CN115068661B - A calcium alginate composite porous biological matrix dressing, its preparation method and application - Google Patents

Abstract

The invention belongs to the technical field of biological materials, and particularly relates to a calcium alginate composite porous biological matrix dressing, a preparation method and application thereof. The calcium alginate composite porous biological matrix dressing is prepared from the following materials: 5-15 parts of cell-free freeze-dried powder digestion product and 85-95 parts of calcium alginate gel, wherein the sum of the weight of the cell-free freeze-dried powder digestion product and the calcium alginate gel is 100 parts. The calcium alginate composite porous biological matrix dressing provided by the invention not only can prolong the release duration of the medicine, but also has better antibacterial and anti-inflammatory effects.

Description

A calcium alginate composite porous biological matrix dressing, its preparation method and application

technical field

The invention belongs to the technical field of biomaterials, and in particular relates to a calcium alginate composite porous biomatrix dressing, its preparation method and application.

Background technique

The epidermis, dermis, and subcutaneous tissue together constitute the skin structure of the human body. As one of the most important organs in the human body, the skin covers the surface of the human body and has defense functions, sensory functions, immune functions, absorption functions, body temperature regulation functions, excretion functions and secretion and other functions. When human skin is damaged, especially skin burns, it is easy to cause various damages to the body after the burn, such as immune system imbalance, excessive loss of water, protein and other cell components, and increased metabolism. Severe burns can even be life-threatening. Therefore, wound dressing must be used to cover the wound surface after injury, which can temporarily protect the skin barrier and promote wound healing, which is especially important for large area burns.

Acellular dermal matrix dressing is a dermal substitute that can be used for full-thickness skin defects and a medical dressing for wound healing of skin injuries such as burns. It is a natural skin that has undergone a series of treatments to remove cellular components in the epidermis and dermis. However, the dermal extracellular matrix components and its three-dimensional spatial structure are retained. Such a structure can provide a "dermal template" for skin regeneration, and induce host cell components such as fibroblasts and endothelial cells to differentiate and grow in the scaffold structure after transplantation.

CN106310352A discloses a preparation method of an antibacterial acellular dermal matrix dressing. The preparation method comprises the following steps: selecting healthy mammals, peeling off the animal skin, removing hair, preparing split skin slices, laser drilling, low temperature freezing and thawing, ultrasonic The antibacterial acellular dermal matrix dressing was obtained after shaking, degreasing treatment, decellularization treatment, soaking in water-soluble chitosan, freeze-drying, packaging and irradiation sterilization. However, due to the limited functional effect of chitosan itself, the effect of the prepared dressing is not ideal. It is necessary to use chitosan in combination with other materials to improve the comprehensive performance of the dressing.

The applicant applied for an invention patent application with the application number 202111545807.X on December 16, 2021, and the title of the invention is "Preparation and Application of a Degradable Placental Acellular Porous Sponge Matrix Dressing". The preparation includes the following steps: (1) material selection of placenta tissue; (2) collection and transportation of placenta tissue; (3) virus inactivation treatment of placenta tissue; (4) removal of virus inactivation solution residue; (6) Decellularization process; (7) Removal of residual decellularized solution; (8) Freeze-drying for the first time; (9) Freezing ball milling; (10) Digesting the obtained decellularized freeze-dried powder; (11) Adjust the pH of the obtained homogeneous digestion product, and pour it into freeze-drying molds of different specifications to obtain a decellularized porous sponge; (12) Sterilization and inactivation of viruses. On this basis, the applicant replaced the placental tissue with animal tissue, such as pig skin, pig small intestine, pig pericardium or pig bladder, and pre-prepared a porous biomatrix dressing by the above method, but unfortunately, the obtained Porous biomatrix dressings have been found to have a short duration of drug release, limited effect, and no antibacterial and anti-inflammatory effects.

In view of this, the present invention is proposed.

Contents of the invention

The object of the present invention is to provide a calcium alginate composite porous biological matrix dressing, its preparation method and application. The calcium alginate composite porous biomatrix dressing provided by the present invention can not only prolong the release duration of medicines, but also have good antibacterial and anti-inflammatory effects.

For realizing the purpose of the present invention, the present invention adopts following technical scheme:

A calcium alginate composite porous biomatrix dressing, wherein the calcium alginate composite porous biomatrix dressing is prepared from the following materials:

Decellularized freeze-dried powder digestion product 5-15 parts by weight

Calcium alginate gel 85-95 parts by weight

The sum of both weights is 100 parts by weight.

As a preferred solution, the calcium alginate composite porous biomatrix dressing is prepared from the following materials:

Decellularized freeze-dried powder digestion product 10 parts by weight

90 parts by weight of calcium alginate gel.

Further, the calcium alginate gel is obtained by reacting sodium alginate, calcium chloride and gluconolactone.

Further, the calcium alginate gel is prepared by the following preparation method: completely dissolve sodium alginate with an appropriate amount of water for injection, add calcium chloride with a mass ratio of 1 to 3:1 to sodium alginate and stir evenly, Then add gluconolactone with a mass ratio of 4-6:1 to sodium alginate calcium chloride mixture, adjust the pH to 4.5-6.5 with hydrochloric acid, stir at a temperature of 45-55°C for 30-45 minutes to obtain the Calcium alginate gel.

In the present invention, the mass ratio of the added calcium chloride to sodium alginate is 1 to 3:1, preferably 2:1; the mass ratio of the added gluconolactone to the sodium alginate calcium chloride mixture 4-6:1, preferably 5:1.

Further, the digested product of decellularized freeze-dried powder is obtained by collecting and transporting animal tissues, virus inactivation treatment, cycle freeze-thaw treatment, decellularization, removal of residual decellularization solution, freeze-drying, freeze-ball milling and pepsin digestion be made of.

In the present invention, the decellularized freeze-dried powder digestion product is prepared by referring to the method in CN114177340A or CN114306749A from animal tissues.

Preferably prepared as follows:

(1) Collection and transportation: Collect fresh animal tissues, cut into small pieces with a length and width no greater than 2cm, and then immerse them in virus inactivation solution (peracetic acid solution). After soaking in the solution for 0.5-4 hours, place them in -8 Long-term storage below ℃; animal tissues are transported in low-temperature cold chain (below 0°C);

(2) Virus inactivation treatment: the virus inactivation solution is mixed with peracetic acid, absolute ethanol (≥99.7%) and purified water in a volume ratio of 3:5:92, and 19L of virus inactivation solution is added to 1kg of animal tissue for treatment 0.5 to 2 hours;

(3) Multiple cycles of freezing and thawing treatment: The tissue after virus inactivation treatment is subjected to multiple cycles of freezing and thawing treatment, the freezing temperature is -80°C to -50°C, and the freezing time is not less than 6 hours; the melting temperature is 23°C ~28°C; after each thaw, discard the supernatant by centrifugation, and re-add an equal amount of purified water to freeze and thaw;

(4) Decellularization: Treat the tissue treated in step (3) with enzyme solution, non-ionic surfactant and ionic surfactant solution for 60-90 minutes in sequence, and keep stirring during the period; wherein, the enzyme solution is the mass percentage concentration 0.20%-0.25% trypsin, the non-ionic surfactant solution is triton solution with a mass percentage concentration of 0.10%-0.15%, and the ionic surfactant solution is dodecane with a mass percentage concentration of 0.10%-0.15% Sodium sulfate;

(5) Remove the residual decellularized solution: wash the tissue treated in step (4) with purified water for 15 minutes, centrifuge and discard the supernatant, and repeat 4 to 6 times;

(6) Freeze-drying: put the tissue treated in step (5) into a freeze-drying tray, pre-freeze at -60 to -45°C for 6 to 8 hours, and then raise the temperature from the pre-freezing temperature gradient to 25°C, each gradient temperature The temperature point was increased by 5°C, each temperature point was maintained for 3 hours at the first 8 temperature points, and each temperature point was maintained for 2 hours, and then vacuum-dried to obtain dry decellularized animal tissue;

(7) Freeze ball milling: the dried decellularized animal tissue obtained in step (6) is subjected to freeze ball milling at a temperature of -20° C. to -50° C. to obtain decellularized freeze-dried powder;

(8) Pepsin digestion: the decellularized freeze-dried powder obtained in step (7) is digested with pepsin solution, wherein the mass-to-volume ratio of the quality of the decellularized freeze-dried powder to the pepsin solution is 10mg: 1ml, and the pepsin solution The concentration is 1mg/ml, and the digestion time is 24-96 hours to obtain the digested product of decellularized freeze-dried powder.

Wherein, the animal tissue is pig skin, pig small intestine, pig pericardium or pig bladder.

The present invention also provides a preparation method of the calcium alginate composite porous biomatrix dressing, the method comprising the steps of:

S1. Preparation of acellular freeze-dried powder digestion product

The animal tissue is prepared into a decellularized freeze-dried powder digestion product;

S2, preparation of calcium alginate gel

Calcium alginate gel is prepared by reacting sodium alginate with calcium chloride and gluconolactone;

S3. Preparation of calcium alginate composite porous biomatrix dressing

The calcium alginate composite porous biomatrix dressing is prepared by preparing the digested product of decellularized freeze-dried powder prepared in step S1 and the calcium alginate gel prepared in step S2.

Further, the process of step S2 is: completely dissolve sodium alginate with an appropriate amount of water for injection, add calcium chloride with a mass ratio of 1 to 3:1 to sodium alginate and stir evenly, and then add sodium alginate and calcium chloride The mass ratio of the mixed solution is 4-6:1 gluconolactone, hydrochloric acid to adjust the pH to 4.5-6.5, and stirred at a temperature of 45-55° C. for 30-45 minutes to obtain the calcium alginate gel.

Further, step S3 includes the following steps:

S3.1. Mix the digested product of decellularized freeze-dried powder and calcium alginate gel at a mass ratio of 5-15:85-95, adjust the pH to 6.5-7.5 with NaOH solution, stir and mix evenly to obtain a mixture;

S3.2. Pour the obtained mixture into a freeze-drying mold to freeze-dry to obtain a freeze-dried product;

S3.3, placing the freeze-dried product in a forming mold and pressing it to obtain a three-layer matrix dressing;

S3.4, placing the three-layer matrix dressing in a perforated forming mold for pressing, uniformly punching holes, and obtaining a perforated matrix dressing;

S3.5, packing the perforated matrix dressing to obtain a packaged matrix dressing;

S3.6. Sterilize the packaged matrix dressing to obtain the calcium alginate composite porous biological matrix dressing.

Further, in step S3.3, the thickness of the uppermost layer and the lowermost layer of the obtained three-layer matrix dressing is 0.5 mm to 1.0 mm, and the thickness of the inner layer is 1.5 mm to 2.0 mm; in step S3.4, the thickness of the punched hole is The hole diameter is 2mm~3mm.

Further, in step S3.6, the sterilization treatment is EB radiation sterilization treatment, and the sterilization dose is controlled within the range of 20-25 kGY.

Further, in step S3.2, the freeze-drying is to pre-freeze at a temperature of -60 to -45°C for 6 to 8 hours, and then increase the temperature from the pre-freezing temperature gradient to 25°C, and each gradient temperature point increases 5°C, each of the first 8 temperature points was kept for 3 hours, and each of the subsequent temperature points was kept for 2 hours, and then vacuum-dried to obtain a freeze-dried product.

In the present invention, the first 8 temperature points are the first 8 temperature points from the pre-freezing temperature, such as the pre-freezing temperature is -55 ° C, and the first 8 gradient temperature points are -55 ° C, -50 ° C, - 45°C, -40°C, -35°C, -30°C, -25°C and -20°C.

The present invention also provides the application of the calcium alginate composite porous biomatrix dressing in the preparation of medical aesthetics, plastic surgery or women's health products.

The calcium alginate composite porous biomatrix dressing provided by the present invention can load a variety of cells and support the growth of cells, such as dermal fibroblasts, endometrial mesenchymal stem cells or human epidermal growth factor (hEGF), etc., for the preparation of Medical aesthetics, plastic surgery or women's health products, such as minimally invasive treatment of diseases caused by local soft tissue structure relaxation, can also be widely used in the fields of aesthetic medicine such as breast augmentation and facial plastic surgery.

Compared with prior art, the present invention has following advantage:

The calcium alginate composite porous biomatrix dressing provided by the present invention can not only prolong the release duration of medicines, but also have good antibacterial and anti-inflammatory effects.

Description of drawings

Fig. 1 is the electron micrograph of the calcium alginate composite porous biomatrix dressing that the embodiment of the present invention 1 makes;

Fig. 2 is the electron micrograph of the porous biomatrix dressing that comparative example 1 makes;

Fig. 3 is the drug release duration figure of product of the present invention and comparative example product;

Fig. 4 is a diagram of drug release duration of calcium alginate composite porous biomatrix dressings with different pore sizes.

Detailed ways

The following are specific embodiments of the present invention, and the described examples are for further describing the present invention, rather than limiting the present invention.

In the following examples, the digested product of decellularized freeze-dried powder is prepared by referring to the method in CN114177340A or CN114306749A of animal tissue, preferably according to the following method:

(1) Collection and transportation: collect fresh animal tissues, such as pig skin, pig small intestine, pig pericardium or pig bladder, cut into small pieces with a length and width not greater than 2cm, and then immerse in virus inactivation solution (peracetic acid solution), After soaking in the solution for 2.5 hours, store it below -8°C for long-term storage; animal tissues are transported in a low-temperature cold chain (below 0°C);

(2) Virus inactivation treatment: the virus inactivation solution is mixed with peracetic acid, absolute ethanol (≥99.7%) and purified water in a volume ratio of 3:5:92, and 19L of virus inactivation solution is added to 1kg of animal tissue for treatment 1.2 hours;

(3) Multiple cycles of freeze-thaw treatment: The tissue after virus inactivation treatment was subjected to multiple cycles of freeze-thaw treatment, the freezing temperature was -65°C, and the freezing time was 6 hours; the melting temperature was 25°C; after each thawing, centrifuged Discard the supernatant, add an equal amount of purified water again, freeze and thaw, and cycle freeze-thaw three times;

(4) Decellularization: the tissue treated in step (3) was treated with enzyme solution, non-ionic surfactant and ionic surfactant solution for 75 minutes in sequence, during which the stirring was continued; wherein, the enzyme solution had a concentration of 0.22% by mass trypsin, the non-ionic surfactant solution is a triton solution with a mass percentage concentration of 0.12%, and the ionic surfactant solution is sodium lauryl sulfate with a mass percentage concentration of 0.12%;

(5) Remove the residual decellularized solution: wash the tissue treated in step (4) with purified water for 15 minutes, centrifuge and discard the supernatant, and repeat 5 times;

(6) Freeze-drying: Put the tissue treated in step (5) into a freeze-drying tray, pre-freeze at -55°C for 7 hours, and then gradually increase the temperature from the pre-freezing temperature -55°C to 25°C. The gradient temperature point was increased by 5°C, each of the first 8 temperature points was maintained for 3 hours, and each subsequent temperature point was maintained for 2 hours, and then vacuum-dried to obtain dry decellularized animal tissue;

(7) Freezing ball milling: the dried decellularized animal tissue obtained in step (6) is subjected to freezing ball milling at a temperature of -35° C. to obtain decellularized freeze-dried powder;

(8) Pepsin digestion: the decellularized freeze-dried powder obtained in step (7) is digested with pepsin solution, wherein the mass-to-volume ratio of the quality of the decellularized freeze-dried powder to the pepsin solution is 10mg: 1ml, and the pepsin solution The concentration is 1 mg/ml, and the digestion time is 60 hours to obtain the digested product of decellularized freeze-dried powder.

Example 1

The calcium alginate composite porous biomatrix dressing of this embodiment is prepared from the following materials:

Decellularized freeze-dried powder digestion product 10 parts by weight

Calcium alginate gel 90 parts by weight

Preparation:

S1. Preparation of acellular freeze-dried powder digestion product

The pig skin is collected and transported according to the above-mentioned preferred method, virus inactivation treatment, cycle freeze-thaw treatment, decellularization, removal of decellularization solution residue, cryoball milling and pepsin digestion to obtain a decellularized freeze-dried powder digestion product.

S2, preparation of calcium alginate gel

Dissolve sodium alginate completely with an appropriate amount of water for injection, add calcium chloride with a mass ratio of 2:1 to sodium alginate and stir evenly, then add glucose with a mass ratio of 5:1 to sodium alginate and calcium chloride mixture lactone and hydrochloric acid to adjust the pH to 5.0, and stirred at a temperature of 50° C. for 40 minutes to obtain the calcium alginate gel.

S3. Preparation of calcium alginate composite porous biomatrix dressing

S3.1. Mix the digested product of decellularized freeze-dried powder and calcium alginate gel at a mass ratio of 10:90, adjust the pH to 7.0 with NaOH solution, stir and mix evenly to obtain a mixture;

S3.2. Pour the obtained mixture into a freeze-drying mold to freeze-dry to obtain a freeze-dried product; wherein, the freeze-drying is to pre-freeze at a temperature of -55°C for 7 hours, and then freeze at a temperature of -55°C Gradiently raise the temperature to 25°C, each gradient temperature point is increased by 5°C, each temperature point of the first 8 temperature points is kept for 3 hours, and each temperature point after that is kept for 2 hours, and then vacuum-dried to obtain a freeze-dried product;

S3.3. Place the freeze-dried product in a forming mold and press it to obtain a three-layer matrix dressing. The thickness of the uppermost layer and the lowermost layer of the obtained three-layer matrix dressing is 0.8mm, and the thickness of the inner layer is 1.8mm;

S3.4, placing the three-layer matrix dressing in a perforated mold for pressing, uniformly punching, the hole diameter of the punched hole is 2mm, and obtaining the matrix dressing after punching;

S3.5, packing the perforated matrix dressing to obtain a packaged matrix dressing;

S3.6. The packaged matrix dressing was sterilized by EB irradiation with a sterilization dose of 22 kGY to obtain the calcium alginate composite porous biological matrix dressing.

The electron micrograph of the obtained calcium alginate composite porous biomatrix dressing is shown in FIG. 1 .

Example 2

The calcium alginate composite porous biomatrix dressing of this embodiment is prepared from the following materials:

Decellularized freeze-dried powder digestion product 5 parts by weight

Calcium alginate gel 95 parts by weight

Preparation:

S1. Preparation of acellular freeze-dried powder digestion product

The pig small intestine is collected and transported according to the above-mentioned preferred method, virus inactivation treatment, cycle freeze-thaw treatment, decellularization, removal of decellularization solution residue, cryogenic ball milling and pepsin digestion to obtain the decellularized freeze-dried powder digestion product.

S2, preparation of calcium alginate gel

Dissolve sodium alginate completely with an appropriate amount of water for injection, add calcium chloride with a mass ratio of 1:1 to sodium alginate and stir evenly, then add glucose with a mass ratio of 4:1 to sodium alginate and calcium chloride mixture lactone and hydrochloric acid to adjust the pH to 4.5, and stirred at a temperature of 45° C. for 30 minutes to obtain the calcium alginate gel.

S3. Preparation of calcium alginate composite porous biomatrix dressing

S3.1. Mix the digested product of decellularized freeze-dried powder and calcium alginate gel at a mass ratio of 5:95, adjust the pH to 6.5 with NaOH solution, stir and mix evenly to obtain a mixture;

S3.2. Pour the obtained mixture into a freeze-drying mold to freeze-dry to obtain a freeze-dried product; wherein, the freeze-drying is to pre-freeze at a temperature of -60°C for 6 hours, and then freeze at a temperature of -60°C Gradiently raise the temperature to 25°C, each gradient temperature point is increased by 5°C, each temperature point of the first 8 temperature points is kept for 3 hours, and each temperature point after that is kept for 2 hours, and then vacuum-dried to obtain a freeze-dried product;

S3.3. Place the freeze-dried product in a forming mold and press to obtain a three-layer matrix dressing. The thickness of the uppermost layer and the lowermost layer of the obtained three-layer matrix dressing is 0.5mm, and the thickness of the inner layer is 1.5mm;

S3.4, placing the three-layer matrix dressing in a perforated molding mold for pressing, uniformly punching, the hole diameter of the punched hole is 3mm, and obtaining the matrix dressing after punching;

S3.5, packing the perforated matrix dressing to obtain a packaged matrix dressing;

S3.6. The packaged matrix dressing is sterilized by EB irradiation with a sterilization dose of 20 kGY to obtain the calcium alginate composite porous biological matrix dressing.

Example 3

The calcium alginate composite porous biomatrix dressing of this embodiment is prepared from the following materials:

Decellularized freeze-dried powder digestion product 15 parts by weight

Calcium alginate gel 85 parts by weight

Preparation:

S1. Preparation of acellular freeze-dried powder digestion product

The pig pericardium is collected and transported according to the above-mentioned preferred method, virus inactivation treatment, cycle freeze-thaw treatment, decellularization, removal of decellularization solution residue, cryogenic ball milling and pepsin digestion to obtain the decellularized freeze-dried powder digestion product.

S2, preparation of calcium alginate gel

Dissolve sodium alginate completely with an appropriate amount of water for injection, add calcium chloride with a mass ratio of 3:1 to sodium alginate and stir evenly, then add glucose with a mass ratio of 6:1 to sodium alginate and calcium chloride mixture lactone and hydrochloric acid to adjust the pH to 6.5, and stirred at a temperature of 55° C. for 45 minutes to obtain the calcium alginate gel.

S3. Preparation of calcium alginate composite porous biomatrix dressing

S3.1. Mix the digested product of decellularized freeze-dried powder and calcium alginate gel at a mass ratio of 15:85, adjust the pH to 7.5 with NaOH solution, stir and mix evenly to obtain a mixture;

S3.2. Pour the obtained mixture into a freeze-drying mold to freeze-dry to obtain a freeze-dried product; wherein, the freeze-drying is to pre-freeze at a temperature of -45°C for 8 hours, and then freeze at a temperature of -45°C Gradiently raise the temperature to 25°C, each gradient temperature point is increased by 5°C, each temperature point of the first 8 temperature points is kept for 3 hours, and each temperature point after that is kept for 2 hours, and then vacuum-dried to obtain a freeze-dried product;

S3.3. Place the freeze-dried product in a forming mold and press it to obtain a three-layer matrix dressing. The thickness of the uppermost layer and the lowermost layer of the obtained three-layer matrix dressing is 1.0mm, and the thickness of the inner layer is 2.0mm;

S3.4, placing the three-layer matrix dressing in a perforated mold for pressing, uniformly punching, the hole diameter of the punched hole is 2.5mm, and obtaining the matrix dressing after punching;

S3.5, packing the perforated matrix dressing to obtain a packaged matrix dressing;

S3.6. Subject the packaged matrix dressing to EB irradiation sterilization with a sterilization dose of 25 kGY to obtain the calcium alginate composite porous biological matrix dressing.

Comparative example 1

The porous biomatrix dressing of this comparative example is prepared from the following materials:

100 parts by weight of acellular freeze-dried powder digestion product

Preparation:

S1. Preparation of acellular freeze-dried powder digestion product

The pig skin is collected and transported according to the above-mentioned preferred method, virus inactivation treatment, cycle freeze-thaw treatment, decellularization, removal of decellularization solution residue, cryoball milling and pepsin digestion to obtain a decellularized freeze-dried powder digestion product.

S2, preparation of porous biomatrix dressing

S2.1. Adjust the pH of the digested product of the decellularized lyophilized powder to 7.0 with NaOH solution to obtain a solution;

S2.2. Pour the solution obtained in step S2.1 into a freeze-drying mold to freeze-dry to obtain a freeze-dried product; wherein, the freeze-drying is to pre-freeze at a temperature of -55°C for 7 hours, and then from the pre-freeze The temperature gradient is increased to 25°C, and each gradient temperature point is increased by 5°C, and each temperature point of the first 8 temperature points is maintained for 3 hours, and each temperature point is maintained for 2 hours, and then vacuum-dried to obtain a freeze-dried product;

S3.3. The freeze-dried product is sterilized by EB irradiation with a sterilization dose of 22 kGY to obtain the porous biomatrix dressing.

The electron micrograph of the obtained porous biomatrix dressing is shown in FIG. 2 .

Comparative example 2

The composite porous biomatrix dressing of this comparative example is prepared from the following materials:

Decellularized freeze-dried powder digestion product 10 parts by weight

Chitosan-calcium alginate gel 90 parts by weight

Preparation:

S1. Preparation of acellular freeze-dried powder digestion product

The pig skin is collected and transported according to the above-mentioned preferred method, virus inactivation treatment, cycle freeze-thaw treatment, decellularization, removal of decellularization solution residue, cryoball milling and pepsin digestion to obtain a decellularized freeze-dried powder digestion product.

S2, preparation of calcium alginate gel

2.0% (w/v) sodium alginate solution was prepared by electrostatic droplet method under the condition of 1.0mol/LCaCl solution as a gel bath to obtain calcium alginate gel microspheres with a particle size of _mainly 400-600 μm, and Calcium alginate gel microspheres were mixed with 0.7% (w/v) chitosan solution at a volume ratio of 1:7, and then mixed with 0.5% (w/v) sodium alginate solution at a volume ratio of 10:20 , to obtain chitosan-calcium alginate gel.

S3. Preparation of calcium alginate composite porous biomatrix dressing

The decellularized freeze-dried powder digestion product was mixed with chitosan-calcium alginate gel at a mass ratio of 10:90, and the calcium alginate composite porous biomatrix dressing was prepared according to the method in Example 1.

Test example 1

This test example investigated the drug release duration of the product of the present invention and the product of the comparative example.

The test method is as follows:

(1) Add physiological saline to the porous biomatrix dressings prepared in Example 1, Comparative Example 1 and Comparative Example 2 respectively to prepare a 10 mg/mL solution, and shake it up and down for 2 minutes to obtain a uniform gel solution;

(2) Mix the hEGF solution with the gel solution so that the final hEGF concentration is about 3 ng/mL to obtain the prepared hEGF-containing gel;

(3) Add the prepared hEGF-containing gel to a 48-well plate (500 μL per well), place the 48-well plate in a cell incubator at 37°C, and incubate for 1 hour to make it gel;

(4) After incubation, take out the 48-well plate, add 500 μL of PBS to each well, and then put the 48-well plate back into the incubator;

(5) Take out the 48-well plate every 24 hours, add new PBS (500 μL) after absorbing the supernatant, and store the absorbed supernatant in a -80°C refrigerator;

(6) Repeatedly;

(7) The ELISA kit measures the concentration of each sample on days 0, 2, 3, 4, 5, 6, 7, 9, 15, 21 and 28, respectively.

The duration of drug release of the porous biomatrix dressings prepared in Example 1, Comparative Example 1 and Comparative Example 2 is shown in Figure 3 .

It can be seen from Figure 3 that, compared with the porous biomatrix dressings prepared in Comparative Example 1 and Comparative Example 2, the hEGF loaded in the porous biomatrix dressing prepared by the present invention can release slowly and sustainably, exerting a lasting curative effect.

Test example 2

This test example investigated the effect of the pore size of the porous biomatrix dressing on the duration of drug release.

Test method: prepare porous biomatrix dressing according to the method of Example 1, the difference is that the apertures of the holes punched in step S3.4 are different, respectively 2mm, 3mm and 5mm, and investigate the influence of different apertures on the duration of drug release . The specific measurement method is the same as that of Test Example 1.

The results of the measured drug release duration of porous biomatrix dressings with different pore sizes are shown in Figure 4.

The results show that with the reduction of the pore size of the porous biomatrix dressing, the drug release duration will be longer, and it can be seen that the pore size of 5mm can be continuously released to about 98% in about 20 days; The release amount is about 82%, and the release amount is about 80% when the aperture is 2mm to 28 days, and it will still release slowly every day in the later stage. Generally, 28 days of drug sustained release is a commonly used observation period. This result shows that the pore size of the porous biomatrix dressing directly affects the drug sustained release function.

Test example 3

This test example investigated the antibacterial performance of the product of the present invention and the product of comparative example.

experiment method:

The porous biomatrix dressings prepared in Examples and Comparative Examples were picked up with sterilized tweezers respectively, and 3 samples (sample A, sample B, sample C) were respectively inoculated with Escherichia coli, Staphylococcus aureus and Candida albicans respectively. Bacteria, the dressing must be laid flat so that the bacteria evenly contact the sample, placed in a sterilized plate, and cultivated at (37±1)°C and relative humidity RH>90% for 24 hours. Take out the samples cultured for 24 hours, add 20ml of eluent respectively, wash sample A, sample B, sample C and cover film repeatedly (it is better to pick up the film with tweezers to rinse), shake well, take a certain amount to inoculate on nutrient agar culture In the medium, count the viable bacteria after culturing at (37±1)°C for (24-48) hours, and determine the number of viable bacteria. The above experiment was repeated twice.

The antibacterial rate calculation formula is as follows:

In the formula:

R——antibacterial rate (%);

B——the average number of bacteria recovered from the blank control sample (cfu/tablet);

C—the average number of bacteria recovered from the sample (cfu/tablet).

The antibacterial rate of the calculated dressings against Escherichia coli, Staphylococcus aureus and Candida albicans were statistically analyzed by APSS.

The test results are shown in Table 1:

Table 1. Antibacterial performance test results

Example 1 Example 2 Example 3 Comparative example 1 Comparative example 2 Escherichia coli 92% 93% 90% 53% 61% Staphylococcus aureus 90% 91% 88% 55% 65% Candida albicans 93% 95% 91% 59% 67%

It can be seen from the above test results that, compared with the porous biomatrix dressing prepared in the comparative example, the calcium alginate composite porous biomatrix dressing prepared in the present invention has better antibacterial properties.

Claims (9)

1. The calcium alginate composite porous biological matrix dressing is characterized by being prepared from the following materials:

5 to 15 weight portions of decellularized freeze-dried powder digestion product

85-95 parts by weight of calcium alginate gel

The sum of the weight of the two is 100 weight parts;

the calcium alginate gel is prepared by reacting sodium alginate, calcium chloride and gluconolactone;

the calcium alginate gel is prepared by the following preparation method: completely dissolving sodium alginate by using a proper amount of water for injection, and adding the sodium alginate with the mass ratio of 1-3: 1, and then adding the sodium alginate and calcium chloride mixed solution with the mass ratio of 4-6: 1, regulating the pH value to 4.5-6.5 by hydrochloric acid, and stirring for 30-45 minutes at the temperature of 45-55 ℃ to obtain the calcium alginate gel.

2. The calcium alginate composite porous biological matrix dressing according to claim 1, wherein the calcium alginate composite porous biological matrix dressing is prepared from the following materials:

10 parts by weight of cell-free freeze-dried powder digestion product

90 parts by weight of calcium alginate gel.

3. The calcium alginate composite porous biological matrix dressing according to claim 2, wherein the decellularized lyophilized powder digestion product is prepared by collecting and transporting animal tissue, virus inactivating treatment, circulating freeze thawing treatment, decellularizing, removing decellularized solution residues, freeze drying, freeze ball milling and pepsin digestion.

4. A method for preparing the calcium alginate composite porous biological matrix dressing according to any one of claims 1 to 3, wherein the preparation method comprises the following steps:

s1, preparing cell-free freeze-dried powder digestion products

Preparing animal tissues into cell-free freeze-dried powder digestion products;

s2, preparing calcium alginate gel

Reacting sodium alginate with calcium chloride and gluconolactone to obtain calcium alginate gel;

s3, preparing calcium alginate composite porous biological matrix dressing

And (3) preparing the acellular freeze-dried powder digestion product prepared in the step (S1) and the calcium alginate gel prepared in the step (S2) into the calcium alginate composite porous biological matrix dressing.

5. The method according to claim 4, wherein the step S2 comprises the steps of: completely dissolving sodium alginate by using a proper amount of water for injection, and adding the sodium alginate with the mass ratio of 1-3: 1, and then adding the sodium alginate and calcium chloride mixed solution with the mass ratio of 4-6: 1, regulating the pH value to 4.5-6.5 by hydrochloric acid, and stirring for 30-45 minutes at the temperature of 45-55 ℃ to obtain the calcium alginate gel.

6. The method according to claim 4, wherein step S3 comprises the steps of:

s3.1, the cell-free freeze-dried powder digestion product and calcium alginate gel are mixed according to the mass ratio of 5-15: 85-95, regulating the pH to 6.5-7.5 by using NaOH solution, and uniformly stirring and mixing to obtain a mixture;

s3.2, pouring the obtained mixture into a freeze-drying mold for freeze-drying to obtain a freeze-dried product;

s3.3, placing the freeze-dried product in a forming mold for pressing to obtain a three-layer matrix dressing;

s3.4, placing the three-layer matrix dressing in a punching forming die for pressing and uniformly punching to obtain a punched matrix dressing;

s3.5, packaging the perforated matrix dressing to obtain a packaged matrix dressing;

and S3.6, sterilizing the packed matrix dressing to obtain the calcium alginate composite porous biological matrix dressing.

7. The method according to claim 6, wherein in step S3.3, the thickness of the uppermost layer and the lowermost layer of the three-layer matrix dressing is 0.5mm to 1.0mm, and the thickness of the inner layer is 1.5mm to 2.0mm; in the step S3.4, the hole diameter of the punched hole is 2 mm-3 mm.

8. The method according to claim 6 or 7, wherein in step S3.6, the sterilization treatment is EB irradiation sterilization treatment, and the sterilization dose is controlled within a range of 20 to 25 kGY.

9. Use of a calcium alginate composite porous biological matrix dressing according to any one of claims 1 to 3 in the preparation of a medical, cosmetic or feminine health product.

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