CN112587711A

CN112587711A - Gelatin/chitosan dressing loaded with platelet-rich plasma and preparation method thereof

Info

Publication number: CN112587711A
Application number: CN202011311025.5A
Authority: CN
Inventors: 周谋; 施琳颖; 林放
Original assignee: Southern Theater Command General Hospital of PLA
Current assignee: Southern Theater Command General Hospital of PLA
Priority date: 2020-11-20
Filing date: 2020-11-20
Publication date: 2021-04-02

Abstract

The invention discloses a gelatin/chitosan dressing loaded with platelet-rich plasma and a preparation method and application thereof. The gelatin/chitosan dressing loaded with the platelet rich plasma is prepared from gelatin microspheres loaded with the platelet rich plasma, carboxymethyl chitosan and a cross-linking agent. The preparation method of the gelatin/chitosan dressing carrying the platelet-rich plasma comprises the following steps: mixing the gelatin microspheres loaded with the platelet-rich plasma and the carboxymethyl chitosan, drying, adding a cross-linking agent, and reacting to obtain the platelet-rich plasma. The gelatin/chitosan dressing carrying the platelet-rich plasma has a good three-dimensional hole structure, the aperture is 20-40 mu m, and gelatin microspheres can be obviously seen to be embedded on the hole wall under an electron microscope, so that the gelatin/chitosan dressing is favorable for adhesion, migration, differentiation and proliferation of cells; the dressing has high porosity, strong water absorption and high tensile strength.

Description

Gelatin/chitosan dressing loaded with platelet-rich plasma and preparation method thereof

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a gelatin/chitosan dressing loaded with platelet-rich plasma and a preparation method thereof.

Background

Hemostasis is an important step in emergency medical treatment, both in normal times and in wartime. Traditional hemostatic dressings such as sterile gauze cannot keep a wound moist for a long time, and can possibly cause delayed healing of the wound, and the soaked dressings are easy to allow pathogens to pass through, so that secondary infection is caused. In addition, the failure material fiber is easy to fall off, causing abnormal reaction and influencing the healing of the wound surface.

Gelatin (Gel) is a water-soluble protein, and is generally obtained by hydrolyzing collagen from animal skin and bone, and converting the triple helix structure of collagen into a random chain by hydrolysis, and is a protein having good compatibility and degradability. The gelatin can be dissolved in water to form gel with good elasticity when heated to a certain temperature, and has the characteristics of good affinity, high dispersibility and the like.

The chitosan dressing is the wound surface covering which is most valued at present, has a loose and porous structure and good water absorption and air permeability, and can ensure enough oxygen content of the wound surface. Meanwhile, the chitosan can promote the proliferation and crawling of epithelial cells, has a certain inhibition effect on the growth of fibroblasts, can promote the healing of wounds, and also has a certain inhibition effect on bacteria and fungi. The advantages and good performance provide a certain foundation for the wound healing promotion material.

Platelet-rich plasma (PRP) is Platelet-rich plasma obtained by centrifugation of whole blood. At present, dressings prepared by combining chitosan and gelatin are common, but the freeze-drying of platelet-rich plasma, the gelatin and the chitosan are not reported.

Disclosure of Invention

In order to overcome the defects of the existing dressing, the first aspect of the invention aims to provide a gelatin/chitosan dressing loaded with platelet-rich plasma.

The second aspect of the invention aims to provide a preparation method of the gelatin/chitosan dressing loaded with the platelet rich plasma.

The third aspect of the invention aims to provide a gelatin/chitosan dressing loaded with platelet rich plasma with an antibacterial function.

The fourth aspect of the present invention is to provide a method for preparing the above platelet rich plasma loaded gelatin/chitosan dressing having an antibacterial function.

The fifth aspect of the present invention aims to provide the use of the platelet rich plasma loaded gelatin/chitosan dressing of the first aspect and/or the platelet rich plasma loaded gelatin/chitosan dressing with an antibacterial function of the third aspect in the preparation of a wound repair dressing.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

in a first aspect of the invention, a gelatin/chitosan dressing loaded with platelet rich plasma is provided, which is prepared from gelatin microspheres loaded with platelet rich plasma, chitosan and a cross-linking agent.

The water absorption rate of the gelatin/chitosan dressing loaded with the platelet-rich plasma is preferably 1800-4000%; more preferably 2500% to 4000%; most preferably 3380% to 3840%.

The porosity of the gelatin/chitosan dressing loaded with the platelet-rich plasma is preferably 79-90%; more preferably 81% to 90%; most preferably 86% to 88.9%.

The preferable mass ratio of the chitosan, the gelatin microsphere loaded with the platelet-rich plasma and the cross-linking agent is 24: (3-32): (960-2400); more preferably 24: (3-12): (960-2000); most preferably 24: 6: 1916.

the concentration of the chitosan is preferably 10-50 mg/mL; more preferably 20-40 mg/mL; most preferably 40 mg/mL.

The chitosan is preferably carboxymethyl chitosan.

The deacetylation degree of the carboxymethyl chitosan is preferably 80-95%.

The concentration of the gelatin microsphere loaded with the platelet rich plasma is preferably 5-20 mg/mL; more preferably 5-15 mg/mL; most preferably 10 mg/mL.

The cross-linking agent is preferably EDC/NHS; more preferably, the molar ratio of EDC to NHS is (1-3): 1 EDC/NHS; most preferably 50mmol/L EDC and 25mmol/L NHS.

The gelatin microsphere loaded with the platelet-rich plasma is preferably obtained by the following method: mixing gelatin microsphere and platelet rich plasma, infiltrating, and freeze drying.

The gelatin microspheres and the platelet-rich plasma are preferably mixed in a mass-to-volume ratio (g: mL) of 1: (4-6); more preferably 1: 5.

the soaking condition is preferably 0-4 ℃ for 20-28 h.

The preparation method of the gelatin microsphere comprises the following steps: adding the gelatin solution into the mixed solution of olive oil and span 80 to obtain a gelatin-olive oil mixed solution, stirring, adding a cross-linking agent, reacting, adding acetone, aging, centrifuging, washing, soaking, and freeze-drying to obtain the gelatin microspheres.

The mass, volume and volume (g: g: mL: mL) ratio of the gelatin, the cross-linking agent, the olive oil and the span 80 is preferably 1 (0.004-0.006): (80-120): (1.5-2.5).

The gelatin solution is preferably added dropwise with stirring.

The stirring condition is preferably stirring for 0.5-1 h at the rotating speed of 300-500 rmp under ice bath.

The cross-linking agent is preferably glutaraldehyde.

The addition mode of the cross-linking agent is divided into 4-6 times, and the interval of each time is 15-25 min.

The reaction condition is preferably reaction for 1.5-2.5 h at a rotating speed of 300-500 rmp.

The aging condition is preferably 0-4 ℃ for 10-16 h.

The washing is preferably carried out by sequentially washing with absolute ethyl alcohol, isopropyl alcohol and deionized water.

The soaking condition is preferably that deionized water is soaked for 10-16 h at 0-4 ℃.

The preparation method of the platelet rich plasma comprises the following steps: whole blood was collected and centrifuged to obtain Platelet Rich Plasma (PRP).

The centrifugation is preferably performed by two-step centrifugation (the two-step centrifugation refers to the experimental steps described in the reference (research progress of technology for preparing platelet-rich plasma from Fuweili-chess-plum-arrow and components thereof) in the journal of China repair and reconstruction surgery, 2014, 12, 28, 12, 1546 and 1550).

The platelet count in the platelet-rich plasma is preferably 800-1200 multiplied by 10⁹/L。

In a second aspect of the present invention, there is provided a method for preparing the above gelatin/chitosan dressing loaded with platelet rich plasma, comprising the following steps: mixing the gelatin microsphere loaded with the platelet-rich plasma and the chitosan, drying, adding a cross-linking agent, and reacting.

The chitosan is preferably carboxymethyl chitosan.

The deacetylation degree of the carboxymethyl chitosan is preferably 80-95%.

The drying is preferably freeze drying.

The reaction condition is preferably 20-30 ℃ for 20-28 h; more preferably, the reaction is carried out for 20 to 26 hours at a temperature of between 25 and 30 ℃.

The preparation method of the gelatin/chitosan dressing loaded with the platelet-rich plasma further comprises the following steps: after the reaction was completed, the solution was removed, washed with ethanol and deionized water, respectively, and freeze-dried.

the soaking condition is preferably 0-4 ℃ for 20-28 h.

The gelatin solution is preferably added dropwise with stirring.

The cross-linking agent is preferably glutaraldehyde.

The aging condition is preferably 0-4 ℃ for 10-16 h.

In a third aspect of the invention, a platelet rich plasma loaded gelatin/chitosan dressing with an antibacterial function is provided, which is prepared from antibiotic-containing platelet rich plasma loaded gelatin microspheres, chitosan and a cross-linking agent.

The antibiotic is preferably at least one of gentamicin and azithromycin; more preferably gentamicin.

The preferable mass ratio of the chitosan, the gelatin microsphere containing antibiotic and loaded with the platelet-rich plasma and the cross-linking agent is 24: (3-32): (960-2400); more preferably 24: (3-12): (960-2000); most preferably 24: 6: 1916.

The chitosan is preferably carboxymethyl chitosan.

The deacetylation degree of the carboxymethyl chitosan is preferably 80-95%.

The concentration of the gelatin microsphere loaded with the platelet-rich plasma containing the antibiotic is preferably 5-20 mg/mL; more preferably 5-15 mg/mL; most preferably 10 mg/mL.

The gelatin microsphere loaded with the platelet rich plasma containing the antibiotic is preferably obtained by the following method: mixing gelatin microsphere containing antibiotic with platelet rich plasma, infiltrating, and freeze drying.

The mass-to-volume ratio (g: mL) of the gelatin microspheres containing antibiotics to the platelet-rich plasma is preferably 1: (4-6); more preferably 1: 5.

the soaking condition is preferably 0-4 ℃ for 20-28 h.

The antibiotic-containing gelatin microspheres are preferably prepared by the following method: and mixing the gelatin microspheres with antibiotics to obtain the gelatin microspheres containing the antibiotics.

The mass ratio of the gelatin microspheres to the antibiotics is preferably (10-30): 1.

The gelatin solution is preferably added dropwise with stirring.

The cross-linking agent is preferably glutaraldehyde.

The aging condition is preferably 0-4 ℃ for 10-16 h.

In a fourth aspect of the present invention, there is provided a method for preparing the platelet rich plasma loaded gelatin/chitosan dressing with antibacterial function, comprising the following steps: mixing gelatin microsphere loaded with platelet rich plasma containing antibiotic and chitosan, drying, adding cross-linking agent, and reacting.

The chitosan is preferably carboxymethyl chitosan.

The deacetylation degree of the carboxymethyl chitosan is preferably 80-95%.

The drying is preferably freeze drying.

The preparation method of the gelatin/chitosan dressing with the antibacterial function and the platelet rich plasma loading capacity further comprises the following steps: after the reaction was completed, the solution was removed, washed with ethanol and deionized water, respectively, and freeze-dried.

the soaking condition is preferably 0-4 ℃ for 20-28 h.

The gelatin microsphere containing the antibiotic is preferably obtained by the following method: and mixing the gelatin microspheres with antibiotics to obtain the gelatin microspheres containing the antibiotics.

The gelatin solution is preferably added dropwise with stirring.

The cross-linking agent is preferably glutaraldehyde.

The aging condition is preferably 0-4 ℃ for 10-16 h.

In a fifth aspect of the invention, there is provided the use of the platelet rich plasma loaded gelatin/chitosan dressing of the first aspect and/or the platelet rich plasma loaded gelatin/chitosan dressing having an antibacterial function of the third aspect in the preparation of a wound repair dressing.

The invention has the beneficial effects that:

the gelatin/chitosan dressing loaded with the platelet-rich plasma and the gelatin/chitosan dressing loaded with the platelet-rich plasma with the antibacterial function have a good three-dimensional hole structure, the aperture is 20-40 mu m, and gelatin microspheres can be obviously seen to be embedded on the hole wall under an electron microscope, so that the gelatin/chitosan dressing is beneficial to adhesion, migration, differentiation and proliferation of cells; the porosity of the dressing is high, which is beneficial to the input of nutrient substances and the discharge of waste; the dressing has strong water absorption, has good body fluid retention capacity, maintains nutrient components, and provides a required microenvironment for the growth of cells and tissues; meanwhile, the dressing has high tensile strength.

The gelatin/chitosan dressing loaded with the platelet-rich plasma and the gelatin/chitosan dressing loaded with the platelet-rich plasma with the antibacterial function have no cytotoxicity to L929 cells and HUVEC cells, and have good biocompatibility; meanwhile, the composition has good blood safety, has no influence on Activated Partial Thromboplastin Time (APTT)/plasma Prothrombin Time (PT), has good antibacterial performance and excellent wound repair performance, and has important significance on the recovery of tissue function and attractiveness.

The dressing has the advantages of wide raw material source, low price, easy processing and forming, and high cost performance.

Drawings

FIG. 1 is a representation of Gelatin Microspheres (GMs) and a platelet rich plasma loaded gelatin/chitosan dressing (CMC/GMs/PRP): wherein, a is the SEM image of GMs prepared in example 1; b is the particle size distribution plot of GMs prepared in example 1; c is the SEM image of the CMC/GMs/PRP prepared in examples 1-9.

FIG. 2 is a graph showing the water absorption of CMC/GMs/PRP prepared in examples 1 to 9.

FIG. 3 is a graph of the porosity of CMC/GMs/PRP prepared in examples 1-9.

FIG. 4 is a graph of tensile strength of CMC/GMs/PRP prepared in examples 1-9.

FIG. 5 is a graph of the degradation rate of CMC/GMs/PRP prepared in examples 1-9: wherein A is a degradation rate graph of CMC/GMs/PRP prepared in examples 1-9 under 0U/mL lysozyme; b is a degradation rate graph of the CMC/GMs/PRP prepared in the example 1-9 under 10000U/mL lysozyme; c is a visual graph of the degradation of the CMC/GMs/PRP prepared in the examples 1-9 under different concentrations of lysozyme.

FIG. 6 is a graphical representation of the gelatin microparticles containing gentamicin (GMs @ GS) prepared in example 11: wherein A is an SEM picture of GMs @ GS; b is a particle size distribution map of GMs @ GS.

FIG. 7 is a graph of the effect of Gentamicin (GS) loaded platelet rich plasma gelatin/chitosan dressing (CMC/GMs @ GS/PRP), gelatin/chitosan dressing (CMC/GMs), Gentamicin (GS) loaded gelatin/chitosan dressing (CMC/GMs @ GS) on cell viability: wherein A is a cell survival rate chart of L929 cells in leaching solutions of different dressings; and B is a cell staining pattern of the L929 cells after adding leaching solutions of different dressings.

FIG. 8 is a graph of the effect of Gentamicin (GS) loaded platelet rich plasma gelatin/chitosan dressing (CMC/GMs @ GS/PRP), gelatin/chitosan dressing (CMC/GMs), Gentamicin (GS) loaded gelatin/chitosan dressing (CMC/GMs @ GS) on cell migration and repair capacity: wherein A is a graph of the influence of leaching solutions of different dressings on scratched HUNEC cells; b is a graph of cell mobility of the scratch-treated HUNEC cells after addition of the leach solutions of the different dressings.

Fig. 9 is a graph of the hemolysis rate of Gentamicin (GS) -containing platelet rich plasma loaded gelatin/chitosan dressing (CMC/GMs @ GS/PRP), gelatin/chitosan dressing (CMC/GMs), Gentamicin (GS) -containing gelatin/chitosan dressing (CMC/GMs @ GS).

FIG. 10 is a graph of Activated Partial Thromboplastin Time (APTT)/plasma Prothrombin Time (PT) for Gentamicin (GS) -containing platelet rich plasma loaded gelatin/chitosan dressing (CMC/GMs @ GS/PRP), gelatin/chitosan dressing (CMC/GMs), Gentamicin (GS) -containing gelatin/chitosan dressing (CMC/GMs @ GS).

Figure 11 is a graph of PDGF cumulative release of Gentamicin (GS) -containing platelet rich plasma loaded gelatin/chitosan dressing (CMC/GMs @ GS/PRP).

Fig. 12 is a graph of the effect of Gentamicin (GS) -containing platelet rich plasma loaded gelatin/chitosan dressing (CMC/GMs @ GS/PRP), gelatin/chitosan dressing (CMC/GMs) on staphylococcus aureus, escherichia coli, pseudomonas aeruginosa: wherein A is a diagram of the inhibition effect of Gentamicin (GS) -containing gelatin/chitosan dressing (CMC/GMs @ GS/PRP) carrying platelet-rich plasma and gelatin/chitosan dressing (CMC/GMs) on staphylococcus aureus, escherichia coli and pseudomonas aeruginosa; b is a diameter diagram of a bacteriostatic circle generated by CMC/GMs @ GS/PRP in a culture dish of staphylococcus aureus, escherichia coli and pseudomonas aeruginosa.

FIG. 13 is a graph of wound bacterial growth 3 days after treatment of the wound with different dressings: wherein A is a bacterial growth condition graph of wound tissue homogenate coated on an agar plate after the wound is treated by different dressings for 3 days; b is a graph of the number of Escherichia coli in the homogenate of the wound tissue after the wound is treated for 3 days by different dressings; c is a graph of the number of staphylococcus aureus in the homogenate of the wound tissue after the wounds are treated by different dressings for 3 days; B. in the C, I, II, III, IV and V respectively represent Gauze group, CMC/GMs group, CMC/GMs @ GS group, CMC/GMs @ GS/PRP group and Aguacel Ag group.

Figure 14 is a graph of H & E staining of skin tissue after treatment of wounds with different dressings.

Fig. 15 is a close-up view of H & E staining of skin tissue after treatment of wounds with different dressings.

Note: groups 1-9 in FIGS. 1-5 represent the platelet rich plasma loaded gelatin/chitosan dressings (CMC/GMs/PRP) prepared in examples 1-9.

Detailed Description

The present invention will be described in further detail with reference to specific examples.

The materials, reagents and the like used in the present examples are commercially available reagents and materials unless otherwise specified.

Some of the test materials and reagents used in this example are shown in Table 1, and some of the test instruments used are shown in Table 2.

TABLE 1 test materials and reagents

TABLE 2 Experimental instruments

Example 1 preparation of a gelatin/chitosan dressing loaded with platelet rich plasma

1. Preparation of gelatin microspheres

(1) Weighing 1g of gelatin, adding 10mL of deionized water, and then placing the gelatin in a 60 ℃ water bath kettle to change the gelatin into a 10% gelatin solution for later use;

(2) transferring 100mL of olive oil into a 250mL beaker, adding magnetons, continuously stirring at the rotating speed of 400rpm, adding 2mL of span 80, and placing the beaker into a water bath kettle at 40 ℃ for later use;

(3) transferring the 10% gelatin solution prepared in the step (1) into a dropping funnel, and slowly dropping the gelatin solution into the olive oil obtained in the step (2) at the rotating speed of 400rmp until the dropping is finished (about 2 hours), so as to obtain a gelatin-olive oil mixed solution;

(4) transferring the mixed solution obtained in the step (3) to an ice bath, keeping the rotating speed of 400rmp, continuing stirring for 0.5h, and then adding 0.1mL of 5 wt% glutaraldehyde as a cross-linking agent (20 uL is taken every time, and the addition is carried out every 20 minutes);

(5) after the cross-linking agent is added, keeping the rotating speed of 400rmp, continuing stirring for reaction for 2 hours, then adding 15ml of acetone with the temperature of 4 ℃, and stirring for 30 min;

(6) placing the mixed solution with complete reaction in a refrigerator at 4 ℃ for standing and aging overnight (12 h);

(7) centrifuging the mixture after aging overnight at 10000rmp to obtain precipitate;

(8) washing the precipitate with anhydrous ethanol, isopropanol and deionized water sequentially, and repeating for 3 times;

(9) soaking the washed precipitate with an appropriate amount of deionized water and placing in a refrigerator at 4 ℃ overnight (12 h);

(10) the soaked precipitate was freeze dried to obtain the desired Gelatin Microspheres (GMs).

2. Preparation of platelet rich plasma

Separating collected fresh 400mL of whole blood of healthy people for 10min by using a quintuplet bag at 4000r/min, separating blood components of the centrifuged whole blood by using a full-automatic blood cell component analyzer, performing secondary centrifugation on platelets in a tunica albuginea bag at 800r/min for 10min, and finally separating by using a splint to obtain platelets with the platelet count of 1000 x 10⁹PRP of/L.

3. Preparation of PRP-loaded gelatin microspheres (GMs/PRP)

Weighing 10mg of the Gelatin Microspheres (GMs) prepared in the step 1, adding 50 μ L of the PRP prepared in the step 2, uniformly mixing, placing in a refrigerator at 4 ℃ for fully infiltrating and expanding for 24h to ensure that the PRP is loaded on the Gelatin Microspheres (GMs) to the maximum extent, then placing in an ultra-low temperature refrigerator at-80 ℃ for pre-freezing overnight (12h), freeze-drying the mixture for 24h the next day, and taking out to obtain the required loaded PRP gelatin microspheres (GMs/PRP).

Preparation of CMC/GMs/PRP dressing

(1) Weighing 2g of carboxymethyl chitosan, and dissolving the carboxymethyl chitosan in 100mL of deionized water to prepare 2%;

(2) weighing 1g of the PRP-loaded gelatin microspheres (GMs/PRP) prepared in the step 3, and dispersing in 100mL of deionized water to form a PRP-loaded gelatin microsphere (GMs/PRP) dispersion liquid with the concentration of 10 mg/mL;

(3) mixing the carboxymethyl chitosan solution obtained in the step (1) and the dispersion liquid of the loaded PRP gelatin microspheres (GMs/PRP) according to a volume ratio of 2: 1, respectively injecting 500uL of the mixture into each hole of a 48-hole plate, and performing vacuum freeze drying to obtain an uncrosslinked PRP-GMs/CMCS dressing;

(4) the uncrosslinked PRP-GMs/CMCS dressing was soaked in MES buffer solution (50mmol/L, 70% ethanol solution) containing 50mL EDC/NHS (50mmol/L EDC, 25mmol/L NHS) and crosslinked at room temperature for 24 h. After the reaction is finished, the excessive crosslinking solution is discarded, and the crosslinking agent is removed by soaking and cleaning for a plurality of times by using 75% ethanol and deionized water. Vacuum freeze drying to obtain CMC/GMs/PRP cross-linked dressing.

Example 2 preparation of a gelatin/chitosan dressing loaded with platelet rich plasma

The preparation method of this example is identical to that of example 1, except that in step 4 (3), the following is specified:

preparation of CMC/GMs/PRP dressing

(3) Mixing the carboxymethyl chitosan solution obtained in the step (1) and the dispersion liquid of the loaded PRP gelatin microspheres (GMs/PRP) according to the volume ratio of 1: 1, respectively injecting 500uL of the mixture into each hole of a 48-hole plate, and performing vacuum freeze drying to obtain the uncrosslinked PRP-GMs/CMCS dressing.

Example 3 preparation of a gelatin/chitosan dressing loaded with platelet rich plasma

preparation of CMC/GMs/PRP dressing

(3) Mixing the carboxymethyl chitosan solution obtained in the step (1) and the dispersion liquid of the loaded PRP gelatin microspheres (GMs/PRP) according to the volume ratio of 1: 2, then respectively injecting 500uL into each hole of a 48-hole plate, and carrying out vacuum freeze drying to obtain the uncrosslinked PRP-GMs/CMCS dressing.

EXAMPLE 4 preparation of a gelatin/Chitosan dressing loaded with platelet-rich plasma

The preparation method of this example is identical to that of example 1, except that in step 4 (1), the following is specified:

preparation of CMC/GMs/PRP dressing

(1) 3g of carboxymethyl chitosan was weighed out and dissolved in 100mL of deionized water to prepare a 3% carboxymethyl chitosan solution (CMCs solution).

Example 5 preparation of a gelatin/chitosan dressing loaded with platelet rich plasma

The preparation method of this example is the same as that of example 2, except that in step 4 (1), the following is specified:

preparation of CMC/GMs/PRP dressing

EXAMPLE 6 preparation of a gelatin/Chitosan dressing loaded with platelet-rich plasma

The preparation method of this example is identical to that of example 3, except that in step 4 (1), the following is specified:

preparation of CMC/GMs/PRP dressing

Example 7 preparation of a gelatin/chitosan dressing loaded with platelet rich plasma

preparation of CMC/GMs/PRP dressing

(1) 4g of carboxymethyl chitosan was weighed out and dissolved in 100mL of deionized water to prepare a 4% carboxymethyl chitosan solution (CMCs solution).

EXAMPLE 8 preparation of a gelatin/Chitosan dressing loaded with platelet-rich plasma

preparation of CMC/GMs/PRP dressing

EXAMPLE 9 preparation of a gelatin/Chitosan dressing loaded with platelet-rich plasma

preparation of CMC/GMs/PRP dressing

EXAMPLE 10 Properties of platelet rich plasma loaded gelatin/Chitosan dressing

1.1 Scanning Electron Microscope (SEM)

GMs obtained in example 1 and platelet-rich plasma-loaded gelatin/chitosan dressings (CMC/GMs/PRP dressing) obtained in examples 1 to 9 were subjected to gold spraying and then observed under a scanning electron microscope (test conditions: 5kV electron beam), and the results are shown in FIG. 1: as can be seen in FIG. 1A, the GMs gelatin microspheres are spherical and have uniform particle size; as seen in fig. 1B, the mean hydrodynamic diameter of GMs, 28.61 μm; as can be seen from C in fig. 1, all the dressings have a good three-dimensional pore structure, wherein the dressing prepared by using a 4% carboxymethyl chitosan solution (CMCs solution) has the smallest pore structure and a pore diameter of 20-40 um, and gelatin microspheres can be obviously seen to be embedded in the pore wall; wherein, the more GMs/PRP content, the more microspheres loaded on the three-dimensional pore wall of the dressing, the more favorable the cell adhesion, migration, differentiation and proliferation, the PRP release, the nutrient input and the waste discharge, and the good microenvironment for the cells can be provided.

1.2 Water absorption Property test

The dressings prepared in examples 1 to 9 were weighed with balance and recorded as M_oThe dressing was placed in a PBS (10mmol/L, pH 7.4) solution, after 30min the surface was quickly blotted with filter paper, and the sample was weighed to record the mass M_w. Each sample was run in parallel for three times and the average value was calculated, and the water absorption of the sample was calculated according to equation (1):

wherein Mo is the weight (g) of the dry dressing; mw is the weight of the dressing after imbibition (g); x is the water absorption (%) of the dressing.

The results are shown in table 3 and fig. 2: the higher the water absorption of the obtained dressing with the increase of the concentration of the CMCs; the water absorption rate of the dressing directly shows the water absorption and water retention capacity of the dressing, and the dressing has enough water absorption rate to have good body fluid retention capacity when being used for wound repair, maintain nutrient components and provide a required microenvironment for the growth of cells and tissues.

Table 3 test results of water absorption properties of dressings prepared in examples 1 to 9

Note: g1 to G9 are dressings prepared in examples 1 to 9, respectively.

1.3 porosity test

The porosity of the sample was determined by medium saturation as follows: the weight of each of the dressings prepared in examples 1 to 9 was measured using a balance and was designated as W₁Soaking the dressing in absolute ethyl alcohol until the sample is adsorbed to saturation, adsorbing the sample on two sides by filter paper for 30s under certain pressure, and quickly weighing and recording as W₂Each sample was run in parallel in triplicate and averaged, and the sample porosity was calculated according to equation (2):

wherein rho is the density of absolute ethyl alcohol and is 0.79g/cm³(ii) a V is the volume of the dressing to be measured, cm³。

The results are shown in table 4 and fig. 3: the porosity of the dressings prepared in examples 1-9 is above 80%, wherein the porosity of the dressings G6-G9 reaches 85%, which shows that: as the concentration of CMCs increases, the resulting dressing has higher porosity; provides larger space for the growth of cells on the dressing and the exchange of nutrient transport body fluid, and the larger porosity is also beneficial to the body fluid entering the dressing, the release of growth factors and the absorption of wound exudate.

TABLE 4 porosity test results for dressings prepared in examples 1-9

Note: g1 to G9 are dressings prepared in examples 1 to 9, respectively.

1.4 mechanical Property testing

The mechanical property of the dressing is tested according to the pharmaceutical industry standard YY/T0471.4-2004, and an electronic tensile testing machine is selected to test the tensile strength of the dressing, and the specific steps are as follows: cutting the dressing prepared in examples 1 to 9 into a size of 90mm in length and 25mm in width; the thickness H (mm) was measured with a vernier caliper and recorded; placing the sample on an electronic tension tester and measuring the test length of the sample by using a ruler; inputting the test length, the sample width and the sample thickness into an electronic tensile testing machine, and then adjusting the rising speed of the electronic tensile testing machine to 300mm/min to obtain the tensile strength of the sample; the air humidity and air temperature at the time of the test were recorded. Three experiments were performed in parallel for each sample and averaged, with the results shown in table 5 and figure 4: the tensile strength of the dressing prepared in the embodiments 1-9 is between 0.08 and 0.27Mpa, so that the content of carboxymethyl chitosan in the dressing has obvious influence on the tensile property of the dressing, and the tensile strength of the dressing is obviously improved along with the increase of the content of carboxymethyl chitosan in the dressing; the content of the gelatin microspheres in the dressing has certain influence on the mechanical property of the dressing; among them, the group G2/G5/G8 has a significantly higher tensile strength than the other corresponding carboxymethyl chitosan group.

TABLE 5 porosity test results for dressings prepared in examples 1-9

Note: g1 to G9 are dressings prepared in examples 1 to 9, respectively.

1.5 in vitro degradation (0% lysozyme, 1% lysozyme)

The degradation behavior of the dressings prepared in examples 1 to 9 was monitored as follows: weighing the initial weight (W) of the dressing₀) Respectively soaking in PBS and PBS solution containing 10000U/mL lysozyme, placing in a constant temperature shaking table (37 deg.C, 70rpm) at 3, 7Taking out the dressing after 14, 21 and 28 days, washing with ultrapure water, freeze-drying and weighing (W)_t). The in vitro degradation rate of the dressing was calculated using equation (3) and the results are shown in figure 5: a, B in FIG. 5 shows that the degradation of each group of dressings was irregular without lysozyme degradation, but the degradation was consistent after lysozyme was added, and lysozyme had a significant effect of promoting the degradation of the dressings; the organism contains lysozyme, and there is an enzyme experiment that reflects the shape of the dressing when the human body degrades. A scanning electron microscope observation is carried out on samples of the dressing of example 8 after being soaked in PBS and PBS solution containing 10000U/mL lysozyme for 7, 14 and 21 days, and the result is shown as C in figure 5.

Percent degradation (%) - (W)_o-W_t)/W_oX 100% formula (3)

By combining the test data of the CMCs/GMs/PRP in the scanning electron microscope, the mechanical property and the swelling rate and the porosity and degradation rate, the preparation proportion of the dressing prepared in the example 8 is considered to be the optimal proportion.

EXAMPLE 11 preparation of Gentamicin (GS) -containing platelet-rich plasma-loaded gelatin/chitosan dressing (CMC/GMs @ GS/PRP)

The preparation method of this example is identical to example 8, except that step 1 further comprises loading Gentamicin (GS) in Gelatin Microspheres (GMs), specifically as follows: taking 10mg of prepared Gelatin Microspheres (GMs), adding 0.51mg of GS to ensure that the drug loading of GMs @ GS microspheres is (50.52 +/-2.10) mu g/mg and the encapsulation rate is 48.08 +/-1.90%, and uniformly mixing to prepare GMs @ GS; GMs was replaced with GMs @ GS in step 3.

EXAMPLE 12 preparation of gelatin/Chitosan dressing (CMC/GMs)

The preparation method of this example is identical to example 8, except that steps 2, 3 are not included, i.e. the gelatin microspheres are not loaded with PRP.

EXAMPLE 13 preparation of Gentamicin (GS) -containing gelatin/chitosan dressing (CMC/GMs @ GS)

The preparation method of this example is identical to example 11, except that steps 2, 3 are not included, i.e. the gelatin microspheres are not loaded with PRP.

Effects of the embodiment

1.1 Scanning Electron Microscope (SEM)

GMs @ GS prepared in example 11 was subjected to metal spraying and observed under a scanning electron microscope (test conditions: 5kV electron beam), and the results are shown in FIG. 6: GMs @ GS gelatin microspheres are spherical as seen in A of FIG. 6, and GMs @ GS has a mean hydrodynamic diameter of 81.80 μm as seen in B of FIG. 6.

1.2 in vitro biocompatibility

(1) CCK8 experiments: adding the dressing prepared in the example 11-13 into DMEM complete medium (0.1g/mL) (adding the same amount of DMEM complete medium into a control group), and leaching for 24 hours; the cultured L929 cells (ATCC) were digested at a cell concentration of 2X 10³100uL of each cell is plated in a 96-well plate, 100uL of leaching liquor is added into each well after 12h, and under the condition that the cells adhere to the wall and the state is good, 100uL of leaching liquor is added into each well, 10uLCCK8 working solution is added into the leaching liquor for 24h, 48h and 72h respectively after the leaching liquor is added, and the cells are incubated for 30-40min at 37 ℃ in an incubator; the cell viability was calculated by measuring the absorbance at 450nm, i.e., the OD value, using a microplate reader, and the results are shown in a of fig. 7 (different numbers in the figure indicate significant differences): CMC/GMs @ GS/PRP, CMC/GMs had no effect on cell viability.

(2) Staining dead and live cells: and evaluating the cell activity at 24h, 48h and 72h after adding the leaching solution, wherein the specific steps are as follows: preparing Live-Dead detection working solution according to the kit instruction; absorbing the culture medium in the sample, washing the sample for 1-2 times by PBS, and operating gently; adding Live-Dead activity detection working solution, and incubating at room temperature in a dark place for 45 min; PBS was washed and excess liquid was aspirated with filter paper, and the YEESPEC cell imager (excitation/emission filter set to 488/530nm for live cells (green) and 530/580nm for dead cells (red)) was photographed with a microscope, and the results are shown in fig. 7, B: CMC/GMs @ GS/PRP, CMC/GMs had no effect on cell viability.

(3) Scratch test: before inoculating cells on the culture plate, drawing a horizontal line mark on the back of the 6-well plate by using a marker pen (the same visual field is conveniently positioned during photographing); HUNEC cells (ATCC) are digested and then inoculated into a 6-well plate, and the number of the HUNEC cells is preferably that the HUNEC cells are paved on the bottom of the plate after being attached to the wall (the HUNEC cells can be cultured for a period of time when the HUNEC cells are small in number until the HUNEC cells are paved on the bottom of the; after the cells are paved on the bottom of the plate, making scratches on the cells by using a 10-microliter gun head to be vertical to the orifice plate, and ensuring the widths of all the scratches to be consistent as much as possible; adding mitomycin (prophylactico) at 4. mu.g/mlToo rapid a proliferation of the resting cells) in a complete/basal medium; after the culture box is cultured for 0h, 4h, 10h and 24h at 37 ℃, photographing and recording are carried out, and the cell mobility is calculated as follows: cell mobility ═ 0h scratch width-post incubation scratch width)/0 h scratch width × 100%; average scratch width is the scratch void area/length; the results are shown in FIG. 8: the results of A in FIG. 8 show that CMC/GM_SGroup, CMC/GM_S@ GS/group, CMC/GM_SThe @ GS/PRP group and the control group showed significant cell proliferation at 4h, gradually extending toward the middle, due to cell proliferation. At 10h, cells in the CMC/GMS @ GS/PRP group are firstly merged, which shows that PRP in the dressing plays a role in promoting cell proliferation; the results of B (different numbers in the figure indicate significant differences) in fig. 8 show that the higher the cell mobility, the better the cell proliferation effect with increasing time.

1.3 blood compatibility test

(1) Hemolysis test

Adding the dressing prepared in examples 11-13 into a DMEM complete medium (0.1g/mL), leaching for 24h, respectively taking 4mL of leaching liquor, deionized water and PBS in a centrifuge tube (the deionized water and the PBS are respectively used as a positive control group and a negative control group), respectively adding 200 mu L of 16% erythrocyte suspension for co-incubation, incubating for 1, 2, 4, 8, 16 and 24h, and then centrifuging for 5min at 1000 Xg to collect supernatant. The absorbance at 540nm of each set of samples was measured by a microplate reader, and the hemolysis rate was calculated by the following formula: the hemolysis ratio (%) - (a-C)/(B-C) × 100%, where A, B and C represent the absorbance of the experimental group, positive control group and negative control group, respectively (three groups of each sample are made in parallel), and the results are shown in fig. 9: the leaching liquor of the dressing reacts with the red blood cells for 24 hours, and no obvious hemolysis condition occurs, so that the dressing is proved to have good blood safety.

(2) Activated Partial Thromboplastin Time (APTT)/plasma Prothrombin Time (PT)

Fresh SD rat whole blood was centrifuged at 1000 Xg for 5min, and the supernatant serum was collected. The dressing prepared in examples 11 to 13 was added to DMEM complete medium (0.1g/mL), extracted for 24 hours, the extract and PBS were mixed with serum in equal amounts, incubated for 10 minutes, and then APTT and PT values were measured using a blood detector, with the results shown in fig. 10: there was no significant change in both APTT and PT values for the dressing group compared to the PBS group, demonstrating that the dressing had no effect on APTT and PT.

(3) Growth factor in vitro release assay

2X 2 of the dressing prepared in example 11 were each placed in a 15mL centrifuge tube, 10mL of PBS buffer solution was added, followed by shaking in a shaker at 37 ℃ at 120rpm, 1mL of the centrifuge tube solution was taken at 0, 3, 6, 12, 24, 48, 72, 96, 120, 144 hours, frozen and then supplemented with an equal amount of PBS solution. After all time points were collected, the PDGF-BB elisa kit was used for testing, and the results are shown in FIG. 11: the PDGF-BB has the accumulated release amount of 52.5 +/-4.03% in 24h and is steadily increased to 69.7 +/-2.31% in a subsequent period of time, and the slow release at the later stage probably causes the high density of cross-linked bonds, so that the PDGF-BB release is blocked.

1.4 in vitro antibacterial test

1) Recovery of strain and preparation of bacterial suspension

The frozen gram-positive bacteria (s. aureus) and gram-negative bacteria (e. coli, p. aeruginosa) strains were thawed and cultured on solid LB medium for recovery. Picking out single colony with good growth after recovery every day, inoculating in liquid LB culture medium at 37 deg.C, culturing for 24 hr, respectively diluting with normal saline, counting by plate colony counting method, and making into bacteria with concentration of 1 × 10⁸CFU/mL of laboratory bacterial suspension.

2) Zone of inhibition test

Round specimens of CMC/GMs @ GS/PRP dressing (10mm diameter) prepared in example 11 were sterilized by UV irradiation on a clean bench for 30 min. Dripping 100 μ L of the bacterial suspension on a solid LB culture medium, uniformly coating and sticking a sample to be detected by using a coating rod, rightly placing the dressing for 15min, and then placing the culture dish in a 37 ℃ biochemical incubator for inverted culture. After 24h of culture, the cells were taken out and observed on the medium and the zone of inhibition diameter (D) was recorded, three replicates per group. The results are shown in FIGS. 12-A and 12-B: the prepared CMC/GMs @ GS/PRP dressing has certain bacteriostasis to escherichia coli, staphylococcus aureus and pseudomonas aeruginosa; the inhibition zone diameters of the CMC/GMs @ GS/PRP dressing to E.coli, S.aureus and P.aeruginosa are respectively (2.61 +/-0.06 mm), (2.21 +/-0.08 mm) and (1.75 +/-0.04 mm).

1.5 establishment of animal infection model and evaluation of antibacterial property

(1) Model building

Rats (SPF-grade rats purchased from the center of medical laboratory animals, Guangdong province) were weighed before surgery, anesthetized by intraperitoneal injection with 3% sodium pentobarbital (60mg/kg), hairs around the surgery were removed with an animal shaver, and the exposed skin was sterilized with 75% alcohol. Surgical instruments were sterilized in an autoclave and subjected to acute injury treatment (1.5cm) with scissors. Then 20. mu.L of mixed bacterial suspension (1X 10)⁸CFU/mL E.coli 10. mu.L and 1X 10⁸10 mul of CFU/mL staphylococcus aureus) was applied to the wound, the skin was challenged with bacteria for 3h, the uv sterilized dressing was covered over the wound, fixed with medical adhesive bandage, and placed back in the corresponding cage for individual feeding. The Aikou hydrophilic fiber silver-containing (Aquacel Ag) dressing is used as a commercial control group, the medical gauze is used as a negative control group, and the dressings prepared in examples 11 to 13 are used as an experimental group.

(2) Wound bacterial count

To determine the bacterial growth in the rat wound, day 3 skin tissue was homogenized with 2mL of sterile physiological saline, the tissue homogenate was serially diluted, 100. mu.L of the homogenate was placed on a gram-negative bacteria selection medium and mannitol agar medium plate, respectively, the triangular bar was spread evenly, and the plate was placed in a 37 ℃ biochemical incubator for inverted culture. After 24h incubation, the number of bacteria on the medium was observed and recorded, three replicates per group. Coli (white colony morphology) was selectively cultured on gram-negative bacteria selection medium, and mannitol agar medium was used for selectively culturing staphylococcus aureus (gold colony morphology), as shown in A, B, C in fig. 13 (different numbers in the figure indicate significant differences): on the third day, a gauze group, a CMC/GMs dressing group and an Ag dressing group can see a large amount of staphylococcus aureus and escherichia coli growth, and the number of bacterial colonies is reduced when the bacteria basically cover the whole panel CMC/GMs @ GS/PRP dressing group; the antibacterial dressing has the function of inhibiting or killing bacteria at the wound, and the antibacterial efficiency of the CMC/GMs @ GS/PRP dressing group is higher than that of the commercial Aquacel Ag group and the CMC/GMs @ GS dressing group.

(3) HE staining

1) Material taking: after the wounds are respectively acted on 3d, 7d, 14d and 21d by the dressing, taking skin tissues, and fixing the cut skin tissues in 4% paraformaldehyde for more than 24 h; the tissue is taken out of the fixative and placed in a dehydration box.

2) And (3) dehydrating: placing the dewatering box into a hanging basket, dewatering with gradient alcohol in a dewatering machine sequentially (75% alcohol for 4h, 85% alcohol for 2h, 90% alcohol for 2h, 95% alcohol for 1h, absolute ethanol I for 30min, and absolute ethanol II for 30min, adding xylene, respectively, and respectively, transparent for 10min and 5min, soaking in soft wax I for 1h, soaking in soft wax II for 1h, and soaking in hard wax III for 1 h.

3) Embedding: embedding the wax-soaked tissue in an embedding machine, firstly putting the melted wax into an embedding frame, taking the tissue out of a dehydration box before the wax is solidified, putting the tissue into the embedding frame according to the requirements of an embedding surface, pasting a corresponding label, freezing and cooling at the temperature of minus 20 ℃, taking the wax block out of the embedding frame after the wax is solidified, and finishing the wax block.

4) Slicing: placing the trimmed wax block on a paraffin slicer to be sliced, wherein the slice thickness is 4 mu m, floating the sliced section on warm water at 40 ℃ of a slice spreading machine to flatten the tissue, taking out the tissue by using a glass slide, placing the tissue into a baking oven at 60 ℃ to bake the slice, and taking out the sliced section to be stored at normal temperature for later use after the water is used for baking the wax.

5) Dewaxing and rehydration: placing the slices in xylene I for 20min, xylene II for 20min, anhydrous ethanol I for 10min, anhydrous ethanol II for 10min, 95% ethanol for 5min, 90% ethanol for 5min, 80% ethanol for 5min, 70% ethanol for 5min, and washing with distilled water.

6) Hematoxylin staining of cell nucleus: and (3) slicing the cut slices into Harris hematoxylin for dyeing for 3-8 min, washing with tap water, differentiating for several seconds by 1% hydrochloric acid alcohol, washing with tap water, returning blue by 0.6% ammonia water, and washing with running water.

7) Eosin staining of cytoplasm: and (5) dyeing the slices in eosin dye liquor for 1-3 min.

8) Dewatering and sealing: and (3) putting the slices into 95% alcohol I for 5min, 95% alcohol II for 5min, absolute ethyl alcohol I for 5min, absolute ethyl alcohol II for 5min, xylene I for 5min, and xylene II for 5min, dehydrating and transparentizing the slices, taking the slices out of the xylene, slightly drying the slices, and sealing the slices with neutral gum.

9) Microscopic examination and image acquisition analysis, the results are shown in fig. 14: on day 3, the CMC/GMs @ GS/PRP dressing group showed a distinct squamous epithelial layer, whereas the gauze group had no epithelial layer. Meanwhile, a large amount of lymphocytes and red blood cells infiltrate the gauze group slices, which represent the inflammatory reaction of the part; in contrast, the CMC/GMs @ GS/PRP dressing group had negligible numbers of lymphocytes; on

days

7 and 14, re-epithelialization of the CMC/GMs @ GS/PRP dressing group was significantly accelerated, while the epithelialization area of the gauze group was smaller; on day 21, both the gauze group and the CMC/GMs @ GS/PRP dressing group showed complete epithelialization, with complete return of the skin layers to normal for each group. However, the microscopic structure of the regenerated tissue is observed at a magnification that is very different, as shown in FIG. 15: the CMC/GMs @ GS/PRP dressing group not only significantly thickened the neoepidermis, but also presented a tight junction of the epidermis and dermis, i.e. a downward bulge of the epidermis (epidermal ridge or epidermal network) and corresponding upward bulge of the dermis layer, which stagger to increase the surface area and thus strengthen the connection between the epidermis and the dermis, which is of great importance for the restoration of tissue function and aesthetics.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims

1. A gelatin/chitosan dressing loaded with platelet rich plasma, which is characterized in that: is prepared from gelatin microspheres loaded with platelet-rich plasma, chitosan and a cross-linking agent.

2. The platelet rich plasma loaded gelatin/chitosan dressing of claim 1, wherein: the chitosan, the platelet-rich plasma-loaded gelatin microspheres and the cross-linking agent are mixed according to the mass ratio of 24: (3-32): (960-2400);

the crosslinker is preferably EDC/NHS.

3. The platelet rich plasma loaded gelatin/chitosan dressing of claim 2, wherein the platelet rich plasma loaded gelatin microspheres are obtained by the following method: mixing gelatin microsphere and platelet-rich plasma, infiltrating, and freeze drying;

the gelatin microspheres and the platelet-rich plasma are preferably mixed according to the mass-volume ratio of 1: (4-6);

the soaking condition is preferably 0-4 ℃ for 20-28 h.

4. The platelet rich plasma loaded gelatin/chitosan dressing of claim 3, wherein the platelet rich plasma is prepared by the following method: taking whole blood, and centrifuging to obtain the platelet-rich plasma.

5. The platelet rich plasma loaded gelatin/chitosan dressing of any of claims 1-4, wherein: the water absorption rate of the gelatin/chitosan dressing loaded with the platelet-rich plasma is 1800-4000%;

the porosity of the gelatin/chitosan dressing loaded with the platelet-rich plasma is preferably 79-90%.

6. The method for preparing the platelet rich plasma loaded gelatin/chitosan dressing of any one of claims 1 to 5, comprising the steps of: mixing gelatin microspheres loaded with platelet-rich plasma and chitosan, drying, adding a cross-linking agent, and reacting;

the reaction condition is preferably 20-30 ℃ for 20-28 h.

7. The gelatin/chitosan dressing with the antibacterial function and loaded with the platelet-rich plasma is characterized in that: is prepared from gelatin microspheres loaded with platelet rich plasma containing antibiotic, chitosan and cross-linking agent.

8. The platelet rich plasma loaded gelatin/chitosan dressing having antibacterial function according to claim 7, wherein: the chitosan, the antibiotic-containing platelet-rich plasma-loaded gelatin microspheres and the cross-linking agent are mixed according to the mass ratio of 24: (3-32): (960-2400).

9. The platelet rich plasma loaded gelatin/chitosan dressing having antibacterial function according to claim 7 or 8, wherein:

the antibiotic is at least one of gentamicin and azithromycin;

the crosslinker is preferably EDC/NHS.

10. The preparation method of the platelet rich plasma loaded gelatin/chitosan dressing with antibacterial function according to any one of claims 7 to 9, characterized by comprising the following steps: mixing gelatin microspheres loaded with platelet-rich plasma and containing antibiotics with chitosan, drying, adding a cross-linking agent, and reacting;

the reaction condition is preferably 20-30 ℃ for 20-28 h.