydrogen peroxide-treated bacterial cellulose gel sheet;

[0063] 2) taking out the bacterial cellulose gel sheet treated with hydrogen peroxide in step 1), immersing it in 100 mL of 0.1 mol / L magnesium nitrate solution for 24 h, and ultrasonically treating it at 100 W for 1 h to obtain a salt-treated bacterial cellulose gel sheet;

[0064] 3) taking out the salt-treated bacterial cellulose gel sheet from step 2), freezing and thawing once at -30°C / room temperature to obtain a freeze-thaw-treated bacterial cellulose gel sheet;

[0065] 4) immersing the freeze-thaw treated bacterial cellulose gel sheet in 100 mL of 0.1 mol / L sodium hydroxide solution for 12 h to obtain an alkali treated bacterial cellulose gel sheet,

[0066] 5) taking out the alkali-treated bacterial cellulose gel sheet from step 4) and stretching it to an elongation of 30% to obtain a stretched bacterial cellulose gel sheet;

[0067] 6) immersing the stretched bacterial cellulose gel sheet in 100 mL of 0.1 mol / L hydrochloric acid for 1 hour to obtain a textured bacterial cellulose gel sheet;

[0068] 7) taking out the textured bacterial cellulose gel sheet from step 6) and washing it thoroughly with deionized water to obtain a washed textured bacterial cellulose gel sheet;

[0069] 8) Then, 1 gram of the washed bacterial cellulose gel sheet was added into 99 mL of water for injection, and the sheet was homogenized and crushed with a homogenizer until it could pass through a 26G syringe needle to obtain a textured bacterial cellulose microgel dispersion.

[0070] Example 3

[0071] 1) taking 10 g of bacterial cellulose gel sheet, immersing it in 100 mL of 10% hydrogen peroxide solution for 2 h, and ultrasonically treating it at 100 W for 30 min to obtain a hydrogen peroxide-treated bacterial cellulose gel sheet;

[0072] 2) taking out the bacterial cellulose gel sheet treated with hydrogen peroxide in step 1), immersing it in 50 mL of 0.2 mol / L zinc chloride solution for 2 h, and ultrasonically treating it at 100 W for 0.5 h to obtain a salt-treated bacterial cellulose gel sheet;

[0073] 3) taking out the salt-treated bacterial cellulose gel sheet from step 2), and freezing and thawing at -20°C / room temperature twice to obtain a freeze-thaw-treated bacterial cellulose gel sheet;

[0074] 4) immersing the freeze-thaw treated bacterial cellulose gel sheet in 50 mL of 0.1 mol / L sodium hydroxide solution for 24 hours to obtain an alkali-treated bacterial cellulose gel sheet;

[0075] 5) taking out the alkali-treated bacterial cellulose gel sheet from step 4) and stretching it to an elongation of 10% to obtain a stretched bacterial cellulose gel sheet;

[0076] 6) immersing the stretched bacterial cellulose gel sheet in 50 mL of 0.1 mol / L hydrochloric acid for 1 hour to obtain a textured bacterial cellulose gel sheet;

[0077] 7) taking out the textured bacterial cellulose gel sheet from step 6) and washing it thoroughly with deionized water to obtain a washed textured bacterial cellulose gel sheet;

[0078] 8) Then, 9 grams of the washed bacterial cellulose gel was added with 1 mL of water for injection, and the mixture was homogenized and crushed with a homogenizer until the gel could pass through a 22G syringe needle to obtain a textured bacterial cellulose microgel dispersion.

[0079] Example 4

[0080] The difference from Example 1 is that the magnesium chloride treatment in step 2) is omitted, that is:

[0081] 1) taking 5 g of bacterial cellulose gel sheet, immersing it in 50 mL of 5% hydrogen peroxide aqueous solution for 1 hour, wherein ultrasonic treatment is performed at 50 W for 30 minutes to obtain a hydrogen peroxide-treated bacterial cellulose gel sheet;

[0082] 2) taking out the bacterial cellulose gel sheet treated with hydrogen peroxide in step 1), and freezing and thawing at -40°C / room temperature for 5 times to obtain a freeze-thawed bacterial cellulose gel sheet;

[0083] 3) immersing the freeze-thaw treated bacterial cellulose gel sheet in 50 mL of 0.1 mol / L sodium hydroxide solution for 6 h to obtain an alkali-treated bacterial cellulose gel sheet;

[0084] 4) taking out the alkali-treated bacterial cellulose gel sheet from step 3) and stretching it to an elongation of 60% to obtain a stretched bacterial cellulose gel sheet;

[0085] 5) Immersing the stretched bacterial cellulose gel sheet in 100 mL of 0.1 mol / L hydrochloric acid for 2 h to obtain a textured bacterial cellulose gel sheet.

[0086] 6) taking out the textured bacterial cellulose gel sheet from step 5) and washing it thoroughly with deionized water to obtain a washed textured bacterial cellulose gel sheet;

[0087] 7) Then, 1 gram of the washed bacterial cellulose gel was added into 99 mL of normal saline, and the mixture was homogenized and crushed until it could pass through a 29G syringe needle to obtain a textured bacterial cellulose microgel dispersion.

[0088] Example 5

[0089] The difference from Example 1 is that the freeze-thaw treatment in step 3) is omitted, that is:

[0090] 1) taking 5 g of bacterial cellulose gel sheet, immersing it in 50 mL of 5% hydrogen peroxide aqueous solution for 1 hour, wherein ultrasonic treatment is performed at 50 W for 30 minutes to obtain a hydrogen peroxide-treated bacterial cellulose gel sheet;

[0091] 2) taking out the bacterial cellulose gel sheet treated with hydrogen peroxide in step 1), immersing it in 50 mL of 0.1 mol / L magnesium chloride solution for 2 h, and ultrasonically treating it at 50 W for 0.5 h to obtain a salt-treated bacterial cellulose gel sheet;

[0092] 3) taking out the salt-treated bacterial cellulose gel sheet from step 2) and immersing it in 50 mL of 0.1 mol / L sodium hydroxide solution for 6 h to obtain an alkali-treated bacterial cellulose gel sheet.

[0093] 4) taking out the alkali-treated bacterial cellulose gel sheet from step 3) and stretching it to an elongation of 60% to obtain a stretched bacterial cellulose gel sheet;

[0094] 5) Immersing the stretched bacterial cellulose gel sheet in 100 mL of 0.1 mol / L hydrochloric acid for 2 h to obtain a textured bacterial cellulose gel sheet.

[0095] 6) taking out the textured bacterial cellulose gel sheet from step 5) and washing it thoroughly with deionized water to obtain a washed textured bacterial cellulose gel sheet;

[0096] 7) Then, 1 gram of the washed bacterial cellulose gel was added into 99 mL of physiological saline, and the mixture was homogenized and broken into pieces by a homogenizer until the pieces could pass through a 29G syringe needle, thereby obtaining a textured bacterial cellulose microgel dispersion.

[0097] Example 6

[0098] The difference from Example 1 is that step 2) and step 3) are omitted at the same time, that is:

[0099] 1) taking 5 g of bacterial cellulose gel sheet, immersing it in 50 mL of 5% hydrogen peroxide aqueous solution for 1 hour, wherein ultrasonic treatment is performed at 50 W for 30 minutes to obtain a hydrogen peroxide-treated bacterial cellulose gel sheet;

[0100] 2) taking out the bacterial cellulose gel sheet treated with hydrogen peroxide in step 1), and immersing it in 50 mL of 0.1 mol / L sodium hydroxide solution for 6 h to obtain an alkali-treated bacterial cellulose gel sheet,

[0101] 3) taking out the alkali-treated bacterial cellulose gel sheet from step 2) and stretching it to an elongation of 60% to obtain a stretched bacterial cellulose gel sheet;

[0102] 4) Immersing the stretched bacterial cellulose gel sheet in 100 mL of 0.1 mol / L hydrochloric acid for 2 h to obtain a textured bacterial cellulose gel sheet.

[0103] 5) taking out the textured bacterial cellulose gel sheet from step 4) and washing it thoroughly with deionized water to obtain a washed textured bacterial cellulose gel sheet;

[0104] 6) Then, 1 gram of the washed bacterial cellulose gel was added into 99 mL of physiological saline, and the mixture was homogenized and broken into pieces by a homogenizer until the pieces could pass through a 29G syringe needle, thereby obtaining a textured bacterial cellulose microgel dispersion.

[0105] Example 7

[0106] The difference from Example 1 is that the stretching process in step 5) is omitted, that is:

[0107] 1) taking 5 g of bacterial cellulose gel sheet, immersing it in 50 mL of 5% hydrogen peroxide aqueous solution for 1 hour, wherein ultrasonic treatment is performed at 50 W for 30 minutes to obtain a hydrogen peroxide-treated bacterial cellulose gel sheet;

[0108] 2) taking out the bacterial cellulose gel sheet treated with hydrogen peroxide in step 1), immersing it in 50 mL of 0.1 mol / L magnesium chloride solution for 2 h, and ultrasonically treating it at 50 W for 0.5 h to obtain a salt-treated bacterial cellulose gel sheet;

[0109] 3) taking out the salt-treated bacterial cellulose gel sheet from step 2), and subjecting the sheet to freeze-thaw treatment at -40°C / room temperature for 5 cycles to obtain a freeze-thaw-treated bacterial cellulose gel sheet;

[0110] 4) immersing the freeze-thaw treated bacterial cellulose gel sheet in 50 mL of 0.1 mol / L sodium hydroxide solution for 6 h to obtain an alkali treated bacterial cellulose gel sheet,

[0111] 5) taking out the alkali-treated bacterial cellulose gel sheet from step 4), and immersing it in 100 mL of 0.1 mol / L hydrochloric acid for 2 h to obtain a textured bacterial cellulose gel sheet.

[0112] 6) taking out the textured bacterial cellulose gel sheet from step 5) and washing it thoroughly with deionized water to obtain a washed textured bacterial cellulose gel sheet;

[0113] 7) Then, 1 gram of the washed bacterial cellulose gel was added into 99 mL of physiological saline, and the mixture was homogenized and broken into pieces by a homogenizer until the pieces could pass through a 29G syringe needle, thereby obtaining a textured bacterial cellulose microgel dispersion.

[0114] Example 8

[0115] The difference from Example 1 is that the freeze-thaw treatment in step 3) and the stretching treatment in step 5) are omitted, namely:

[0116] 1) taking 5 g of bacterial cellulose gel sheet, immersing it in 50 mL of 5% hydrogen peroxide aqueous solution for 1 h, wherein ultrasonic treatment is performed at 50 W for 0.5 h to obtain a hydrogen peroxide-treated bacterial cellulose gel sheet;

[0117] 2) taking out the bacterial cellulose gel sheet treated with hydrogen peroxide in step 1), immersing it in 50 mL of 0.1 mol / L magnesium chloride solution for 2 h, and ultrasonically treating it at 50 W for 0.5 h to obtain a salt-treated bacterial cellulose gel sheet;

[0118] 3) taking out the salt-treated bacterial cellulose gel sheet from step 2) and immersing it in 50 mL of 0.1 mol / L sodium hydroxide solution for 6 h to obtain an alkali-treated bacterial cellulose gel sheet.

[0119] 4) taking out the alkali-treated bacterial cellulose gel sheet from step 3), and immersing it in 100 mL of 0.1 mol / L hydrochloric acid for 2 h to obtain a textured bacterial cellulose gel sheet.

[0120] 5) taking out the textured bacterial cellulose gel sheet from step 4) and washing it thoroughly with deionized water to obtain a washed textured bacterial cellulose gel sheet;

[0121] 6) Then, 1 gram of the washed bacterial cellulose gel was added into 99 mL of physiological saline, and the mixture was homogenized and broken into pieces by a homogenizer until the pieces could pass through a 29G syringe needle, thereby obtaining a textured bacterial cellulose microgel dispersion.

[0122] Comparative Example 1

[0123] 1 gram of commercially available bacterial cellulose paste was added to 99 mL of normal saline and homogenized with a homogenizer until the paste could pass through a 29G syringe needle to obtain a homogenized bacterial cellulose microgel dispersion.

[0124] Preparation of dermal fillers

[0125] Example 9

[0126] Taking the mass of the textured bacterial cellulose microgel dispersion as 100%, 0.2% sodium carboxymethyl cellulose was added to the textured bacterial cellulose microgel dispersion prepared in Example 1, stirred evenly, filled into a syringe, sterilized by wet heat, and packaged. Figure 1 shown.

[0127] Example 10

[0128] Taking the mass of the textured bacterial cellulose microgel dispersion as 100%, 1% by mass of sodium carboxymethyl cellulose was added to the textured bacterial cellulose microgel dispersion prepared in Example 1, stirred evenly, filled into a syringe, sterilized by wet heat, and packaged.

[0129] Embodiment 11

[0130] Taking the mass of the textured bacterial cellulose microgel dispersion as 100%, 0.5% by mass of sodium hyaluronate was added to the textured bacterial cellulose microgel dispersion prepared in Example 1, stirred evenly, filled into a syringe, sterilized by wet heat, and packaged.

[0131] Example 12

[0132] Taking the mass of the textured bacterial cellulose microgel dispersion as 100%, 0.2% by mass of sodium hyaluronate was added to the textured bacterial cellulose microgel dispersion prepared in Example 1, stirred evenly, filled into a syringe, sterilized by wet heat, and packaged.

[0133] Embodiment 13

[0134] Taking the mass of the textured bacterial cellulose microgel dispersion as 100%, 1% by mass of sodium hyaluronate was added to the textured bacterial cellulose microgel dispersion prepared in Example 1, stirred evenly, filled into a syringe, sterilized by wet heat, and packaged.

[0135] Embodiment 14

[0136] Taking the mass of the textured bacterial cellulose microgel dispersion as 100%, 0.2% by mass of sodium carboxymethyl chitosan was added to the textured bacterial cellulose microgel dispersion prepared in Example 1, stirred evenly, filled into a syringe, sterilized by wet heat, and packaged.

[0137] Embodiment 15

[0138] Taking the mass of the textured bacterial cellulose microgel dispersion as 100%, 1% by mass of sodium carboxymethyl chitosan was added to the textured bacterial cellulose microgel dispersion prepared in Example 1, stirred evenly, filled into a syringe, sterilized by wet heat, and packaged.

[0139] Example 16

[0140] Taking the mass of the textured bacterial cellulose microgel dispersion as 100%, 0.2% by mass of sodium hyaluronate was added to the textured bacterial cellulose microgel dispersion prepared in Example 2, stirred evenly, filled into a syringe, sterilized by wet heat, and packaged.

[0141] Embodiment 17

[0142] Taking the mass of the textured bacterial cellulose microgel dispersion as 100%, 0.2% by mass of sodium hyaluronate was added to the textured bacterial cellulose microgel dispersion prepared in Example 3, stirred evenly, filled into a syringe, sterilized by wet heat, and packaged.

[0143] Embodiment 18

[0144] Taking the mass of the textured bacterial cellulose microgel dispersion as 100%, 0.2% by mass of sodium hyaluronate was added to the textured bacterial cellulose microgel dispersion prepared in Example 4, stirred evenly, filled into a syringe, sterilized by wet heat, and packaged.

[0145] Embodiment 19

[0146] Taking the mass of the textured bacterial cellulose microgel dispersion as 100%, 0.2% by mass of sodium hyaluronate was added to the textured bacterial cellulose microgel dispersion prepared in Example 5, stirred evenly, filled into a syringe, sterilized by wet heat, and packaged.

[0147] Embodiment 20

[0148] Taking the mass of the textured bacterial cellulose microgel dispersion as 100%, 0.2% by mass of sodium hyaluronate was added to the textured bacterial cellulose microgel dispersion prepared in Example 6, stirred evenly, filled into a syringe, sterilized by wet heat, and packaged.

[0149] Embodiment 21

[0150] Taking the mass of the textured bacterial cellulose microgel dispersion as 100%, 0.2% by mass of sodium hyaluronate was added to the textured bacterial cellulose microgel dispersion prepared in Example 7, stirred evenly, filled into a syringe, sterilized by wet heat, and packaged.

[0151] Embodiment 22

[0152] Taking the mass of the textured bacterial cellulose microgel dispersion as 100%, 0.2% by mass of sodium hyaluronate was added to the textured bacterial cellulose microgel dispersion prepared in Example 8, stirred evenly, filled into a syringe, sterilized by wet heat, and packaged.

[0153] Comparative Example 2

[0154] Taking the mass of bacterial cellulose microgel dispersion as 100%, 0.2% sodium carboxymethyl cellulose was added to the homogenized bacterial cellulose microgel dispersion prepared in Comparative Example 1, stirred evenly, filled into a syringe, sterilized by wet heat, and packaged.

[0155] Morphological observation of bacterial cellulose microgel aggregation state

[0156] The textured bacterial cellulose microgel dispersions prepared in Examples 1 to 8 and the homogenized bacterial cellulose microgel dispersion in Comparative Example 1 were injected into a mold through a syringe needle, freeze-dried, and the morphology of the freeze-dried cross-section was observed using a scanning electron microscope (SEM). The textured bacterial cellulose prepared in Examples 1 to 8 can be observed to have an obvious three-dimensional porous scaffold structure, indicating that it can provide a three-dimensional mechanical support structure and sufficient cell proliferation space for tissue regeneration, but the regularity and order of the three-dimensional porous scaffold structure of the textured bacterial cellulose obtained by omitting the treatment step is relatively low, and the aggregation state of Comparative Example 1 is not ideal.

[0157] Among them, the SEM images of the aggregation states of Example 1, Example 8 and Comparative Example 1 are shown in Figures 2 to 4 .

[0158] Evaluation of collagen production

[0159] Human dermal fibroblasts (HSF) were used as an in vitro model, and the effect of promoting collagen production was evaluated by detecting the synthesis of type I collagen in HSF cells co-cultured with dermal fillers.

[0160] Appropriate amounts of the skin fillers prepared in Examples 9 to 22 and Comparative Example 2 of the same mass were respectively placed in a 12-well cell culture plate, and fresh DMEM containing 10% calf serum was used as the culture medium. HSF cells (ATCC, catalog number: CRL-1568) were cultured at a rate of 5×10 5 The cells were inoculated at a density of 100 cells / well and cultured for 48 hours to 14 days. The culture medium was collected every two days. The blank control group was HSF cell culture medium without skin fillers. The type I collagen content (ng / mL) in the supernatant was detected using an ELISA kit. The results are shown in Tables 1 and 2.

[0161] Table 1

[0162] Example 48h 2d 4d 6d Example 9 <![CDATA[2.07±0.09 a ]]> <![CDATA[3.65±0.08 b ]]> <![CDATA[4.77±0.08 c ]]> <![CDATA[5.62±0.04 c ]]> Example 10 <![CDATA[2.13±0.21 a ]]> <![CDATA[3.73±0.06 b ]]> <![CDATA[4.87±0.10 c ]]> <![CDATA[5.79±0.09 c ]]> Embodiment 11 <![CDATA[1.99±0.15 a ]]> <![CDATA[3.60±0.14 b ]]> <![CDATA[4.52±0.23 c ]]> <![CDATA[5.34±0.27 c ]]> Example 12 <![CDATA[1.98±0.17 a ]]> <![CDATA[3.58±0.03 b ]]> <![CDATA[4.76±0.04 c ]]> <![CDATA[5.67±0.07 c ]]> Embodiment 13 <![CDATA[2.10±0.04 a ]]> <![CDATA[3.70±0.14 b ]]> <![CDATA[4.64±0.17 c ]]> <![CDATA[5.71±0.17 c ]]> Embodiment 14 <![CDATA[2.11±0.07 a ]]> <![CDATA[3.68±0.07 b ]]> <![CDATA[4.70±0.06 c ]]> <![CDATA[5.56±0.01 c ]]> Embodiment 15 <![CDATA[2.07±0.14 a ]]> <![CDATA[3.72±0.21 b ]]> <![CDATA[4.45±0.16 c ]]> <![CDATA[5.51±0.13 c ]]> Example 16 <![CDATA[2.06±0.07 a ]]> <![CDATA[3.67±0.37 b ]]> <![CDATA[4.66±0.29 c ]]> <![CDATA[5.61±0.01 c ]]> Embodiment 17 <![CDATA[2.11±0.16 a ]]> <![CDATA[3.91±0.04 b ]]> <![CDATA[4.86±0.11 c ]]> <![CDATA[5.49±0.09 c ]]> Embodiment 18 <![CDATA[2.02±0.12 a ]]> <![CDATA[2.47±0.14 a ]]> <![CDATA[3.42±0.19 b ]]> <![CDATA[3.87±0.26 b ]]> Embodiment 19 <![CDATA[2.00±0.17 a ]]> <![CDATA[2.47±0.06 a ]]> <![CDATA[3.60±0.12 b ]]> <![CDATA[3.96±0.05 b ]]> Embodiment 20 <![CDATA[2.09±0.17 a ]]> <![CDATA[2.47±0.12 a ]]> <![CDATA[3.38±0.28 b ]]> <![CDATA[3.92±0.06 b ]]> Embodiment 21 <![CDATA[2.13±0.20 a ]]> <![CDATA[2.35±0.05 a ]]> <![CDATA[3.24±0.12 b ]]> <![CDATA[3.76±0.16 b ]]> Embodiment 22 <![CDATA[1.96±0.16 a ]]> <![CDATA[2.33±0.13 a ]]> <![CDATA[3.28±0.14 b ]]> <![CDATA[3.73±0.05 b ]]> Comparative Example 2 <![CDATA[2.06±0.24 a ]]> <![CDATA[2.09±0.17 a ]]> <![CDATA[2.13±0.14 a ]]> <![CDATA[2.11±0.12 a ]]>

[0163] Note: Different lowercase letters in the same column of data indicate significant differences at the α=0.05 level (IBM SPSS statistics 25 software T test for statistical analysis).

[0164] Table 2

[0165] Example 8d 10d 12d 14d Example 9 <![CDATA[6.78±0.13 d ]]> <![CDATA[6.73±0.03 d ]]> <![CDATA[6.93±0.13 d ]]> <![CDATA[6.9±0.23 d ]]> Example 10 <![CDATA[6.84±0.17 d ]]> <![CDATA[6.8±0.09 d ]]> <![CDATA[6.83±0.16 d ]]> <![CDATA[6.8±0.39 d ]]> Embodiment 11 <![CDATA[6.57±0.14 d ]]> <![CDATA[6.57±0.33 d ]]> <![CDATA[6.97±0.27 d ]]> <![CDATA[6.76±0.22 d ]]> Example 12 <![CDATA[6.79±0.08 d ]]> <![CDATA[6.67±0.23 d ]]> <![CDATA[6.76±0.19 d ]]> <![CDATA[6.53±0.18 d ]]> Embodiment 13 <![CDATA[6.67±0.23 d ]]> <![CDATA[6.94±0.12 d ]]> <![CDATA[6.89±0.24 d ]]> <![CDATA[6.86±0.21 d ]]> Embodiment 14 <![CDATA[6.8±0.02 d ]]> <![CDATA[6.6±0.30 d ]]> <![CDATA[6.97±0.30 d ]]> <![CDATA[6.71±0.30 d ]]> Embodiment 15 <![CDATA[6.69±0.19 d ]]> <![CDATA[6.75±0.27 d ]]> <![CDATA[6.87±0.11 d ]]> <![CDATA[6.73±0.27 d ]]> Example 16 <![CDATA[6.15±0.57 d ]]> <![CDATA[6.1±0.35 d ]]> <![CDATA[6.81±0.26 d ]]> <![CDATA[6.75±0.30 d ]]> Embodiment 17 <![CDATA[5.48±0.14 c ]]> <![CDATA[6.74±0.26 d ]]> <![CDATA[6.78±0.18 d ]]> <![CDATA[6.79±0.23 d ]]> Embodiment 18 <![CDATA[4.50±0.18 b ]]> <![CDATA[4.8±0.09 c ]]> <![CDATA[5.23±0.16 c ]]> <![CDATA[5.43±0.22 c <!-- 9 -->]]> Embodiment 19 <![CDATA[4.43±0.14 b ]]> <![CDATA[4.87±0.13 c ]]> <![CDATA[5.16±0.20 c ]]> <![CDATA[5.25±0.17 c ]]> Embodiment 20 <![CDATA[4.24±0.10 b ]]> <![CDATA[4.27±0.32 b ]]> <![CDATA[4.38±0.10 b ]]> <![CDATA[4.42±0.09 b ]]> Embodiment 21 <![CDATA[4.27±0.10 b ]]> <![CDATA[4.45±0.06 b ]]> <![CDATA[4.44±0.03 b ]]> <![CDATA[4.39±0.28 b ]]> Embodiment 22 <![CDATA[4.38±0.22 b ]]> <![CDATA[4.42±0.21 b ]]> <![CDATA[4.47±0.13 b ]]> <![CDATA[4.38±0.34 b ]]> Comparative Example 2 <![CDATA[2.24±0.21 a ]]> <![CDATA[2.26±0.31 a ]]> <![CDATA[2.36±0.33 a ]]> <![CDATA[2.53±0.34 a ]]>

[0166] Note: Different lowercase letters in the same column of data indicate significant differences at the α=0.05 level (IBM SPSS statistics 25 software T test for statistical analysis).

[0167] According to Table 1 and Table 2, the dermal fillers prepared in Examples 9 to 17 of the present invention have a better effect of promoting HSF cells to synthesize type I collagen.

Claims

1. A method for preparing a textured bacterial cellulose gel sheet, the method comprising the following steps: 1) treating the bacterial cellulose gel sheet with a hydrogen peroxide aqueous solution to obtain a hydrogen peroxide-treated bacterial cellulose gel sheet, wherein the bacterial cellulose gel sheet is immersed in the hydrogen peroxide aqueous solution for 1 to 2 hours, wherein the bacterial cellulose gel sheet is ultrasonicated for 10 to 30 minutes; 2) treating the hydrogen peroxide-treated bacterial cellulose gel sheet with an aqueous solution of an alkaline earth metal salt and / or a transition metal salt to obtain a salt-treated bacterial cellulose gel sheet, wherein the hydrogen peroxide-treated bacterial cellulose gel sheet is immersed in the aqueous salt solution for 2 to 24 hours, wherein the hydrogen peroxide-treated bacterial cellulose gel sheet is subjected to ultrasound for 0.5 to 1 hour; 3) subjecting the salt-treated bacterial cellulose gel sheet to freeze-thaw treatment to obtain a freeze-thaw-treated bacterial cellulose gel sheet; 4) treating the freeze-thaw treated bacterial cellulose gel sheet with alkali to obtain an alkali treated bacterial cellulose gel sheet, wherein the freeze-thaw treated bacterial cellulose gel sheet is immersed in the alkali aqueous solution for 6 to 24 hours; 5) stretching the alkali-treated bacterial cellulose gel sheet to obtain a stretched bacterial cellulose gel sheet; 6) treating the stretched bacterial cellulose gel sheet with acid to obtain a textured bacterial cellulose gel sheet, wherein the stretched bacterial cellulose gel sheet is immersed in the aqueous solution of the acid for 1 to 2 hours; or I) treating the bacterial cellulose gel sheet with a hydrogen peroxide aqueous solution to obtain a hydrogen peroxide-treated bacterial cellulose gel sheet, wherein the bacterial cellulose gel sheet is immersed in the hydrogen peroxide aqueous solution for 1 to 2 hours, wherein the bacterial cellulose gel sheet is ultrasonicated for 10 to 30 minutes; II) subjecting the hydrogen peroxide-treated bacterial cellulose gel sheet to freeze-thaw treatment to obtain a freeze-thaw-treated bacterial cellulose gel sheet; III) treating the freeze-thaw treated bacterial cellulose gel sheet with alkali to obtain an alkali treated bacterial cellulose gel sheet, wherein the freeze-thaw treated bacterial cellulose gel sheet is immersed in the alkali aqueous solution for 6 to 24 hours; IV) stretching the alkali-treated bacterial cellulose gel sheet to obtain a stretched bacterial cellulose gel sheet; V) treating the stretched bacterial cellulose gel sheet with an acid to obtain a textured bacterial cellulose gel sheet, wherein the stretched bacterial cellulose gel sheet is immersed in the aqueous solution of the acid for 1 to 2 hours; or 1) treating the bacterial cellulose gel sheet with a hydrogen peroxide aqueous solution to obtain a hydrogen peroxide-treated bacterial cellulose gel sheet, wherein the bacterial cellulose gel sheet is immersed in the hydrogen peroxide aqueous solution for 1 to 2 hours, wherein the bacterial cellulose gel sheet is ultrasonicated for 10 to 30 minutes; 2) treating the bacterial cellulose gel sheet treated with hydrogen peroxide with an aqueous solution of an alkaline earth metal salt and / or a transition metal salt to obtain a salt-treated bacterial cellulose gel sheet, wherein the bacterial cellulose gel sheet treated with hydrogen peroxide is immersed in the aqueous solution of the salt for 2 to 24 hours, wherein the hydrogen peroxide-treated bacterial cellulose gel sheet is subjected to ultrasonic treatment for 0.5 to 1 hour; 3) treating the salt-treated bacterial cellulose gel sheet with alkali to obtain an alkali-treated bacterial cellulose gel sheet, wherein the salt-treated bacterial cellulose gel sheet is immersed in the alkali aqueous solution for 6 to 24 hours; 4) stretching the alkali-treated bacterial cellulose gel sheet to obtain a stretched bacterial cellulose gel sheet; 5) treating the stretched bacterial cellulose gel sheet with acid to obtain a textured bacterial cellulose gel sheet, wherein the stretched bacterial cellulose gel sheet is immersed in the aqueous solution of the acid for 1 to 2 hours.

2. The preparation method according to claim 1, characterized in that: The alkaline earth metal salt and / or transition metal salt is at least one of magnesium chloride, calcium chloride, strontium chloride, zinc chloride, magnesium nitrate, calcium nitrate, strontium nitrate and zinc nitrate.

3. The preparation method according to claim 1, characterized in that: In step 1), step I) or step a), the mass / volume ratio of the bacterial cellulose gel sheet to the aqueous hydrogen peroxide solution is 1 g:(1-10 mL).

4. The preparation method according to claim 1, characterized in that: The volume concentration of the hydrogen peroxide aqueous solution is 2% to 10%.

5. The preparation method according to claim 1, characterized in that: In step 2) or step 2), the mass / volume ratio of the bacterial cellulose gel sheet to the salt aqueous solution is 1 g:(1-10 mL).

6. The preparation method according to claim 1, characterized in that: The molar concentration of the aqueous salt solution is 0.01 to 1 mol / L.

7. The preparation method according to claim 1, characterized in that: In step 4), step III) or step III), the base is sodium hydroxide.

8. The preparation method according to claim 1, characterized in that The mass / volume ratio of the bacterial cellulose gel sheet to the aqueous solution of the alkali is 1 g:(1-10 mL); the alkali concentration is 0.1 mol / L.

9. The preparation method according to claim 1, characterized in that: In step 6), step V) or step V), the acid is hydrochloric acid.

10. The preparation method according to claim 1, characterized in that The mass / volume ratio of the bacterial cellulose gel sheet to the acid aqueous solution is 1 g:(1-20 mL); the acid concentration is 0.1 mol / L.

11. The preparation method according to claim 1, characterized in that: In step 3) or step II), freeze and thaw 1 to 5 times.

12. The preparation method according to claim 1, characterized in that: In step 5), step IV) or step IV), the alkali-treated bacterial cellulose gel sheet is stretched to an elongation of 10% to 60%.

13. The preparation method according to any one of claims 1 to 12, characterized in that: The method further comprises washing the textured bacterial cellulose gel sheet to obtain a washed textured bacterial cellulose gel sheet.

14. The preparation method according to claim 13, characterized in that: The textured bacterial cellulose gel sheet was washed with water.

15. A method for preparing a textured bacterial cellulose microgel, the method comprising homogenizing and crushing the cleaned textured bacterial cellulose gel sheet in the preparation method according to claim 13 or 14 to obtain a homogenized textured bacterial cellulose microgel.

16. The preparation method according to claim 15, characterized in that: The cleaned textured bacterial cellulose gel sheet is homogenized and crushed in physiological saline or water for injection to obtain a homogenized textured bacterial cellulose microgel dispersion.

17. The preparation method according to claim 16, characterized in that: In the textured bacterial cellulose microgel dispersion, the mass content of the physiological saline or water for injection is 10% to 99%.

18. A skin filling composition comprising the textured bacterial cellulose microgel prepared by the preparation method according to any one of claims 15 to 17, and optionally other fillers.

19. The skin filling composition according to claim 18, characterized in that The skin filling composition also includes growth factors that promote skin growth.

20. The skin filling composition according to claim 19, characterized in that The growth factors include epidermal growth factor and / or fibroblast growth factor.

21. The skin filling composition according to claim 18, characterized in that The other fillers include at least one of sodium carboxymethylcellulose, sodium hyaluronate and carboxymethyl chitosan.

22. The skin filling composition according to any one of claims 18 to 21, characterized in that Taking the mass of the textured bacterial cellulose microgel dispersion in the preparation method according to claim 16 or 17 as 100%, the mass fraction of the other filler is 0.2% to 20%.

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