CN115068661A

CN115068661A - Calcium alginate composite porous biological matrix dressing, preparation method and application thereof

Info

Publication number: CN115068661A
Application number: CN202210709176.9A
Authority: CN
Inventors: 黄志军; 黄涵
Original assignee: Hangzhou Beirong Biotechnology Co ltd
Current assignee: Hangzhou Beirong Biotechnology Co ltd
Priority date: 2022-06-21
Filing date: 2022-06-21
Publication date: 2022-09-20
Anticipated expiration: 2042-06-21
Also published as: CN115068661B

Abstract

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

Description

Calcium alginate composite porous biological matrix dressing, preparation method and application thereof

Technical Field

The invention belongs to the technical field of biological materials, and particularly relates to a calcium alginate composite porous biological matrix dressing, and a preparation method and application thereof.

Background

The epidermis, the dermis and the subcutaneous tissue together form the skin structure of a human body, and the skin, which is one of the most important organs in the human body, covers the surface of the human body and has the functions of defense, perception, immunity, absorption, body temperature regulation, excretion, secretion and the like. When human skin is damaged, especially skin burns, various damages of organisms are easily caused after the burns, such as immune system disorder, excessive loss of moisture, protein and other cell components, and metabolism aggravation, and the serious burns can also threaten life. Therefore, the wound must be covered with a wound dressing after injury, temporarily protect the skin barrier, and promote healing of the wound, which is especially important for large area burns.

The acellular dermal matrix dressing is a dermal substitute which can be used for full-layer skin defects and a medical dressing for healing skin injury wound surfaces such as burns, and is a medical dressing which is used for natural skin to remove cell components in epidermis and dermis through a series of treatments, but keeps dermal extracellular matrix components and three-dimensional space structures thereof, and the structure can provide a 'dermal template' for skin regeneration and induces host cell components such as fibroblasts and endothelial cells to differentiate and grow in a scaffold structure after transplantation.

CN106310352A discloses a preparation method of an antibacterial acellular dermal matrix dressing, which comprises the following steps: selecting healthy mammals, peeling the skin of the animals, removing hairs, preparing a faulted skin sheet, preparing holes by laser, freezing and thawing at low temperature, oscillating by ultrasonic waves, degreasing, decellularizing, soaking water-soluble chitosan, freeze-drying, packaging and irradiating for sterilization to obtain the antibacterial acellular dermal matrix dressing. However, the chitosan has limited functional effect, so the prepared dressing has less ideal effect, and the chitosan and other materials are required to be compounded for use so as to improve the comprehensive performance of the dressing.

The applicant filed an invention patent application with the application number of 202111545807.X, entitled "preparation and application of degradable placenta decellularized porous sponge matrix dressing" on 12/16/2021. The preparation method comprises the following steps: (1) selecting materials from placenta tissues; (2) collecting and transporting placenta tissues; (3) inactivating the viruses of the placenta tissues; (4) removing the virus inactivation solution residue; (5) performing multiple circulating freeze thawing treatment; (6) a cell removal process; (7) removing the cell-free solution residue; (8) carrying out primary freeze drying; (9) freezing and ball milling; (10) digesting the obtained acellular freeze-dried powder; (11) adjusting the pH value of the obtained uniform digestion product, and pouring the uniform digestion product into freeze-drying molds of different specifications to obtain the acellular porous sponge; (12) sterilizing and inactivating viruses. On the basis of the substitution of the placenta tissue by the animal tissue, such as pigskin, pig small intestine, pig pericardium or pig bladder, the applicant prepares a porous biological matrix dressing by adopting the method, but unfortunately, the obtained porous biological matrix dressing has short release duration, limited action effect and no antibacterial and anti-inflammatory effects in tests.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The invention aims to provide a calcium alginate composite porous biological matrix dressing, a preparation method and application thereof. The calcium alginate composite porous biological matrix dressing provided by the invention not only can prolong the release duration of the medicament, but also has better antibacterial and anti-inflammatory effects.

In order to realize the purpose of the invention, the invention adopts the following technical scheme:

the calcium alginate composite porous biological matrix dressing is prepared from the following materials:

5-15 parts by weight of digestion product of acellular freeze-dried powder

85-95 parts by weight of calcium alginate gel

The sum of the weight of the two is 100 parts by weight.

As a preferable scheme, the calcium alginate composite porous biological matrix dressing is prepared from the following materials:

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

90 parts of calcium alginate gel.

Furthermore, the calcium alginate gel is obtained by the reaction of sodium alginate, calcium chloride and gluconolactone.

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

In the invention, the mass ratio of the added calcium chloride to the sodium alginate is (1-3): 1, preferably 2: 1; the mass ratio of the added gluconolactone to the mixed solution of sodium alginate and calcium chloride is 4-6: 1, preferably 5: 1.

further, the digestion product of the cell-free freeze-dried powder is prepared by collecting and transporting animal tissues, inactivating viruses, circularly freezing and thawing, removing cells, removing the residue of a cell-free solution, freeze-drying, freezing and ball-milling and digesting pepsin.

In the invention, the digestion product of the decellularized lyophilized powder is prepared by referring to the method in CN114177340A or CN114306749A for animal tissues.

The preparation method is preferably as follows:

(1) collecting and transporting: collecting fresh animal tissues, shearing the fresh animal tissues into small pieces with the length and width not more than 2cm, then immersing the small pieces into a virus inactivation solution (peroxyacetic acid solution), immersing the small pieces in the virus inactivation solution for 0.5 to 4 hours, and then storing the small pieces at the temperature of below 8 ℃ below zero for a long time; the animal tissue is in low-temperature cold chain transportation (below 0 ℃);

(2) virus inactivation treatment: the virus inactivation solution is prepared from peroxyacetic acid, absolute ethyl alcohol (more than or equal to 99.7 percent) and purified water according to a volume ratio of 3: 5: 92, adding 19L of virus inactivation solution into 1kg of animal tissue, and treating for 0.5-2 hours;

(3) multiple-cycle freeze-thaw treatment: performing multiple circulating freeze-thawing treatment on the tissue subjected to virus inactivation treatment, wherein the freezing temperature is-80 to-50 ℃, and the freezing time is not less than 6 hours; the melting temperature is 23-28 ℃; centrifuging and discarding the supernatant after each melting, adding purified water with the same amount again, and freezing and melting;

(4) and (3) cell removal: treating the tissue treated in the step (3) with an enzyme solution, a non-ionic surfactant and an ionic surfactant solution for 60-90 min in sequence, and continuously stirring the mixture; wherein, the enzyme solution is trypsin with the mass percent concentration of 0.20-0.25%, the non-ionic surfactant solution is triton with the mass percent concentration of 0.10-0.15%, and the ionic surfactant solution is lauryl sodium sulfate with the mass percent concentration of 0.10-0.15%;

(5) removing the residue of the cell removing solution: cleaning the tissue treated in the step (4) with purified water for 15min, centrifuging and removing supernatant, and repeating for 4-6 times;

(6) and (3) freeze drying: placing the tissue treated in the step (5) into a freeze-drying tray, pre-freezing for 6-8 hours at-60 to-45 ℃, then raising the temperature from the pre-freezing temperature gradient to 25 ℃, raising the temperature of each gradient temperature point by 5 ℃, keeping each temperature point of the first 8 temperature points for 3 hours, keeping each temperature point of the later 8 temperature points for 2 hours, and then carrying out vacuum pumping to obtain a dried acellular animal tissue;

(7) freezing and ball milling: carrying out frozen ball milling on the dried acellular animal tissue obtained in the step (6) at the temperature of-20 to-50 ℃ to obtain acellular freeze-dried powder;

(8) and (3) pepsin digestion: digesting the cell-free freeze-dried powder obtained in the step (7) by using a pepsin solution, wherein the mass-to-volume ratio of the cell-free freeze-dried powder to the pepsin solution is 10 mg: 1ml, the concentration of the pepsin solution is 1mg/ml, and the digestion time is 24-96 hours, so that a digestion product of the acellular freeze-dried powder is obtained.

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

The invention also provides a preparation method of the calcium alginate composite porous biological matrix dressing, which comprises the following steps:

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

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

s2, preparing calcium alginate gel

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

s3, preparing the calcium alginate composite porous biological matrix dressing

And (4) preparing the acellular freeze-dried powder digestion product obtained in the step (S1) and the calcium alginate gel obtained in the step (S2) into the calcium alginate composite porous biological matrix dressing.

Further, the process of step S2 is: completely dissolving sodium alginate in a proper amount of water for injection, and adding sodium alginate in a mass ratio of 1-3: 1, uniformly stirring, and adding a mixed solution of sodium alginate and calcium chloride in a 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.

Further, step S3 includes the following steps:

s3.1, mixing the cell-free freeze-dried powder digestion product and the calcium alginate gel according to the mass ratio of 5-15: 85-95, adjusting the pH to 6.5-7.5 with NaOH solution, and stirring and mixing uniformly 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 die to be pressed to obtain a three-layer matrix dressing;

s3.4, placing the three layers of matrix dressings in a punching forming die for pressing and uniformly punching to obtain punched matrix dressings;

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

and S3.6, sterilizing 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 0.5mm to 1.0mm, and the thickness of the inner layer is 1.5mm to 2.0 mm; in step S3.4, the aperture of the punched hole is 2 mm-3 mm.

Further, in step S3.6, the sterilization treatment is performed by EB irradiation, and the sterilization amount is controlled within a range of 20 to 25 kGY.

Further, in step S3.2, the freeze-drying is performed by pre-freezing at-60 to-45 ℃ for 6 to 8 hours, then increasing the temperature from the pre-freezing temperature gradient to 25 ℃, increasing the temperature of each gradient by 5 ℃, maintaining each temperature point of the first 8 temperature points for 3 hours, maintaining each temperature point of the next 8 temperature points for 2 hours, and then performing vacuum-drying to obtain the freeze-dried product.

In the invention, the first 8 temperature points are the first 8 temperature points from the pre-freezing temperature, for example, the pre-freezing temperature is-55 ℃, the first 8 gradient temperature points are-55 ℃, 50 ℃, 45 ℃, 40 ℃, 35 ℃, 30 ℃, 25 ℃ and-20 ℃.

The invention also provides application of the calcium alginate composite porous biological matrix dressing in preparation of medical and beauty, face-lifting or female health products.

The calcium alginate composite porous biological matrix dressing provided by the invention can load various cells and support the growth of the cells, such as dermal fibroblasts, endometrial mesenchymal stem cells or human epidermal growth factor (hEGF) and the like, is used for preparing medical, cosmetic or female health products, such as minimally invasive treatment of diseases caused by the relaxation of local soft tissue structures, and can also be widely used in the beauty medical fields of breast augmentation, facial reshaping and the like.

Compared with the prior art, the invention has the following advantages:

the calcium alginate composite porous biological matrix dressing provided by the invention not only can prolong the release duration of the medicament, but also has better antibacterial and anti-inflammatory effects.

Drawings

FIG. 1 is an electron microscope image of a calcium alginate composite porous bio-matrix dressing prepared in example 1 of the present invention;

FIG. 2 is an electron micrograph of the porous biomatrix dressing prepared in comparative example 1;

FIG. 3 is a graph of the duration of drug release for the product of the present invention and the comparative product;

fig. 4 is a graph of the duration of drug release for calcium alginate composite porous biomatrix dressing with different pore sizes.

Detailed Description

The following are specific embodiments of the present invention, which are intended to further illustrate the invention and not to limit it.

In the following examples, the cell-free lyophilized powder digestion product is prepared by the method of animal tissue reference CN114177340A or CN114306749A, and is preferably prepared according to the following method:

(1) collecting and transporting: collecting fresh animal tissue such as Corii Sus Domestica, small intestine of pig, pericardium of pig or bladder of pig, cutting into small pieces with length and width not more than 2cm, soaking in virus inactivating solution (peroxyacetic acid solution) for 2.5 hr, and storing at-8 deg.C for a long time; the animal tissue is in low-temperature cold chain transportation (below 0 ℃);

(2) and (3) virus inactivation treatment: the virus inactivation solution is prepared from peroxyacetic acid, absolute ethyl alcohol (more than or equal to 99.7 percent) and purified water according to a volume ratio of 3: 5: 92, adding 19L of virus inactivation solution into 1kg of animal tissue for treatment for 1.2 hours;

(3) multiple-cycle freeze-thaw treatment: performing multiple circulating freeze thawing treatment on the tissue subjected to virus inactivation treatment, wherein the freezing temperature is-65 ℃, and the freezing time is 6 hours; the melting temperature is 25 ℃; centrifuging and discarding the supernatant after each melting, adding purified water with the same amount again, freezing and melting, and performing cyclic freeze thawing treatment for three times;

(4) and (3) cell removal: treating the tissue treated in the step (3) with an enzyme solution, a non-ionic surfactant and an ionic surfactant solution for 75min in sequence, and continuously stirring the solution; wherein the enzyme solution is trypsin with the mass percent concentration of 0.22%, the non-ionic surfactant solution is triton with the mass percent concentration of 0.12%, and the ionic surfactant solution is lauryl sodium sulfate with the mass percent concentration of 0.12%;

(5) removing the residue of the cell removing solution: cleaning the tissue treated in the step (4) with purified water for 15min, centrifuging and removing supernatant, and repeating for 5 times;

(6) and (3) freeze drying: placing the tissue processed in the step (5) into a freeze-drying tray, pre-freezing for 7 hours at the temperature of minus 55 ℃, then raising the temperature from the pre-freezing temperature of minus 55 ℃ to 25 ℃ in a gradient manner, raising the temperature of each gradient temperature point by 5 ℃, keeping each temperature point of the first 8 temperature points for 3 hours, keeping each temperature point of the later 8 temperature points for 2 hours, and then carrying out vacuum pumping to obtain dried acellular animal tissue;

(7) freezing and ball milling: carrying out frozen ball milling on the dried acellular animal tissue obtained in the step (6) at the temperature of-35 ℃ to obtain acellular freeze-dried powder;

(8) and (3) pepsin digestion: digesting the cell-free freeze-dried powder obtained in the step (7) by using a pepsin solution, wherein the mass-to-volume ratio of the cell-free freeze-dried powder to the pepsin solution is 10 mg: 1ml, the concentration of the pepsin solution is 1mg/ml, and the digestion time is 60 hours, thus obtaining the digestion product of the acellular freeze-dried powder.

Example 1

The calcium alginate composite porous biological matrix dressing of the embodiment is prepared from the following materials:

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

90 parts by weight of calcium alginate gel

The preparation method comprises the following steps:

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

Collecting and transporting pigskin according to the preferred method, inactivating viruses, circularly freezing and thawing, removing cells, removing the residue of a cell-free solution, freezing, ball-milling and digesting pepsin to obtain a cell-free freeze-dried powder digestion product.

S2, preparing calcium alginate gel

Completely dissolving sodium alginate in a proper amount of water for injection, and adding sodium alginate in a mass ratio of 2: 1, stirring uniformly, and adding a mixed solution of sodium alginate and calcium chloride in a mass ratio of 5: 1, regulating the pH value to 5.0 by hydrochloric acid, and stirring for 40 minutes at the temperature of 50 ℃ to obtain the calcium alginate gel.

S3, preparing the calcium alginate composite porous biological matrix dressing

S3.1, mixing the cell-free freeze-dried powder digestion product and the calcium alginate gel according to the mass ratio of 10: 90, regulating the pH value to 7.0 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; wherein the freeze-drying is to pre-freeze for 7 hours at the temperature of-55 ℃, then to increase the temperature from the pre-freezing temperature of-55 ℃ to 25 ℃ in a gradient manner, each gradient temperature point is increased by 5 ℃, each temperature point of the first 8 temperature points is kept for 3 hours, each temperature point of the later 8 temperature points is kept for 2 hours, and then to obtain a freeze-dried product by vacuum pumping;

s3.3, placing the freeze-dried product in a forming die for pressing to obtain a three-layer matrix dressing, wherein the thickness of the uppermost layer and the lowermost layer of the three-layer matrix dressing is 0.8mm, and the thickness of the inner layer is 1.8 mm;

s3.4, placing the three layers of matrix dressings in a punching forming die for pressing and uniformly punching, wherein the aperture of the punched hole is 2mm, and obtaining the punched matrix dressings;

and S3.6, performing EB (Electron Beam) radiation sterilization treatment on the packaged matrix dressing, wherein the sterilization dose is 22kGY, and thus obtaining the calcium alginate composite porous biological matrix dressing.

The electron micrograph of the obtained calcium alginate composite porous biological matrix dressing is shown in figure 1.

Example 2

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

95 parts by weight of calcium alginate gel

The preparation method comprises the following steps:

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

Collecting and transporting the small intestines of the pigs according to the optimal method, inactivating viruses, circularly freezing and thawing, removing cells, removing the residue of a cell-free solution, freezing, ball-milling and digesting pepsin to obtain a cell-free freeze-dried powder digestion product.

S2, preparing calcium alginate gel

Completely dissolving sodium alginate in a proper amount of water for injection, and adding sodium alginate in a mass ratio of 1: 1, uniformly stirring, and adding a mixed solution of sodium alginate and calcium chloride in a mass ratio of 4: 1, regulating the pH value to 4.5 with hydrochloric acid, and stirring for 30 minutes at the temperature of 45 ℃ to obtain the calcium alginate gel.

S3, preparing the calcium alginate composite porous biological matrix dressing

S3.1, mixing the cell-free freeze-dried powder digestion product and the calcium alginate gel according to the mass ratio of 5: 95, mixing, adjusting the pH to 6.5 by using NaOH solution, and stirring and mixing uniformly to obtain a mixture;

s3.2, pouring the obtained mixture into a freeze-drying mold for freeze-drying to obtain a freeze-dried product; wherein the freeze-drying is to pre-freeze for 6 hours at the temperature of 60 ℃ below zero, then to increase the temperature from the pre-freezing temperature of 60 ℃ below zero to 25 ℃ in a gradient manner, each gradient temperature point is increased by 5 ℃, each temperature point of the first 8 temperature points is kept for 3 hours, each temperature point of the later 8 temperature points is kept for 2 hours, and then to vacuum-dry to obtain a freeze-dried product;

s3.3, placing the freeze-dried product in a forming die for pressing to obtain a three-layer matrix dressing, wherein the thickness of the uppermost layer and the lowermost layer of the three-layer matrix dressing is 0.5mm, and the thickness of the inner layer is 1.5 mm;

s3.4, placing the three layers of matrix dressings in a punching forming die for pressing and uniformly punching, wherein the aperture of the punched hole is 3mm, and obtaining the punched matrix dressings;

and S3.6, performing EB (Electron Beam) radiation sterilization treatment on the packaged matrix dressing, wherein the sterilization dose is 20kGY, and thus obtaining the calcium alginate composite porous biological matrix dressing.

Example 3

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

85 parts by weight of calcium alginate gel

The preparation method comprises the following steps:

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

Collecting and transporting the pig heart envelope according to the preferred method, performing virus inactivation treatment, circulating freeze-thawing treatment, decellularization, removing the residue of a decellularized solution, performing freeze ball milling and pepsin digestion to obtain a decellularized freeze-dried powder digestion product.

S2, preparing calcium alginate gel

Completely dissolving sodium alginate in a proper amount of water for injection, and adding the mixture in an amount which is 3: 1, uniformly stirring, and adding a mixed solution of sodium alginate and calcium chloride in a mass ratio of 6: 1, regulating the pH value to 6.5 with hydrochloric acid, and stirring for 45 minutes at the temperature of 55 ℃ to obtain the calcium alginate gel.

S3, preparing the calcium alginate composite porous biological matrix dressing

S3.1, mixing the cell-free freeze-dried powder digestion product and the calcium alginate gel according to the mass ratio of 15: 85, regulating the pH value to 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; wherein the freeze-drying is to pre-freeze for 8 hours at the temperature of minus 45 ℃, then to increase the temperature from the pre-freezing temperature of minus 45 ℃ to 25 ℃ in a gradient way, each gradient temperature point is increased by 5 ℃, each temperature point of the former 8 temperature points is kept for 3 hours, each temperature point of the latter is kept for 2 hours, and then to vacuum-dry to obtain a freeze-dried product;

s3.3, placing the freeze-dried product in a forming die for pressing to obtain a three-layer matrix dressing, wherein the thickness of the uppermost layer and the lowermost layer of the three-layer matrix dressing is 1.0mm, and the thickness of the inner layer is 2.0 mm;

s3.4, placing the three layers of matrix dressings in a punching forming die for pressing and uniformly punching, wherein the aperture of the punched hole is 2.5mm, and obtaining the punched matrix dressings;

and S3.6, performing EB (Electron Beam) radiation sterilization treatment on the packaged matrix dressing, wherein the sterilization dose is 25kGY, and thus obtaining the calcium alginate composite porous biological matrix dressing.

Comparative example 1

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

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

The preparation method comprises the following steps:

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

S2 preparation of porous biological matrix dressing

S2.1, adjusting the pH of the cell-free freeze-dried powder digestion product to 7.0 by using a NaOH solution to obtain a solution;

s2.2, pouring the solution obtained in the step S2.1 into a freeze-drying mold for freeze-drying to obtain a freeze-dried product; wherein the freeze-drying is to pre-freeze for 7 hours at the temperature of-55 ℃, then heat up to 25 ℃ from the pre-freezing temperature gradient, raise the temperature of each gradient by 5 ℃, keep each temperature point of the first 8 temperature points for 3 hours, keep each temperature point of the later 2 hours, and then carry out vacuum pumping to obtain a freeze-dried product;

and S3.3, performing EB irradiation sterilization treatment on the freeze-dried product, wherein the sterilization dose is 22kGY, and thus obtaining the porous biological matrix dressing.

The electron micrograph of the resulting porous biomatrix dressing is shown in fig. 2.

Comparative example 2

The composite porous biological matrix dressing of the comparative example is prepared from the following materials:

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

90 parts by weight of chitosan-calcium alginate gel

The preparation method comprises the following steps:

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

S2, preparing calcium alginate gel

2.0% (w/v) sodium alginate solution in 1.0mol/LCaCl ₂ Preparing calcium alginate gel microspheres with the particle size of mainly 400-600 mu m by an electrostatic liquid drop method under the condition that the solution is gel bath, and mixing the calcium alginate gel microspheres with 0.7% (w/v) chitosan solution in a ratio of 1: 7 volume ratio, then mixed with 0.5% (w/v) sodium alginate solution at 10: mixing at a volume ratio of 20 to obtain the chitosan-calcium alginate gel.

S3, preparing the calcium alginate composite porous biological matrix dressing

Mixing a cell-free freeze-dried powder digestion product and chitosan-calcium alginate gel according to a mass ratio of 10: 90, and preparing the calcium alginate composite porous biological matrix dressing according to the method of the example 1.

Test example 1

The test example examined the duration of drug release for the inventive product and the comparative product.

The test method is as follows:

(1) respectively adding physiological saline into the porous biological matrix dressings prepared in the example 1, the comparative example 1 and the comparative example 2 to prepare 10mg/mL solution, and oscillating up and down for 2min to obtain uniform gel liquid;

(2) mixing the hEGF solution with the gel liquid to ensure that the final concentration of the hEGF is about 3ng/mL, so as to obtain the prepared gel containing the hEGF;

(3) adding the prepared gel containing hEGF into 48-well plate (500 μ L per well), placing 48-well plate in 37 deg.C cell incubator, incubating for 1 hr, and gelatinizing;

(4) after the incubation is finished, taking out the 48-hole plate, adding 500 mu L of PBS into each hole, and then putting the 48-hole plate back to the incubator;

(5) taking out the 48-well plate every 24 hours, sucking the supernatant, adding new PBS (500 mu L), and putting the sucked supernatant into a refrigerator at the temperature of minus 80 ℃ for freezing;

(6) the process is repeated in cycles;

(7) the ELISA kits measured the concentration of each sample on

days

0, 2, 3, 4, 5, 6, 7, 9, 15, 21 and 28, respectively.

The drug release duration time graphs of the porous biomatrix dressings manufactured in example 1, comparative example 1, and comparative example 2 are shown in fig. 3.

As can be seen from fig. 3, compared to the porous biomatrix dressings prepared in comparative examples 1 and 2, the hEGF supported by the porous biomatrix dressing prepared in the present invention can be slowly and continuously released to exert a lasting therapeutic effect.

Test example 2

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

The test method comprises the following steps: porous biomatrix dressings were prepared as in example 1, except that the pores punched in step S3.4 were of different pore sizes, 2mm, 3mm and 5mm respectively, and the effect of the different pore sizes on the duration of drug release was examined. The specific measurement method was the same as in test example 1.

The measured results of the drug release duration graphs of the porous biomatrix dressing with different pore sizes are shown in fig. 4.

The result shows that the release duration of the medicine is longer along with the reduction of the aperture of the porous biological matrix dressing, and that the aperture of 5mm is continuously released to about 20 days to reach about 98 percent; the release amount is about 82% in the case of aperture of 3mm to 28 days, about 80% in the case of aperture of 2mm to 28 days, and the slow release is still carried out in the later period every day. The result that the sustained release of the drug for 28 days is a common observation period shows that the pore diameter of the porous biological matrix dressing directly influences the sustained release function of the drug.

Test example 3

The test examples examined the antibacterial properties of the inventive and comparative products.

The test method comprises the following steps:

the porous bio-matrix dressings prepared in examples and comparative examples were respectively clamped by a pair of sterilization forceps, 3 samples (sample A, sample B, sample C) were respectively inoculated with Escherichia coli, Staphylococcus aureus and Candida albicans, the dressings were laid flat to allow the bacteria to uniformly contact the samples, and the samples were placed in a sterilization plate and cultured for 24 hours at (37. + -. 1) ℃ and a relative humidity RH of more than 90%. And taking out the samples cultured for 24h, respectively adding 20ml of eluent, repeatedly washing the sample A, the sample B, the sample C and the cover film (preferably using tweezers to clamp the film for washing), fully shaking uniformly, inoculating a certain amount of the mixture into a nutrient agar culture medium, culturing at the temperature of 37 +/-1 ℃ for 24-48 h, counting viable bacteria, and determining the viable bacteria number. The above experiment was repeated twice.

The antibacterial rate calculation formula is as follows:

in the formula:

r-antibacterial ratio (%);

b-average number of recovered bacteria (cfu/patch) for placebo;

c-average number of recovered bacteria (cfu/patch) of the sample.

And (4) respectively carrying out statistical analysis on the antibacterial rates of the obtained dressing on escherichia coli, staphylococcus aureus and candida albicans by using APSS.

The test results are shown in table 1:

TABLE 1 results of antibacterial property test

	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％

From the test results, compared with the porous biological matrix dressing prepared by the comparative example, the calcium alginate composite porous biological matrix dressing prepared by the invention has better antibacterial performance.

Claims

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

5-15 parts by weight of digestion product of acellular freeze-dried powder

85-95 parts by weight of calcium alginate gel

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

preferably, the calcium alginate composite porous biological matrix dressing is prepared from the following materials:

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

90 parts of calcium alginate gel.

2. The calcium alginate composite porous biomatrix dressing of claim 1, wherein the calcium alginate gel is obtained by reacting sodium alginate, calcium chloride and gluconolactone.

3. The calcium alginate composite porous biological matrix dressing as claimed in claim 2, wherein the calcium alginate gel is prepared by the following preparation method: completely dissolving sodium alginate in a proper amount of water for injection, and adding sodium alginate in a mass ratio of 1-3: 1, uniformly stirring, and adding a mixed solution of sodium alginate and calcium chloride in a mass ratio of 4-6: 1, regulating the pH value to 4.5-6.5 with hydrochloric acid, and stirring for 30-45 minutes at the temperature of 45-55 ℃ to obtain the calcium alginate gel.

4. The calcium alginate composite porous biological matrix dressing as claimed in claim 3, wherein the said cell-free lyophilized powder digestion product is prepared by collecting and transporting animal tissue, inactivating virus, circulating freeze-thawing, removing cells, removing cell-free solution residue, freeze-drying, freeze-ball milling and pepsin digestion.

5. The preparation method of the calcium alginate composite porous biological matrix dressing as set forth in any one of claims 1 to 4, characterized in that the preparation method comprises the following steps:

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

s2, preparing calcium alginate gel

s3, preparing the calcium alginate composite porous biological matrix dressing

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

7. The method according to claim 5, wherein the step S3 includes the steps of:

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

s3.4, placing the three layers of matrix dressings in a punching forming die for pressing and uniformly punching to obtain the matrix dressings after punching;

8. The method according to claim 7, 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 2.0 mm; in step S3.4, the aperture of the punched hole is 2 mm-3 mm.

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

10. Use of the calcium alginate composite porous biomatrix dressing according to any one of claims 1-4 for the preparation of a medical, cosmetic or feminine health product.