Composition for wound microenvironment healing material

Abstract

The present invention relates to the field of biomaterials, and particularly to a composition for a wound microenvironment healing material. The composition includes collagen particles and a medical biofilm; wherein, the collagen particles at least include precursor particles, secondary particles and framework particles; the porosity index of the medical biofilm is 0.02 - 0.05 mL / (min·cm 2 ). The combined use of the collagen particles and the medical biofilm not only plays a role in the hemostasis stage of wound healing, but also inhibits excessive inflammatory responses in the inflammatory reaction stage, contributing to the uniform formation of the basement membrane thickness; it continuously acts on the basement membrane in the proliferation stage and the granulation connective tissue generation and epithelialization stage, facilitating the thickening of the basement membrane, and finally achieving the effects of precise structural reconstruction - dynamic function regulation - long-term stability maintenance in the process of wound healing, and particularly showing remarkable performance in improving the DEJ structure, restoring skin physiological functions and inhibiting scar contracture.

Description

Technical Field

[0001] The present invention relates to the field of biomaterials, and particularly to a composition for a wound microenvironment healing material. Background Art

[0002] Wound healing not only includes the covering of the damaged area by newly formed epidermis, but also includes the hemostasis stage, the inflammatory response stage, the proliferation stage, the formation of granulation connective tissue, and epithelialization. In wound materials, acellular matrix (ECM) provides an ideal regenerative environment for healing due to its natural three-dimensional topological structure.

[0003] It has been reported in the literature that small particle-sized ECM plays a role in the hemostasis stage of wound healing. The patent with the patent number US8835174B2 discloses a hemostatic device. The sheet-like ECM is ground into particles of about 400 microns, and after laminating or freeze-drying the particles into a three-dimensional structure, the obtained three-dimensional structure is connected and fixed to the ECM sheet for wound hemostasis. On the basis of the foregoing research, the patent with the patent number CN114632019A studied a composition for treating or improving the condition and appearance of the skin, and proposed using acellular tissue as a carrier and combining it with a peptide having a specific amino acid sequence of QHREDGS (SEQ ID NO: 1) as a skin care product to delay or prevent the appearance of dermatological signs caused by photoaging (manifested as the flattening of the dermo-epidermal junction DEJ band). Summary of the Invention

[0004] Based on the foregoing research, in order to obtain a material composed only of ECM that can act on the DEJ band during wound healing, in a first aspect of the present invention, there is provided a composition for a wound microenvironment healing material, the composition is derived from at least one of the small intestinal submucosa, pericardium, bladder, peritoneum, and dermis of mammals, and the composition includes collagen particles and a medical biofilm sheet; wherein, the collagen particles at least include precursor particles, secondary particles, and framework particles; the porosity index of the medical biofilm sheet is 0.02 - 0.05 mL / (min·cm 2 )

[0005] The porosity index (PI) is the amount of water passing through a unit cross-sectional area per unit time, which reflects the density and permeability of the material. The lower the value, the denser the material.

[0006] Preferably, the collagen particles are derived from one or more of the small intestinal submucosa, pericardium, bladder, peritoneum, and dermis of mammals.

[0007] Preferably, the collagen particles are derived from the bladder of mammals.

[0008] Preferably, the medical biofilm sheet is derived from one of the small intestinal submucosa, pericardium, bladder, peritoneum, and dermis of mammals.

[0009] More preferably, the medical biofilm sheet is derived from the bladder of a mammal.

[0010] Preferably, the porosity index of the medical biofilm sheet is 0.02 - 0.04 mL / (min·cm 2 ).

[0011] Preferably, the medical biofilm sheet is an acellular matrix material.

[0012] In the present invention, the medical biofilm sheet is prepared by the perfusion-pressure difference method (the specific method of the perfusion-pressure difference method is detailed in the patent number CN106075583B) from the small intestinal submucosa, pericardium, bladder, peritoneum or dermis of a mammal to prepare a single-layer film, and then 3 - 16 single-layer films are repeatedly stacked and placed, and vacuum laminated to obtain.

[0013] Due to individual differences in mammals, even if the decellularization treatment conditions of the bladders of mammals are exactly the same, the porosity indices of the medical biofilm sheets derived from mammals still have certain differences, such as 0.02 mL / (min·cm 2 ), 0.03 mL / (min·cm 2 ), 0.04 mL / (min·cm 2 ), etc. It can be understood that the porosity index of the medical biofilm sheet derived from the bladder of a mammal has an error of about ±0.01.

[0014] Through a large number of thoughtful experiments, the inventors found that the combination of three kinds of particles and a medical biofilm sheet with a porosity index of 0.02 - 0.05 mL / (min·cm 2 ). can affect the reconstruction and functional recovery of the DEJ area, making the basement membrane (BM) thicken and the thickness be evenly distributed. The inventors speculated that the DEJ area needs to bear the shear force between the epidermis and the dermis, requiring it to have certain mechanical properties, and the three kinds of particles can provide a progressive mechanical transition, reduce stress concentration, provide a good remodeling atmosphere for the proliferative stage of wound repair, and are beneficial to the generation of granulation connective tissue and the maintenance of the shape and integrity of the DEJ area during the epithelialization stage; at the same time, the porosity index is 0.02 - 0.05 mL / (min·cm 2) guides keratinocytes to migrate along the pores, and affects the migration speed and cellular nutrition osmotic supply through a network with a specific density. BM is a sheet structure located between the dermis and the epidermis. It has a certain physical barrier effect during the remodeling of the DEJ area, and has a certain effect on the BM by affecting the proliferation stage, granulation connective tissue generation and epithelialization stage. If the porosity index of the medical biofilm is too large, although it has a certain BM thickening effect, the uneven thickness distribution can be clearly observed. This may be because the increase in pores affects the tensile strength, toughness and other mechanical strengths of the material network, thereby affecting the cell migration process. At the same time, the increase in voids causes the material to degrade faster, which also affects the repair process. If the porosity index of the medical biofilm is too small, the thickness of the remodeled DEJ area BM is observed to be thinner. This may be because the limited migration space affects cell migration. At the same time, the limited space also makes it impossible to fully respond to the signal transmission provided by collagen particles in the proliferation stage, resulting in a decrease in the BM assembly rate.

[0015] As a preferred embodiment, in the collagen particles, the weight ratio of the precursor particles, the secondary particles and the framework particles is (0.9-1.8):1:(5.5-10.5).

[0016] Collagen particles composed of three types of particles, namely precursor particles, secondary particles and framework particles, in a specific weight ratio can reduce stress concentration caused by point contact between particles in the system to a certain extent and reduce the possibility of accelerated migration of particles.

[0017] As a preferred embodiment, the preparation of the collagen particles sequentially includes the steps of tissue fixation, acid-base continuous regulation, pulverization and deionization.

[0018] Preferably, tissue fixation is specifically to take the small intestinal submucosa, pericardium, bladder, peritoneum or dermis of a mammal, wash it in a 0.1%-0.3% peracetic acid aqueous solution for 3-4 hours, and then rinse it with a buffer solution.

[0019] The tissue fixation is specifically to take the small intestinal submucosa, pericardium, bladder, peritoneum or dermis of a mammal, wash it in a 0.1%-0.3% peracetic acid aqueous solution for 3-4 hours, and then rinse it with a buffer solution to obtain a first processed material.

[0020] As a preferred embodiment, the acid-base continuous regulation is specifically to immerse the tissue-fixed mammalian bladder in an acidic aqueous solution, then continue to add an alkaline aqueous solution, let it stand and then take it out to obtain the second treated product.

[0021] The acid-base continuous regulation specifically involves preparing a container, adding an acidic aqueous solution, immersing the submucosa, pericardium, bladder, peritoneum, or dermis of a mammalian tissue-fixed small intestine in the acidic aqueous solution. After 10 - 60 minutes, add an alkaline aqueous solution to the above container until the pH reaches 6.9 - 8.3. After standing for 10 - 30 minutes, take it out to obtain a second treated product.

[0022] The acid-base continuous regulation specifically involves preparing a container, adding an acidic aqueous solution, immersing the first treated product in the acidic aqueous solution. After 10 - 60 minutes, add an alkaline aqueous solution to the above container until the pH reaches 6.9 - 8.3. After standing for 10 - 30 minutes, take it out to obtain a second treated product.

[0023] Preferably, the acidic aqueous solution is selected from one or more of hydrochloric acid aqueous solution, phosphoric acid aqueous solution, citric acid aqueous solution, acetic acid aqueous solution, and tartaric acid aqueous solution.

[0024] Preferably, the pH of the acidic aqueous solution is 2.7 - 4.8.

[0025] When the acidic aqueous solution is one of hydrochloric acid aqueous solution, phosphoric acid aqueous solution, citric acid aqueous solution, acetic acid aqueous solution, and tartaric acid aqueous solution, during the acid-base continuous regulation, directly add an acidic aqueous solution with a pH of 2.7 - 4.8.

[0026] When the acidic aqueous solution is multiple of hydrochloric acid aqueous solution, phosphoric acid aqueous solution, citric acid aqueous solution, acetic acid aqueous solution, and tartaric acid aqueous solution, the acidic aqueous solution at least includes hydrochloric acid aqueous solution.

[0027] As a preferred embodiment, when adding the alkaline aqueous solution, the dropping rate of the alkaline aqueous solution is (0.2 - 3) mL / min.

[0028] Preferably, the dropping rate of the alkaline aqueous solution is (0.5 - 2) mL / min.

[0029] Preferably, the alkaline aqueous solution is an inorganic alkaline aqueous solution.

[0030] More preferably, the alkaline aqueous solution is sodium hydroxide aqueous solution.

[0031] Preferably, in the alkaline aqueous solution, the concentration of hydroxide ion is 0.4 - 1.35 mol / L.

[0032] As a preferred embodiment, the comminution is selected from one of impact comminution, shear comminution, and compression comminution.

[0033] Preferably, the comminution is impact comminution.

[0034] Preferably, the environment during comminution is comminution in liquid nitrogen.

[0035] As a preferred embodiment, during the processes of tissue fixation, acid-base continuous regulation, pulverization, and deionization, the precursor particles, secondary particles, and framework particles are respectively obtained by setting different pulverization speeds and pulverization times during pulverization.

[0036] During pulverization, the precursor particles have a pulverization speed of 120 - 175 m / s and a pulverization time of 2 - 5 min during the processes of tissue fixation, acid-base continuous regulation, pulverization, and deionization.

[0037] During pulverization, the secondary particles have a pulverization speed of 70 - 100 m / s and a pulverization time of 1 - 4 min during the processes of tissue fixation, acid-base continuous regulation, pulverization, and deionization.

[0038] During pulverization, the framework particles have a pulverization speed of 60 - 80 m / s and a pulverization time of 40 - 80 s during the processes of tissue fixation, acid-base continuous regulation, pulverization, and deionization.

[0039] Preferably, the pulverization is specifically pulverizing the second treatment product after freeze-drying to obtain the third treatment product.

[0040] As a preferred embodiment, deionization is specifically putting the second treatment product after pulverization into pure water, performing ultrasonic treatment, and filtering.

[0041] Deionization is specifically putting the second treatment product after pulverization into pure water, performing ultrasonic treatment, filtering, discarding the filtrate, and taking the filter residue for vacuum drying.

[0042] Preferably, the deionization process does not include freeze-drying.

[0043] Preferably, the deionization process includes vacuum drying.

[0044] When removing endotoxin from tissues, weak acids or weak bases are often used to soak or wash the tissues to remove it, which inevitably introduces some ions. Especially after treatment with weak acids or weak bases, further neutralization is required to wash away the acidic / alkaline environment created in the previous step. Since tissues are mainly composed of collagen fibers, this will cause some salt crystals to be embedded in the helical grooves of the collagen fibers, interfering with the hydrogen bonds and electrostatic interactions between collagen molecules, causing instability in the structure of the collagen fibers, and thus affecting the performance of the product during application. Further, it may affect the rupture of the DEJ area during the inflammatory reaction stage of wound repair, exacerbating the unevenness of the BM thickness. To reduce the above effects, the inventors have tried continuous acid-base regulation and directly used a large amount of water for ultrasonic treatment to remove salts. However, it was found that this would affect the mechanical properties of the tissues. It is speculated that this may be because the removal of the previously embedded salt ions makes the original protein fiber structure become loose, resulting in more severe changes in this structure during subsequent crushing, thus affecting its performance. During the experiment, the inventors unexpectedly found that the preparation of collagen particles successively undergoes the steps of tissue fixation, continuous acid-base regulation, crushing, and deionization, that is, deionization is placed after crushing, and during deionization, vacuum drying instead of freeze-drying is used, which can improve the above problems to a certain extent. The collagen particles treated in this way, when used in combination with a medical biofilm with a porosity index of 0.02 - 0.05 mL / (min·cm 2 ), greatly reduce the influence of the aforementioned ion treatment, especially reducing the probability of rupture of the DEJ area during the inflammatory reaction stage of wound repair. This may be because, during continuous acid-base regulation, the acidic environment weakens the hydrogen bonds and electrostatic repulsion between collagen fiber molecules through protonation. During the process from acidic to nearly neutral, the intermolecular hydrophobic interaction is enhanced through deprotonation, further affecting the helical structure of the collagen fibers. Further, when standing in an environment with a pH of 6.9 - 8.3, based on the aforementioned protonation-deprotonation, the potential on the surface of the collagen fibers is further induced, acting on the structural network. Finally, combined with vacuum drying, the natural orientation arrangement of the collagen fibers is maintained, avoiding the damage to the collagen microstructure caused by the formation of ice crystals that may occur during freeze-drying, and enhancing the synergistic cooperation between the collagen particles and the medical biofilm with a porosity index of 0.02 - 0.05 mL / (min·cm 2 ).

[0045] Preferably, the precursor particles, secondary particles, and framework particles that successively undergo tissue fixation, continuous acid-base regulation, crushing, and deionization are mixed in a weight ratio of (0.9 - 1.8):1:(5.5 - 10.5) to obtain collagen particles.

[0046] The second aspect of the present invention provides a method for using a composition for a wound microenvironment healing material. The medical biofilm needs to be hydrated for 10 - 30 min before use.

[0047] Hydration refers to the immersion of medical biofilm in deionized water. Beneficial effects

[0048] In the present invention, the combined use of collagen particles and medical biofilm not only plays a role in the hemostasis stage of wound healing, but also inhibits excessive inflammatory response in the inflammatory reaction stage, reduces the risk of rupture in the DEJ area, and helps to form a uniform basement membrane thickness; it continuously acts on the basement membrane in the proliferation stage and the granulation connective tissue generation and epithelialization stage, which is beneficial to the thickening of the basement membrane. Finally, in the process of wound healing, the effects of precise structural reconstruction - dynamic function regulation - long-term stability maintenance are achieved. Especially, it shows significant effects in improving the structure of the dermal-epidermal junction (DEJ), restoring skin physiological functions and inhibiting scar contracture, providing an optimized solution for complex wound repair and having important clinical transformation value.

[0049] Collagen particles can flexibly fill complex or deep defect areas, fit the shape of the wound surface, reduce voids. The medical biofilm, as a stable three-dimensional scaffold, provides mechanical support and guides the orderly arrangement of cells. Its directional fiber structure simulates the mechanical anisotropy of the natural DEJ area, further promoting the formation of wavy folds in the basement membrane of the DEJ area, reducing α-SMA expression and contractile collagen deposition, and improving skin scars. Moreover, the combined design of collagen particles and medical biofilm realizes the dynamic balance of the wound microenvironment from macroscopic exudate management to microscopic interface humidity through a differential water management mechanism. While achieving active drainage of excess tissue fluid and rapid water absorption, it effectively locks in moisture and maintains the local humidity of the wound surface within the range of 40 - 80% within 48 hours. This not only prevents the microenvironment from drying due to excessive water absorption by collagen particles, but also promotes the migration of keratinocytes and the deposition of basement membrane (BM) through continuous moisturization. Description of the drawings

[0050] Figure 1 Staining diagrams of the examples and comparative examples in Performance Test 3;

[0051] Among them, Figure 1 (a) is the staining diagram of Example 1; Figure 1 (b) is the staining diagram of Comparative Example 1; Figure 1 (c) is the staining diagram of Comparative Example 2; Figure 1 (d) is the staining diagram of Comparative Example 3; Figure 1 (e) is the staining diagram of Comparative Example 4; Figure 1 (f) is the staining diagram of Comparative Example 5; Figure 1 (g) is the staining diagram of Comparative Example 6; Figure 1 (h) 8 is the staining diagram of Comparative Example 7; Figure 1 (i) is the staining diagram of the control group;

[0052] Figure 2Staining diagrams of Comparative Examples 8-9 in Performance Test 3, where Figure 2 (a) is the staining diagram of Comparative Example 8; Figure 2 (b) is the staining diagram of Comparative Example 9. Detailed implementation method

[0053] Example 1

[0054] This example provides a composition for a wound microenvironment healing material. The composition is derived from the bladder of a mammal and includes collagen particles and a medical biofilm sheet.

[0055] The medical biofilm sheet is derived from a pig bladder. After preparing a single-layer film sheet using the perfusion-pressure difference method (the specific method of the perfusion-pressure difference method is detailed in Patent No. CN106075583B), 6 single-layer film sheets are repeatedly stacked and placed, and a vacuum lamination is obtained.

[0056] The collagen particles include precursor particles, secondary particles, and framework particles.

[0057] The preparation of the collagen particles is as follows:

[0058] S1: Tissue fixation (specifically, the fresh pig bladder within half an hour after the death of a closed-breeding pig weighing about 120 kg. After stripping the bladder basement membrane, it is washed in an aqueous solution of 0.15 wt% peracetic acid for 4 h, and then rinsed with PBS buffer solution for 20 min to obtain a first processed product);

[0059] S2: Acid-base continuous regulation (specifically, prepare a container and add a hydrochloric acid aqueous solution with a pH of 3.6. Immerse the first processed product in the hydrochloric acid aqueous solution. After 30 min, continue to add a sodium hydroxide aqueous solution with a hydroxide concentration of 0.4 mol / L to the above container at a rate of 1 mL / min until the pH reaches 7.3. After standing for 30 min, take it out to obtain a second processed product);

[0060] S3: Crushing:

[0061] After the second processed product is freeze-dried, it is impact-crushed in liquid nitrogen at a crushing speed of 140 m / s and a crushing time of 4 min to obtain a third processed product A (finally obtaining precursor particles after deionization).

[0062] After the second processed product is freeze-dried, it is impact-crushed in liquid nitrogen at a crushing speed of 100 m / s and a crushing time of 2 min to obtain a third processed product B (finally obtaining secondary particles after deionization).

[0063] After the second processed product is freeze-dried, it is impact-crushed in liquid nitrogen at a crushing speed of 60 m / s and a crushing time of 40 s to obtain a third processed product C (finally obtaining framework particles after deionization).

[0064] S4: Deionization:

[0065] The third processed material A is put into pure water, ultrasonicated, filtered, the filtrate is discarded, and the filter residue is taken for vacuum drying to obtain precursor particles;

[0066] The third processed material B is put into pure water, ultrasonicated, filtered, the filtrate is discarded, and the filter residue is taken for vacuum drying to obtain secondary particles;

[0067] The third processed material C is put into pure water, ultrasonicated, filtered, the filtrate is discarded, and the filter residue is taken for vacuum drying to obtain framework particles.

[0068] The precursor particles, secondary particles and framework particles are mixed according to a weight ratio of 1:1:7.5 to obtain collagen particles.

[0069] Comparative Example 1

[0070] This example provides a composition for a wound microenvironment healing material. Different from Example 1, the medical biofilm is derived from porcine small intestinal submucosa. After preparing a single-layer film by the perfusion-pressure difference method (the specific method of the perfusion-pressure difference method has been described in detail in Patent No. CN106075583B), 6 single-layer films are repeatedly stacked and placed, and a vacuum lamination is obtained.

[0071] During the preparation of collagen particles, during pulverization:

[0072] After the second processed material is freeze-dried, it is shock-pulverized in liquid nitrogen at a pulverization speed of 140 m / s and a pulverization time of 4 min to obtain the third processed material A (which finally obtains precursor particles after deionization).

[0073] After the second processed material is freeze-dried, it is shock-pulverized in liquid nitrogen at a pulverization speed of 100 m / s and a pulverization time of 2 min to obtain the third processed material B (which finally obtains secondary particles after deionization).

[0074] After the second processed material is freeze-dried, it is shock-pulverized in liquid nitrogen at a pulverization speed of 60 m / s and a pulverization time of 60 s to obtain the third processed material C (which finally obtains framework particles after deionization).

[0075] Comparative Example 2

[0076] This example provides a composition for a wound microenvironment healing material. Different from Example 1, the medical biofilm is derived from porcine pericardium. After preparing a single-layer film by the perfusion-pressure difference method (the specific method of the perfusion-pressure difference method has been described in detail in Patent No. CN106075583B), 6 single-layer films are repeatedly stacked and placed, and a vacuum lamination is obtained.

[0077] During the preparation of collagen particles, during pulverization:

[0078] After the second processed material is freeze-dried, it is impact-crushed in liquid nitrogen at a crushing speed of 150 m / s for 4 minutes to obtain the third processed material A (finally obtaining precursor particles after deionization).

[0079] After the second processed material is freeze-dried, it is impact-crushed in liquid nitrogen at a crushing speed of 100 m / s for 3 minutes to obtain the third processed material B (finally obtaining secondary particles after deionization).

[0080] After the second processed material is freeze-dried, it is impact-crushed in liquid nitrogen at a crushing speed of 80 m / s for 40 seconds to obtain the third processed material C (finally obtaining framework particles after deionization).

[0081] Comparative Example 3

[0082] This example provides a composition for a wound microenvironment healing material. Different from Example 1, the precursor particles, secondary particles, and framework particles are all screened. During the preparation of collagen particles, after the third processed material A is deionized and passed through a 200-mesh sieve, the particles smaller than 200 mesh are collected and passed through a 230-mesh sieve again, and the particles larger than 230 mesh are collected and denoted as precursor particles. After the third processed material B is deionized and passed through a 40-mesh sieve, the particles larger than 40 mesh are collected and denoted as secondary particles. After the third processed material C is deionized and passed through a 20-mesh sieve, the particles smaller than 20 mesh are collected and denoted as framework particles. The precursor particles, secondary particles, and framework particles are mixed in a weight ratio of 1.3:1:8.2 to obtain collagen particles.

[0083] Comparative Example 4

[0084] This example provides a composition for a wound microenvironment healing material. Different from Example 1, during the preparation of collagen particles, deionization is carried out before crushing after acid-base continuous regulation. The preparation of collagen particles is as follows:

[0085] S1: Tissue fixation (specifically, fresh pig bladder within half an hour after the death of a closed-fed pig weighing about 120 kg. After stripping the bladder basement membrane, it is washed in an aqueous solution of peracetic acid with a concentration of 0.15 wt% for 4 hours, and then rinsed with PBS buffer solution for 20 minutes to obtain the first processed material);

[0086] S2: Acid-base continuous regulation (specifically, prepare a container, add a hydrochloric acid aqueous solution with a pH of 5.5, immerse the first processed material in the hydrochloric acid aqueous solution. After 30 minutes, continue to add a sodium hydroxide aqueous solution with a hydroxide concentration of 0.4 mol / L to the above container at a rate of 1 mL / min until the pH reaches 7.3. After standing for 30 minutes, take it out to obtain the second processed material);

[0087] S3: Deionization: Put it into pure water, ultrasonicate, filter, discard the filtrate, and take the filter residue for vacuum drying;

[0088] S4: Crushing:

[0089] After the second processed material is deionized, it is shock-crushed in liquid nitrogen at a crushing speed of 120 m / s for 5 min to obtain precursor particles.

[0090] After the second processed material is deionized, it is shock-crushed in liquid nitrogen at a crushing speed of 70 m / s for 4 min to obtain secondary particles.

[0091] After the second processed material is freeze-dried, it is shock-crushed in liquid nitrogen at a crushing speed of 80 m / s for 60 s to obtain framework particles.

[0092] The precursor particles, secondary particles and framework particles are mixed in a weight ratio of 1.1:1:7.9 to obtain collagen particles.

[0093] Comparative Example 5

[0094] This example provides a composition for a wound microenvironment healing material. Different from Example 1, in the preparation process of collagen particles, instead of continuous acid-base regulation, the first processed material obtained after tissue fixation is treated with a salt solution (specifically, the first processed material is immersed in an aqueous sodium chloride solution of 0.4 mol / L for 30 min and then taken out to obtain the second processed material), and then the same crushing and deionization steps as in Example 1 are carried out.

[0095] The obtained precursor particles, secondary particles and framework particles are mixed in a weight ratio of 0.9:1:9.1 to obtain collagen particles.

[0096] Comparative Example 6

[0097] This example provides a composition for a wound microenvironment healing material. Different from Example 1, in the preparation process of collagen particles, freeze-drying is used during deionization, specifically as follows:

[0098] The third processed material A is put into pure water, ultrasonicated, filtered, the filtrate is discarded, and the filter residue is freeze-dried to obtain precursor particles;

[0099] The third processed material B is put into pure water, ultrasonicated, filtered, the filtrate is discarded, and the filter residue is freeze-dried to obtain secondary particles;

[0100] The third processed material C is put into pure water, ultrasonicated, filtered, the filtrate is discarded, and the filter residue is freeze-dried to obtain framework particles.

[0101] Comparative Example 7

[0102] This example provides a composition for a wound microenvironment healing material. Different from Example 1, the collagen particles are obtained by mixing precursor particles and secondary particles in a weight ratio of 1:1.

[0103] Test results

[0104] 1. Porosity index (PI): After hydrating the medical biofilm sheets obtained in Example 1 and Comparative Examples 1-2 in deionized water for 30 minutes, tightly wrap one end of a glass tube and place the glass tube vertically. Inject 10 cm of water into it, record the amount of water flowing out every three hours, and take the average of three measurements. The formula is: PI = V / S×T, where V is the volume of water flowing out, S is the inner cross-section of the glass tube, and T is the time. The results are shown in Table 1.

[0105] Table 1

[0106] Examples and Comparative Examples <![CDATA[PI (mL / (min·cm 2 ))]]> Example 1 0.0317 Comparative Example 1 0.0833 Comparative Example 2 0.0121

[0107] Combining Table 1 and Table 2, it can be seen that different from the example, in Comparative Example 1, a medical biofilm sheet with a larger porosity index was used. The results show that the composition of Example 1 has an in vitro water retention rate of >95% at both 24 h and 48 h, while in Comparative Example 1, the in vitro water retention rate is <50% both at 24 h and 48 h. It can be known that when the porosity index of the medical biofilm sheet is too high, its in vitro water retention rate is too low when used in combination with collagen particles, and it is not suitable for practical application on wounds, especially skin wounds.

[0108] 2. In vitro water retention rate test: Simulate the body fluid exuded from a wound to prepare wound exudate. Specifically, dissolve fatty acids, albumin, globulin, and triglycerides in 100 g of PBS buffer solution respectively, where the fatty acid accounts for 0.2 wt%, albumin accounts for 4 wt%, globulin accounts for 2.5 wt%, and triglyceride accounts for 0.05 wt%. Take 10 g of wound exudate, drop it on a glass slide, and then weigh the total mass at this time as Wx. Take 100 mg of collagen particles and spread them on the wound exudate so that the 10 g of wound exudate added is completely absorbed by the collagen particles. After weighing the medical biofilm sheet (recorded as W0), hydrate it for 30 minutes and then take it out, dry the surface moisture with filter paper, and weigh it (recorded as W1). Spread it on the collagen particles. At this time, a whole device is formed. Keep the test environment atmosphere at a temperature of 37 °C and a relative humidity of 50%. Weigh the whole device every 2 h (recorded as W2). The water retention rate = (W2 - W0 - Wx) / (W1 - W0 + 10)×100%. Calculate the water retention rates at 24 h and 48 h. The results are shown in Table 2.

[0109] Table 2

[0110] Examples and Comparative Examples Water retention rate at 24 h (%) Water retention rate at 48 h (%) Example 1 >95% >95% Comparative Example 1 ＜50% ＜50% Comparative Example 2 >95% >95% Comparative Example 7 ＜50% ＜50%

[0111] A high water retention rate indicates that the material sample ensures the long-term stability of the moist wound environment.

[0112] The collagen particles of Example 1 include precursor particles, secondary particles and framework particles. Compared with Example 1, the collagen particles in Comparative Example 7 only include precursor particles and secondary particles. The results show that the composition of Example 1 has an in vitro water retention rate > 95% at both 24 h and 48 h, while in Comparative Example 7, the in vitro water retention rate is < 50% whether at 24 h or 48 h. It can be seen that the composition must include specific collagen particles, that is, the collagen particles should at least include precursor particles, secondary particles and framework particles, in order to effectively maintain the in vitro water retention rate and ensure the long-term stability of the moist wound environment.

[0113] In addition, combined with Table 1, Table 2 and Table 3 and Figure 1 , it can be seen that compared with Example 1, Comparative Example 2 uses a medical biofilm sheet with a lower porosity index. Although its in vitro water retention rate reaches more than 95% at both 24 h and 48 h, it cannot effectively restore the structure of the skin DEJ region. Compared with the control group, after the application of the composition in Comparative Example 2, the papillae in the DEJ region are of different sizes, the density decreases, the distribution is uneven, the BM becomes thinner, uneven in thickness, and there are a small number of discontinuous breaks. Therefore, when the porosity index of the medical biofilm sheet in the composition is too low, it cannot (combined with the specifically treated collagen particles in this scheme) effectively improve the restoration of the DEJ region.

[0114] 3. Wound model of diabetic rats: Rats aged 8 - 10 weeks and weighing about 250 g were used to induce a type 2 diabetes model by a high-fat diet (60% fat) combined with streptozotocin (STZ, 50 mg / kg, intraperitoneal injection) and confirm the diabetic state (fasting blood glucose > 16.7 mmol / L) for 1 week. A full-thickness skin defect wound of 2 cm × 2 cm was made on the back surface of the rats. The samples of the examples and comparative examples were respectively used for the wound model of diabetic rats (the method of using the composition is to first spray and cover the collagen particles on the wound, and then hydrate the medical biofilm sheet for 20 min and then superpose and cover it on the collagen particles), and the collagen particles in Example 1 were used alone for the wound model of diabetic rats, denoted as Comparative Example 8. The medical biofilm sheet in Example 1 was used alone for the wound model of diabetic rats (the usage method is to directly cover the hydrated medical biofilm sheet on the wound skin), denoted as Comparative Example 9. In addition, diabetic rats without established defect wounds were taken as the control group. A handheld hygrometer was used to measure the wound humidity at 24 h and 48 h respectively. Four weeks after the operation, the wound tissues were taken, fixed in 4% paraformaldehyde, and stained with HE and Col IV respectively to observe the size and morphology of the papillae in the inner epidermis DEJ region and the structure of the basement membrane (BM). As Figure 1-2 The results are shown in Table 3:

[0115] Table 3

[0116]

Claims

1. A composition for a wound microenvironment healing material, characterized in that, The composition comprises collagen particles and a medical biofilm sheet; the medical biofilm sheet is derived from the bladder of a mammal; the collagen particles are derived from the bladder of a mammal; Among them, the collagen particles at least include precursor particles, secondary particles and framework particles; in the collagen particles, the weight ratio of the precursor particles, secondary particles and framework particles is (0.9 - 1.8):1:(5.5 - 10.5); The preparation of the collagen particles successively undergoes the steps of tissue fixation, acid-base continuous regulation, pulverization and deionization; During the processes of tissue fixation, acid-base continuous regulation, pulverization and deionization, the precursor particles, secondary particles and framework particles are respectively obtained by setting different pulverization speeds and pulverization times during pulverization; during pulverization, the precursor particles have a pulverization speed of 120 - 175 m / s and a pulverization time of 2 - 5 min during the processes of tissue fixation, acid-base continuous regulation, pulverization and deionization; during pulverization, the secondary particles have a pulverization speed of 70 - 100 m / s and a pulverization time of 1 - 4 min during the processes of tissue fixation, acid-base continuous regulation, pulverization and deionization; during pulverization, the framework particles have a pulverization speed of 60 - 80 m / s and a pulverization time of 40 - 80 s during the processes of tissue fixation, acid-base continuous regulation, pulverization and deionization; The specific acid-base continuous regulation is to immerse the bladder of a mammal that has undergone tissue fixation in an acidic aqueous solution, then continue to add an alkaline aqueous solution, take it out after standing, and obtain a second treated product; Deionization is specifically to put the second treated product after pulverization into pure water, perform ultrasonic treatment, and filter; The porosity index of the medical biofilm is 0.02 - 0.05 mL / (min·cm 2 ).

2. The composition for a wound microenvironment healing material according to claim 1, wherein When adding the alkaline aqueous solution, the dropping speed of the alkaline aqueous solution is (0.2 - 3) mL / min.

3. The composition for a wound microenvironment healing material according to claim 1, wherein The pulverization is selected from one of impact pulverization, shear pulverization and compression pulverization.

4. A method of using the composition for wound microenvironment healing material according to any one of claims 1-3, characterized in that, The medical biofilm sheet needs to be hydrated for 10 - 30 min before use.

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