Composition for wound surface microenvironment healing material

Abstract

The invention relates to the field of biological materials, in particular to a composition for a wound surface microenvironment healing material. The composition comprises collagen particles and a medical biological membrane, wherein the collagen particles at least comprise precursor particles, secondary particles and framework particles; and the porosity index of the medical biological membrane is 0.02 to 0.05 mL / (min.cm < 2 >). The collagen particles and the medical biological membrane are combined for use, not only play a role in a hemostasis stage of wound healing, but also inhibit excessive inflammatory reaction in an inflammatory reaction stage, so that formation of the thickness of a basement membrane is uniform; according to the present invention, the application has the following characteristics that the effect is good, the effect continuously acts on the basilar membrane during the proliferation stage and the granulation connective tissue generation and epithelization stage, the basilar membrane thickening is easily achieved, the effects of accurate structure reconstruction, dynamic function regulation and long-term stability maintenance are finally achieved during the wound healing process, and the effect is particularly significant in the aspects of DEJ structure improvement, skin physiological function recovery and scar contracture inhibition.

Description

Technical Field

[0001] The invention relates to the field of biomaterials, and in particular to a composition of wound microenvironment healing materials. Background Art

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

[0003] It has been reported in the literature that small-size ECM particles play a role in the hemostasis stage of wound healing. Patent No. US8835174B2 discloses a hemostasis device, which grinds the sheet ECM into particles of about 400 microns, laminates or freeze-dries the particles into a three-dimensional structure, and then connects and fixes the obtained three-dimensional structure to the ECM sheet for wound hemostasis; based on the above research, patent No. CN114632019A studies a composition for treating or improving skin condition and appearance, and proposes using decellularized tissue as a carrier and combining it with a peptide having a specific amino acid sequence of QHREDGS (SEQID NO: 1) to use as a skin care product to delay or prevent the appearance of dermatological signs caused by photoaging (manifested as flattening of the dermis-epidermis junction DEJ band). Summary of the invention

[0004] Based on the above research, in order to obtain a material composed only of ECM that can act on the DEJ band during wound healing, the first aspect of the present invention provides a composition for wound microenvironment healing material, the composition is derived from at least one of the small intestinal submucosa, pericardium, bladder, peritoneum, and dermis of a mammal, and the composition includes collagen particles and a medical biofilm; wherein the collagen particles include at least precursor particles, secondary particles, and framework particles; the porosity index of the medical biofilm 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, reflecting 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 a mammal.

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

[0008] Preferably, the medical biological membrane sheet is derived from one of the following: the submucosa of the small intestine, the pericardium, the bladder, the peritoneum, and the dermis of a mammal.

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

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

[0011] Preferably, the medical biofilm is a decellularized matrix material.

[0012] In the present invention, the medical biological membrane sheet is prepared from the small intestinal submucosa, pericardium, bladder, peritoneum or dermis of a mammal by a perfusion-pressure difference method (the specific method of the perfusion-pressure difference method has been described in detail in patent number CN106075583B) to prepare a single-layer membrane sheet, and then 3-16 layers of the single-layer membrane sheets are repeatedly stacked and placed, and vacuum lamination is obtained.

[0013] Due to individual differences in mammals, even if the decellularization conditions of the mammalian bladder are exactly the same, the porosity index of the medical biofilm from mammals still varies to a certain extent, such as 0.02 mL / (min·cm 2 )、0.03mL / (min·cm 2 )、0.04mL / (min·cm 2 ), etc., it can be understood that the porosity index of the medical biological membrane sheet derived from the mammalian bladder has an error of about ±0.01.

[0014] Through a large number of thoughtful experiments, the inventors found that the three particles and the porosity index are 0.02-0.05mL / (min·cm 2 ) combined with a medical biological membrane can affect the reconstruction and functional recovery of the DEJ area, resulting in thickening of the basement membrane (BM) and uniform thickness distribution. The inventors speculate that the DEJ area needs to withstand the shear force between the epidermis and the dermis, requiring it to have certain mechanical properties. The three particles can provide a gradual mechanical transition, reduce stress concentration, and provide a good remodeling atmosphere for the proliferation stage of wound repair, and are conducive to the formation 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 0.02-0.05mL / (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 is specifically to prepare a container, add an acidic aqueous solution, immerse the small intestinal submucosal layer, pericardium, bladder, peritoneum or dermis of a mammal that has undergone tissue fixation in the acidic aqueous solution, and after 10-60 minutes, continue to add an alkaline aqueous solution to the above container until the pH is 6.9-8.3, let it stand for 10-30 minutes, and then take it out to obtain the second processed product.

[0022] The acid-base continuous regulation is specifically to prepare a container, add an acidic aqueous solution, immerse the first treated product in the acidic aqueous solution, and after 10-60 minutes, continue to add an alkaline aqueous solution to the above container until the pH is 6.9-8.3, let it stand for 10-30 minutes, and then take it out to obtain the second treated product.

[0023] Preferably, the acidic aqueous solution is selected from one or more of a hydrochloric acid aqueous solution, a phosphoric acid aqueous solution, a citric acid aqueous solution, an acetic acid aqueous solution, and a 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, the acidic aqueous solution with a pH of 2.7-4.8 can be directly added.

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

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

[0028] Preferably, the dropping speed 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 a sodium hydroxide aqueous solution.

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

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

[0033] Preferably, the pulverization is impact pulverization.

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

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

[0036] During the crushing, the precursor particles are in the process of tissue fixation, acid-base continuous regulation, crushing, and deionization, with a crushing speed of 120-175m / s and a crushing time of 2-5min.

[0037] During the crushing, the secondary particles are in the process of tissue fixation, acid-base continuous regulation, crushing, and deionization, with a crushing speed of 70-100m / s and a crushing time of 1-4min.

[0038] During the crushing, the framework particles are in the process of tissue fixation, acid-base continuous regulation, crushing, and deionization, with a crushing speed of 60-80m / s and a crushing time of 40-80s.

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

[0040] As a preferred embodiment, the deionization is specifically to put the crushed second processed product into pure water, perform ultrasound treatment, and filter.

[0041] The deionization specifically includes the following steps: the second treated product is crushed, placed in pure water, subjected to ultrasound, filtered, the filtrate is discarded, and the filter residue is vacuum dried.

[0042] Preferably, the deionization process does not include lyophilization.

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

[0044] When removing endotoxin from tissues, weak acids or weak bases are often used to soak or wash the tissues to remove them, which will inevitably introduce some ions, especially after being treated with weak acids or weak bases, further neutralization is required to wash away the acid / alkaline environment created by the previous steps. The tissues are mainly composed of collagen fibers, which will cause some salt crystals to be embedded in the spiral grooves of the collagen fibers, interfering with the hydrogen bonds and electrostatic interactions between collagen molecules, causing the instability of the collagen fiber structure, thereby affecting the performance of the product during application. Further, it may affect the rupture of the DEJ area during the inflammatory response stage during wound repair, exacerbating the uneven thickness of the BM. In order to reduce the above-mentioned effects, the inventors have tried acid-base continuous regulation, directly using a large amount of water ultrasound to remove salt, but found that this would affect the mechanical properties of the tissue. It is speculated that this may be due to the removal of the previously embedded salt ions, which makes the original protein fiber structure loose, resulting in the subsequent crushing further exacerbating the changes in this structure, causing its performance to be affected. In the experiment, the inventor unexpectedly found that the preparation of collagen particles can improve the above problems to a certain extent by sequentially undergoing tissue fixation, acid-base continuous regulation, crushing and deionization steps, that is, deionization is placed after crushing, and vacuum drying is used instead of freeze-drying during deionization. The collagen particles treated in this way have a porosity index of 0.02-0.05mL / (min·cm 2 ) greatly reduced the impact of the aforementioned ion treatment, especially reduced the probability of rupture in the DEJ zone during the inflammatory response stage of wound repair. This may be due to the fact that, in the acid-base continuous regulation, the acid environment weakened the hydrogen bonds and electrostatic repulsion between collagen fiber molecules through protonation. In the process from acidic to near-neutral, the intermolecular hydrophobic interaction was enhanced through deprotonation, further affecting the helical structure of the collagen fibers. Furthermore, when the membrane was placed in an environment with a pH of 6.9-8.3, the potential on the surface of the collagen fibers was further induced on the basis of the aforementioned protonation-deprotonation, acting on the structural network. Finally, combined with vacuum drying, the natural orientation of the collagen fibers was maintained, avoiding the damage to the collagen microstructure caused by the formation of ice crystals that may be produced by freeze-drying, and enhancing the collagen particles and the porosity index of 0.02-0.05mL / (min·cm 2 )'s synergistic cooperation with medical biological membranes.

[0045] Preferably, the precursor particles, secondary particles and framework particles that have been sequentially subjected to tissue fixation, acid-base continuous 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 of wound microenvironment healing material. The medical biofilm needs to be hydrated for 10-30 minutes before use.

[0047] Hydration refers to soaking the medical biofilm in deionized water. Beneficial Effects

[0048] In the present invention, the combination of collagen particles and medical biological membrane sheets not only plays a role in the hemostasis stage of wound healing, but also inhibits excessive inflammatory response in the inflammatory response stage, reduces the risk of rupture in the DEJ area, and helps to form a uniform thickness of the basement membrane; 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, and ultimately achieves the effect of precise structural reconstruction-dynamic function regulation-long-term stability maintenance in the wound healing process, especially in improving the dermal-epidermal junction (DEJ) structure, restoring skin physiological function 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 wound surface morphology, and reduce gaps. The medical biofilm sheet serves as a stable three-dimensional scaffold, providing mechanical support and guiding cells to arrange in an orderly manner. Its directional fiber structure simulates the mechanical anisotropy of the natural DEJ area, further promoting the formation of wavy wrinkles in the basement membrane of the DEJ area, reducing α-SMA expression and contractile collagen deposition, and improving skin scars. In addition, the combined design of collagen particles and medical biofilm sheets achieves a dynamic balance of the wound microenvironment from macroscopic exudate management to microscopic interface wetness through a differentiated moisture management mechanism, while actively draining excess tissue fluid to quickly absorb water, effectively locking in moisture, and maintaining the local humidity of the wound surface in the range of 40-80% within 48 hours, which not only prevents the microenvironment from drying out due to excessive water absorption by collagen particles, but also promotes keratinocyte migration and basement membrane (BM) deposition through continuous moisturizing. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 It is the dyeing diagram of the embodiment and comparative example in performance test 3; in, Figure 1 (a) is the staining diagram of Example 1; Figure 1 (b) Staining image of Comparative Example 1; Figure 1 (c) Staining image of Comparative Example 2; Figure 1 (d) is the dyeing picture of comparative example 3; Figure 1 (e) is the dyeing picture of comparative example 4; Figure 1 (f) is the dyeing picture of comparative example 5; Figure 1 (g) is the dyeing picture of comparative example 6; Figure 1 (h) 8 is the dyeing picture of comparative example 7; Figure 1 (i) is the staining image of the control group; Figure 2 is the dyeing diagram of Comparative Examples 8-9 in Performance Test 3, wherein Figure 2(a) is the dyeing diagram of Comparative Example 8; Figure 2 (b) Staining image of comparative example 9. DETAILED DESCRIPTION

[0051] Example 1

[0052] This example provides a composition for wound microenvironment healing material, the composition is derived from the bladder of a mammal, and the composition includes collagen particles and medical biological membrane sheets.

[0053] The medical biological membrane comes from pig bladder. After preparing a single-layer membrane by the perfusion-pressure difference method (the specific method of the perfusion-pressure difference method has been detailed in patent number CN106075583B), 6 layers of single-layer membranes are repeatedly stacked and placed, and vacuum lamination is obtained.

[0054] Collagen particles include precursor particles, secondary particles and framework particles.

[0055] Collagen particles were prepared as follows: S1: tissue fixation (specifically, fresh pig bladders within half an hour after death of a closed-housed pig weighing about 120 kg, after peeling off the bladder basement membrane, were washed in a 0.15wt% peracetic acid aqueous solution for 4 hours, and then rinsed with a PBS buffer solution for 20 minutes to obtain a first treatment material); S2: Acid-base continuous regulation (specifically, prepare a container, add hydrochloric acid aqueous solution with a pH of 3.6, immerse the first treated product in the hydrochloric acid aqueous solution, and after 30 minutes, continue to add 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 is 7.3, let it stand for 30 minutes, then take it out to obtain the second treated product); S3: Smash: After freeze-drying, the second treated product was impact-pulverized in liquid nitrogen at a pulverization speed of 140 m / s for 4 min to obtain the third treated product A (precursor particles were finally obtained after deionization).

[0056] After freeze-drying, the second processed product was impact-pulverized in liquid nitrogen at a pulverizing speed of 100 m / s for 2 min to obtain the third processed product B (secondary particles were finally obtained after deionization).

[0057] After freeze-drying, the second processed product was impact-pulverized in liquid nitrogen at a pulverization speed of 60 m / s for 40 s to obtain a third processed product C (framework particles were finally obtained after deionization).

[0058] S4: Deionization: The third treatment product A is placed in pure water, ultrasonicated, filtered, the filtrate is discarded, and the filter residue is vacuum dried to obtain precursor particles; The third treatment product B is placed in pure water, ultrasonicated, filtered, the filtrate is discarded, and the filter residue is vacuum dried to obtain secondary particles; The third treatment product C is placed in pure water, ultrasonicated, filtered, the filtrate is discarded, and the filter residue is vacuum dried to obtain framework particles.

[0059] S5: Precursor particles, secondary particles and framework particles are mixed in a weight ratio of 1:1:7.5 to obtain collagen particles.

[0060] Comparative Example 1 This example provides a composition for wound microenvironment healing materials. Different from Example 1, the medical biological membrane sheet is derived from the submucosal layer of the porcine small intestine. After a single-layer membrane sheet is prepared by the perfusion-pressure difference method (the specific method of the perfusion-pressure difference method is described in detail in Patent No. CN106075583B), 6 layers of single-layer membrane sheets are repeatedly stacked and placed, and vacuum lamination is obtained.

[0061] In the process of preparing collagen particles, during pulverization: After freeze-drying, the second treated product was impact-pulverized in liquid nitrogen at a pulverization speed of 140 m / s for 4 min to obtain the third treated product A (precursor particles were finally obtained after deionization).

[0062] After freeze-drying, the second processed product was impact-pulverized in liquid nitrogen at a pulverizing speed of 100 m / s for 2 min to obtain the third processed product B (secondary particles were finally obtained after deionization).

[0063] After freeze-drying, the second processed product was impact-pulverized in liquid nitrogen at a pulverization speed of 60 m / s for 60 s to obtain a third processed product C (framework particles were finally obtained after deionization).

[0064] Comparative Example 2 This example provides a composition for wound microenvironment healing materials. Different from Example 1, the medical biological membrane is derived from porcine pericardium. After a single-layer membrane is prepared by the perfusion-pressure difference method (the specific method of the perfusion-pressure difference method is described in detail in Patent No. CN106075583B), 6 layers of single-layer membranes are repeatedly stacked and placed, and vacuum lamination is performed to obtain the result.

[0065] In the process of preparing collagen particles, during pulverization: After freeze-drying, the second treated product was impact-pulverized in liquid nitrogen at a pulverizing speed of 150 m / s for 4 min to obtain the third treated product A (precursor particles were finally obtained after deionization).

[0066] After freeze-drying, the second processed product was impact-pulverized in liquid nitrogen at a pulverizing speed of 100 m / s for 3 min to obtain the third processed product B (secondary particles were finally obtained after deionization).

[0067] After freeze-drying, the second processed product was impact-pulverized in liquid nitrogen at a pulverization speed of 80 m / s for 40 s to obtain a third processed product C (framework particles were finally obtained after deionization).

[0068] Comparative Example 3 This example provides a composition for wound microenvironment healing materials. Different from Example 1, the precursor particles, secondary particles and framework particles are all screened. In the process of preparing collagen particles, the third treated material A is deionized and passed through a 200-mesh sieve to collect particles with a mesh size below 200, and then passed through a 230-mesh sieve again to collect particles with a mesh size above 230, which are recorded as precursor particles. The third treated material B is deionized and passed through a 40-mesh sieve to collect particles with a mesh size above 40, which are recorded as secondary particles. The third treated material C is deionized and passed through a 20-mesh sieve to collect particles with a mesh size below 20, which are recorded 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.

[0069] Comparative Example 4 This example provides a composition for wound microenvironment healing materials. The difference from Example 1 is that when preparing collagen particles, deionization is performed after acid-base continuous regulation and before crushing. The preparation of collagen particles is as follows: S1: tissue fixation (specifically, fresh pig bladders within half an hour after death of a closed-housed pig weighing about 120 kg, after peeling off the bladder basement membrane, were washed in a 0.15wt% peracetic acid aqueous solution for 4 hours, and then rinsed with a PBS buffer solution for 20 minutes to obtain a first treatment material); S2: Acid-base continuous regulation (specifically, prepare a container, add hydrochloric acid aqueous solution with a pH of 5.5, immerse the first treated product in the hydrochloric acid aqueous solution, and after 30 minutes, continue to add 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 is 7.3, let it stand for 30 minutes, then take it out to obtain the second treated product); S3: Deionization: put into pure water, sonicate, filter, discard the filtrate, and vacuum dry the residue; S4: Smash: After the second treated product was deionized, it was impact-pulverized in liquid nitrogen at a pulverizing speed of 120 m / s for 5 min to obtain precursor particles.

[0070] After the second treated product was deionized, it was impact-pulverized in liquid nitrogen at a pulverizing speed of 70 m / s for 4 min to obtain secondary particles.

[0071] After the second treated product was freeze-dried, it was impact-pulverized in liquid nitrogen at a pulverization speed of 80 m / s and a pulverization time of 60 s to obtain framework particles.

[0072] S5: Precursor particles, secondary particles and framework particles are mixed in a weight ratio of 1.1:1:7.9 to obtain collagen particles.

[0073] Comparative Example 5 This example provides a composition for wound microenvironment healing materials. Different from Example 1, in the preparation process of collagen particles, no acid-base continuous regulation is performed, but the first treated material obtained after tissue fixation is treated with a saline solution (specifically, the first treated material is immersed in a 0.4 mol / L sodium chloride aqueous solution for 30 minutes and then taken out to obtain a second treated material), followed by the same crushing and deionization steps as the operation process in Example 1.

[0074] 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.

[0075] Comparative Example 6 This example provides a composition for wound microenvironment healing materials. The difference from Example 1 is that during the preparation of collagen particles, freeze-drying is used during deionization, as follows: The third treatment product A is placed in pure water, ultrasonicated, filtered, the filtrate is discarded, and the filter residue is freeze-dried to obtain precursor particles; The third treatment product B is placed in pure water, ultrasonicated, filtered, the filtrate is discarded, and the filter residue is freeze-dried to obtain secondary particles; The third treatment product C is placed in pure water, ultrasonicated, filtered, the filtrate is discarded, and the filter residue is freeze-dried to obtain framework particles.

[0076] Comparative Example 7 This example provides a composition for 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.

[0077] Test Results 1. Porosity index (PI): The medical biofilm sheets obtained in Example 1 and Comparative Examples 1-2 were hydrated in deionized water for 30 min, then tightly wrapped around one end of a glass tube and placed vertically. 10 cm of water was injected into the glass tube. The amount of water flowing out was recorded every three hours. The average value of three measurements was taken. The formula is: PI=V / S×T, where V is the volume of the flowing water, S is the inner cross-section of the glass tube, and T is the time. The results are shown in Table 1.

[0078] Table 1 Examples and Comparative Examples <![CDATA[PI(mL / (min·cm 2 ))]]> Example 1 0.0317 Comparative Example 1 0.0833 Comparative Example 2 0.0121 Combining Table 1 and Table 2, it can be seen that, unlike the embodiment, Comparative Example 1 uses a medical biofilm with a larger porosity index. The results show that the composition of Example 1 has an in vitro water retention rate of >95% at both 24h and 48h, while the in vitro water retention rate of Comparative Example 1 is <50% at both 24h and 48h. It can be seen that the porosity index of the medical biofilm is too high, and when it is used in combination with collagen particles, the in vitro water retention rate is too low, and it is not suitable for practical application on wounds, especially skin wounds.

[0079] 2. In vitro water retention rate test: The wound exudate was prepared by simulating the body fluid exuded from the wound. Specifically, fatty acids, albumin, globulin and triglycerides were dissolved in 100g of PBS buffer, of which fatty acids accounted for 0.2wt%, albumin accounted for 4wt%, globulin accounted for 2.5wt%, and triglycerides accounted for 0.05wt%. Take 10g of wound exudate, drop it on a glass slide, and weigh the mass Wx as a whole. Take 100mg of collagen particles and spread them on the wound exudate so that the 10g of wound exudate added is completely absorbed by the collagen particles. The medical biological membrane is weighed (recorded as W0) and hydrated for 30 minutes before being taken out. The surface moisture is absorbed with filter paper, weighed (recorded as W1), and spread on the collagen particles. At this time, an overall device is formed. The test environment atmosphere is maintained at a temperature of 37°C and a relative humidity of 50%. The overall device is weighed every 2h (recorded as W2). The water retention rate = (W2-W0-Wx) / (W1-W0+10)×100%. The water retention rate of 24h and 48h is calculated. The results are shown in Table 2.

[0080] Table 2 Examples and Comparative Examples 24h water retention rate (%) 48h water retention rate (%) Example 1 >95% >95% Comparative Example 1 ＜50% ＜50% Comparative Example 2 >95% >95% Comparative Example 7 ＜50% ＜50% The high water retention rate indicates that the material sample ensures the long-term stability of the moist environment on the wound surface.

[0081] The collagen particles in 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 of >95% at both 24h and 48h, while the in vitro water retention rate of Comparative Example 7 is <50% at both 24h and 48h. It can be seen that the composition must include specific collagen particles, that is, the collagen particles include at least 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 environment of the wound.

[0082] In addition, combining Table 1, Table 2 and Table 3 and Figure 1It can be seen that compared with Example 1, Comparative Example 2 uses a medical biofilm with a lower porosity index. Although its in vitro water retention rate reaches more than 95% at 24h and 48h, it cannot effectively restore the structure of the skin DEJ area. Compared with the control group, after the application of the composition of Comparative Example 2, the papillae in the DEJ area are of different sizes, the density decreases, the distribution is uneven, the BM becomes thinner, the thickness is uneven, and a small amount of discontinuous fractures occur. Therefore, when the porosity index of the medical biofilm in the composition is too low, it cannot (combined with the collagen particles that have been specifically treated in this solution) achieve effective improvement in the recovery of the DEJ area.

[0083] 3. Diabetic rat wound model: 8-10 weeks old rats 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 2cm×2cm full-thickness skin defect wound was made on the back surface of rats, and the samples of the embodiment and comparative example were used in the diabetic rat wound model (the composition was used by spraying the collagen particles on the wound first, and then hydrating the medical biofilm for 20 minutes and then covering the collagen particles), and the collagen particles in Example 1 were used alone in the diabetic rat wound model, which was recorded as comparative example 8. The medical biofilm in Example 1 was used alone in the diabetic rat wound model (the method of use was to directly cover the medical biofilm that had been hydrated for 20 minutes on the wound skin), which was recorded as comparative example 9. Diabetic rats without defect wounds were taken as the control group, and the wound humidity was measured at 24h and 48h using a handheld humidity meter. The wound tissue was taken 4 weeks after surgery, fixed in 4% paraformaldehyde, and stained with HE and Col IV, respectively, to observe the size and morphology of the papilla in the DEJ area of ​​the inner surface of the epidermis and the basement membrane (BM) structure. Figure 1-2 , the results are shown in Table 3: Table 3

Claims

1. A composition for wound microenvironment healing material, characterized in that: The composition comprises collagen particles and medical biological membrane sheets; the medical biological membrane sheets are derived from the bladder of mammals; the collagen particles are derived from the bladder of mammals; Wherein, the collagen particles at least include precursor particles, secondary particles and framework particles; The preparation of collagen particles sequentially involves the steps of tissue fixation, acid-base continuous regulation, pulverization and deionization; During tissue fixation, acid-base continuous regulation, pulverization, and deionization, precursor particles, secondary particles, and framework particles are obtained by setting different pulverization speeds and pulverization times during pulverization; The acid-base continuous regulation is specifically to immerse the bladder of a mammal after tissue fixation in an acidic aqueous solution, then continue to add an alkaline aqueous solution, and then take it out after standing to obtain a second processed product; Deionization specifically includes placing the crushed second processed product into pure water, ultrasonicating, and filtering; The porosity index of medical biofilm is 0.02-0.05mL / (min·cm 2 ).

2. The composition for wound microenvironment healing material according to claim 1, characterized in that: In the collagen particles, the weight ratio of precursor particles, secondary particles and framework particles is (0.9-1.8):1:(5.5-10.5).

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

4. The composition for wound microenvironment healing material according to claim 1, characterized in that: The pulverization is selected from one of impact pulverization, shear pulverization and compression pulverization.

5. The composition for wound microenvironment healing material according to claim 4, characterized in that: During crushing, the precursor particles are in the process of tissue fixation, acid-base continuous regulation, crushing, and deionization, with a crushing speed of 120-175m / s and a crushing time of 2-5min; during crushing, the secondary particles are in the process of tissue fixation, acid-base continuous regulation, crushing, and deionization, with a crushing speed of 70-100m / s and a crushing time of 1-4min; during crushing, the framework particles are in the process of tissue fixation, acid-base continuous regulation, crushing, and deionization, with a crushing speed of 60-80m / s and a crushing time of 40-80s.

6. A method for using the composition for wound microenvironment healing material according to any one of claims 1 to 5, characterized in that: Medical biofilm sheets need to be hydrated for 10-30 minutes before use.

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