Natural biological particle composition and application

Abstract

The invention relates to the field of biological materials, in particular to a natural biological particle composition and application. The natural biological particle combination comprises at least three kinds of natural biological particles with different particle size ranges, and the particle size range of the first kind of natural biological particles is 40-100 microns; the particle size range of the second natural biological particles is 270-400 [mu] m; the particle size range of the third kind of natural biological particles is 750-1000 [mu] m; the mass ratio of the first natural biological particles to the second natural biological particles to the third natural biological particles is (0.9-1.7): 1: (5.6-10.8); the natural biological particle combination is derived from natural biological tissues. The obtained natural biological particle composition can be directly applied to various wounds during use, the complexity of using the particles in the prior art is reduced, and the natural biological particle composition can directly cover full-thickness skin defect wounds (such as deep wet wounds) and superficial wounds (such as dry epidermis injury).

Description

Technical Field

[0001] The present invention relates to the field of biomaterials, and in particular to a natural bioparticle combination and application. Background Art

[0002] Decellularized matrices obtained after decellularization of natural tissues are widely used in the field of regenerative medicine, and currently there are various product forms such as powders and sheets. Among them, decellularized matrices in powder form usually have a higher surface area and can expose more active sites, but the carrying capacity they provide is very limited. Therefore, sometimes the powder is pressed again or loaded in a carrier such as a gel for use. However, in this way, not only will the production cost increase significantly, but the advantages brought by the higher surface area of ​​the powder will also be reduced to a certain extent.

[0003] Patent No. CN102573939B discloses a granular tissue graft having components of different densities. By controlling the density of the second particles to be at least 150% of the density of the first particles, the low-density particles are absorbed first and the high-density particles are absorbed later, thereby prolonging the degradation time of the decellularized matrix. At the same time, the document also records that before the preparation and production of this kind of particle mixture, the site to be used for repair must be evaluated first, and after obtaining the optimal absorption rate of the particle composition, the specific density value that the first density particles should actually have is calculated, and then the specific density value of the second density is deduced again. Summary of the invention

[0004] In order to reduce the cumbersomeness of using such particles in the prior art and make it easier to use, a natural biological particle combination is intended. When using the natural biological particle combination, it is not necessary to calculate the absorption rate according to the wound to match it, but it can be directly applied to various wounds. In order to solve the above problems, the first aspect of the present invention provides a natural biological particle combination, which comprises at least three natural biological particles with different particle size ranges, wherein the particle size range of the first natural biological particle is 40-100 μm; the particle size range of the second natural biological particle is 270-400 μm; the particle size range of the third natural biological particle is 750-1000 μm; the mass ratio of the first natural biological particle, the second natural biological particle and the third natural biological particle is (0.9-1.7): 1: (5.6-10.8); the natural biological particle combination is derived from natural biological tissue, and the natural biological tissue is selected from one or two of the small intestinal submucosal layer, pericardium, bladder basement membrane, peritoneum, and dermis of mammals.

[0005] Each natural biological particle has a certain degree of dispersion in its particle size range.

[0006] Generally speaking, for a biomaterial system of the same type that can be degraded, large particles always degrade slower than small particles. By using small particles to degrade first, the active factors are preferentially released to promote healing, and large particles are degraded later, playing a certain scaffolding role, so that the synergistic system of small and large particles has a certain self-regulating ability, so it can be directly applied to wounds of different depths. However, experiments have found that this gradient system not only accelerates the formation of scars and tissue contractures, but also because of the existence of the gradient, the particles are not easy to mix evenly when they are finally mixed, resulting in a certain degree of stability risk in the application of the gradient particle system. In order to overcome the above problems, after a lot of thinking and practice, it was found that when the natural biological particle combination includes at least three natural biological particles with particle size ranges of 40-100μm, 270-400μm and 750-1000μm, and the above particles have a mass ratio of (0.9-1.7): 1: (5.6-10.8), the above problems can be overcome, and the stability of the final particle system is significantly improved, and it is not easy to have excessive local concentration during application, which can effectively inhibit tissue contraction or hyperplasia and reduce scars and contractures. Especially when each natural biological particle range has a certain degree of dispersion, it shows a significant inhibitory effect, making the new tissue have good elasticity. It is speculated that such a design may affect the immune response, and the release and regulation of pathway conversion factors during tissue repair are affected. At the same time, this system has a specific multi-level pore structure, forming pore heterogeneity, guiding the adhesion and arrangement of fibroblasts, regulating the interface stress through surface roughness, improving the balance between the shape maintenance and degradation of the new tissue, and realizing the synergistic cooperation of the three particles. The relevant mechanism still needs to be further confirmed.

[0007] Preferably, the particle size dispersion index (PDI) of the first natural biological particles, the second natural biological particles and the third natural biological particles is 0.04-0.54.

[0008] The particle size dispersion index (PDI) is used to describe the dispersion of particle size distribution. The calculation formula is PDI = (D90- D10) / D50, where D90 represents the diameter of particles that account for 90% of the diameter, D50 represents the diameter of particles that account for 50% of the diameter, and D10 represents the diameter of particles that account for 10% of the diameter. The smaller the PDI value, the more concentrated the particle size distribution.

[0009] As a preferred embodiment, the natural biological particle combination has not been treated with enzymes.

[0010] In this solution, the natural bioparticle combination has not been enzymatically treated, has a three-dimensional helical protein structure, retains the natural fiber network, and can be used to support cell regeneration.

[0011] Preferably, the natural biological particle combination has not been cross-linked.

[0012] As a preferred embodiment, the natural biological particle combination is obtained by decellularizing natural biological tissue, and then successively undergoing ion induction treatment and powderization to obtain a powdered product, and the powdered product is surface-finished and mixed to obtain the natural biological particle combination.

[0013] Preferably, the decellularization of natural biological tissues is specifically as follows: after physically treating the natural biological tissues, washing them in a 0.1%-0.3% peracetic acid aqueous solution for 3-4 hours, and then rinsing them with a buffer solution for 3-10 minutes.

[0014] As a preferred embodiment, the ion-induced treatment is specifically as follows: the decellularized natural biological tissue is immersed in an acid solution for 15-30 minutes, and after immersion, an alkaline solution is immediately added to neutralize it to a neutral pH, and then the solution is allowed to stand for 10-15 minutes, and the treated natural biological tissue is taken out to obtain a pre-treated material.

[0015] Preferably, the ion-induced treatment is specifically as follows: take a container, add an acid solution into the container, and immerse the decellularized natural biological tissue in the acid solution for 15-30 minutes. During the immersion, the acid solution completely immerses the decellularized natural biological tissue. After immersion for 15-30 minutes, immediately continue to add an alkaline solution to the container to neutralize it to a neutral pH, then let it stand for 10-15 minutes, take out the treated natural biological tissue, and obtain a pre-treated product.

[0016] Preferably, the acid solution is an inorganic acid aqueous solution and / or an organic acid aqueous solution.

[0017] Preferably, the acid solution is an inorganic acid aqueous solution or a mixture of an inorganic acid aqueous solution and an organic acid aqueous solution.

[0018] Preferably, the acid solution is a mixture of an inorganic acid aqueous solution and an organic acid aqueous solution.

[0019] In the acid solution, the volume ratio of the inorganic acid aqueous solution to the organic acid aqueous solution is (8-20):(3-10).

[0020] More preferably, the inorganic acid aqueous solution is a hydrochloric acid aqueous solution or a phosphoric acid aqueous solution.

[0021] Furthermore, the inorganic acid aqueous solution is a hydrochloric acid aqueous solution.

[0022] Preferably, the organic acid aqueous solution is selected from one of a citric acid aqueous solution, an acetic acid aqueous solution and a tartaric acid aqueous solution.

[0023] Preferably, the organic acid aqueous solution is acetic acid aqueous solution.

[0024] Preferably, when the acid solution is a mixture of an inorganic acid aqueous solution and an organic acid aqueous solution, the acid solution is prepared by mixing 0.025-0.055 g / mL inorganic acid aqueous solution and 0.025-0.055 g / mL organic acid aqueous solution in a volume ratio of (8-20):(3-10), and then diluting with water to adjust the pH to 2.7-5.5.

[0025] As a preferred embodiment, the pH of the acid solution is 2.7-5.5.

[0026] Preferably, the pH of the acid solution is 4.1-5.5.

[0027] In the process of tissue decellularization, it is a common technical means to use a single type of acid solution or a single type of alkaline solution to remove endotoxins from biological tissues, and the use of such a single acid or alkali is usually to wash the biological tissue with an acid or alkaline solution, followed by rinsing with a buffer solution or water. In the present invention, the decellularized natural biological tissue is immersed in an acid solution. In order to remove cellular endotoxins more quickly, in a large number of experiments, it was unexpectedly found that the acid-soaked tissue was not taken out after immersion, but the alkaline solution was continued to be added to the neutral environment, and it was allowed to stand again. After this treatment, it was powdered, so that the product not only effectively removed endotoxins, but also the natural biological particles obtained by this treatment method (i.e., ion induction treatment) combined with powdering and surface finishing had a more regular particle surface and a more uniform particle size, which means that the particles have higher stability, which is conducive to its further application and reduces the risk of uncontrollable local application effects. It is speculated that this is because the triple helical structure of the decellularized natural tissue after acid treatment is embedded with the salt produced during particle induction, which affects the subsequent powdering. This phenomenon is particularly obvious when the acid solution is a mixture of an inorganic acid aqueous solution and an organic acid aqueous solution. At the same time, through scanning electron microscopy characterization, it was found that the natural biological particles obtained by the ion-induced treatment combined with powderization and surface finishing of the present invention have a fluffy and rough microscopic surface structure. This may be due to the large amount of fibers gathered on the surface of the particles, which will also provide more sites for cell attachment and facilitate tissue repair.

[0028] As a preferred embodiment, the powdering is specifically to freeze-dry the pre-treated product and then grind it in liquid nitrogen.

[0029] Preferably, the powderization is performed by freeze-drying the pre-treated product and then pulverizing it in liquid nitrogen to obtain a powdered product.

[0030] As a preferred embodiment, the pulverization method during pulverization in liquid nitrogen is selected from one of impact pulverization, shear pulverization and compression pulverization.

[0031] Preferably, the pulverization method during pulverization in liquid nitrogen is impact pulverization.

[0032] When pulverizing in liquid nitrogen, powder products with different particle size ranges can be obtained by setting the pulverizing speed and pulverizing time of the liquid nitrogen pulverizer, thereby obtaining natural biological particles with different particle size ranges (i.e., powder products after surface finishing). In the present invention, when pulverizing in liquid nitrogen, the pulverizing speed is 60-200 m / s, and the pulverizing time is 10s-5min.

[0033] As a preferred embodiment, the surface finishing is specifically to put the powdered product into pure water, ultrasonicate for 10-20 minutes, filter, discard the filtrate, and dry the filter residue to obtain natural biological particles.

[0034] Preferably, the surface finishing is specifically to put the powdered product into pure water, ultrasonicate for 10-20 minutes, filter, discard the filtrate, and vacuum dry the filter residue to obtain natural biological particles.

[0035] The powdered product includes at least a first powdered product, a second powdered product, and a third powdered product.

[0036] When the powder is pulverized in liquid nitrogen, the pulverization speed is 120-175 m / s and the pulverization time is 2-5 min. The first powder product is surface-treated to obtain the first natural biological particles.

[0037] When the pulverization is carried out in the liquid nitrogen, the pulverization speed is 70-100 m / s and the pulverization time is 1-4 min. The second powdered product is surface treated to obtain the second natural biological particles.

[0038] When the powder is pulverized in liquid nitrogen, the pulverization speed is 60-80 m / s and the pulverization time is 40-80 s. The third powder product is surface-treated to obtain the third natural biological particles.

[0039] The first natural biological particles, the second natural biological particles and the third natural biological particles are mixed in a mass ratio of (0.9-1.7):1:(5.6-10.8) to obtain the natural biological particle combination.

[0040] The second aspect of the present invention provides an application of a natural biological particle combination on a wound surface material, wherein the wound surface material includes a partial-layer skin wound surface material and a full-layer skin wound surface material.

[0041] When applied on wound surface materials, the natural biological particle combination can be used alone or in combination with a biological patch.

[0042] As a preferred embodiment, the wound surface material is in a dry state or a wet state.

[0043] Beneficial effects:

[0044] The natural biological particle combination obtained by the present invention can be directly applied to various wound surfaces when used, reducing the cumbersomeness of using such particles in the prior art, and can be directly covered on full-thickness skin defect wound surfaces (such as deep wet wounds) and superficial wound surfaces (such as dry epidermal injuries); the particle system of the present invention simulates the dynamic evolution law of the natural extracellular matrix through adaptive moisture regulation and gradient degradation characteristics, can match the requirements of different wound microenvironments, significantly inhibit tissue contracture during wound healing, and reduce scar hyperplasia caused by abnormal collagen arrangement. The stability of this natural biological particle combination is significantly improved when used, and it is not easy to have the problem of excessive local concentration. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 This is a scanning electron microscope image of the natural biological particle combination of Example 1. Figure 1 (a) and (b) are electron microscope images of the same position at different magnifications, where: Figure 1 (a) is the field of view at a magnification of 500 times. Figure 1 (b) is the field of view at a magnification of 1000 times; Figure 2 This is a scanning electron microscope image of the natural biological particle combination of Comparative Example 3; Figure 3 This is a scanning electron microscope image of the natural biological particle combination of Comparative Example 4. DETAILED DESCRIPTION

[0046] The reagents used in the present invention are all commercially available. The natural tissues in the examples are obtained from fresh tissues of pigs (weighing about 120 kg) raised in enclosures within half an hour after death.

[0047] Example 1

[0048] This example provides a natural biological particle combination, which includes three natural biological particles with different particle size ranges. Each particle size range of the natural biological particles has a certain degree of dispersion. The natural biological particle combination is derived from natural biological tissues and has not been treated with enzymes or cross-linking.

[0049] The natural biological particle combination is obtained by decellularizing natural biological tissue, and then undergoing ion induction treatment and powderization in succession to obtain a powdered product, and the powdered product is obtained after surface finishing and mixing.

[0050] Specifically: Decellularization of natural biological tissue (specifically bladder basement membrane): The bladder basement membrane was obtained from fresh pig bladders within half an hour after death of closed-feeding pigs (weighing about 120 kg), washed with 0.15% peracetic acid aqueous solution for 3 hours, and then rinsed with PBS buffer solution for 10 minutes.

[0051] Ion induction treatment: Take a container, add an acid solution (specifically, a hydrochloric acid aqueous solution with a pH of 4.3) into the container, and immerse the decellularized natural biological tissue in the acid solution for 25 minutes. During the immersion, the acid solution completely immerses the decellularized natural biological tissue. After immersion for 25 minutes, immediately continue to add an alkaline solution (specifically, a 3wt% sodium hydroxide aqueous solution) into the container to neutralize it to a neutral pH, then let it stand for 10 minutes, take out the treated natural biological tissue, and obtain a pre-treated product.

[0052] Powdering: After the pre-treated product was freeze-dried, it was impact-pulverized in liquid nitrogen to obtain a powdered product. The first powdered product was obtained at a pulverizing speed of 130 m / s and a pulverizing time of 4 min; the second powdered product was obtained at a pulverizing speed of 100 m / s and a pulverizing time of 2 min; the third powdered product was obtained at a pulverizing speed of 60 m / s and a pulverizing time of 50 s.

[0053] Surface finishing: The first powdered product, the second powdered product and the third powdered product are respectively placed in pure water for ultrasonic treatment for 15 minutes, filtered, the filtrate is discarded, and the filter residue is vacuum dried to obtain the first natural biological particles, the second natural biological particles and the third natural biological particles.

[0054] The first natural biological particles, the second natural biological particles and the third natural biological particles are mixed in a mass ratio of 1:1:7.4 to obtain a natural biological particle combination.

[0055] Example 2

[0056] This example provides a natural biological particle combination, which is different from Example 1 in that, ion induction treatment: take a container, add an acid solution into the container (the acid solution is a mixture of hydrochloric acid aqueous solution and acetic acid aqueous solution, which is a mixture of 0.025g / mL inorganic acid aqueous solution and 0.037g / mL organic acid aqueous solution in a volume ratio of 14:6, and then diluted with water to adjust the pH to 4.2), and immerse the decellularized natural biological tissue in the acid solution for 25 minutes. During the immersion, the acid solution completely immerses the decellularized natural biological tissue. After immersion for 25 minutes, immediately continue to add an alkaline solution (specifically 3wt% sodium hydroxide aqueous solution) to the container to neutralize it to a neutral pH, then let it stand for 10 minutes, take out the treated natural biological tissue, and obtain a pre-treated product.

[0057] After surface finishing, the first natural biological particles, the second natural biological particles and the third natural biological particles are mixed in a mass ratio of 1.1:1:6.9 to obtain a natural biological particle combination.

[0058] Comparative Example 1 This example provides a natural biological particle combination. Different from Example 1, the natural biological particle combination includes two natural biological particles with different particle size ranges. Each particle size range of the natural biological particles has a certain degree of dispersion. The natural biological particle combination is derived from natural biological tissues and has not been subjected to enzyme treatment and cross-linking treatment.

[0059] The natural biological particle combination is obtained by decellularizing natural biological tissue, and then undergoing ion induction treatment and powderization in succession to obtain a powdered product, and the powdered product is obtained after surface finishing and mixing.

[0060] Specifically: Decellularization of natural biological tissue (specifically bladder basement membrane): The bladder basement membrane was obtained from fresh pig bladders within half an hour after death of closed-reared pigs (weighing about 120 kg), washed with 0.2% peracetic acid aqueous solution for 3 hours, and then rinsed with PBS buffer solution for 10 minutes.

[0061] Ion induction treatment: Take a container, add an acid solution (specifically, a hydrochloric acid aqueous solution with a pH of 4.3) into the container, and immerse the decellularized natural biological tissue in the acid solution for 25 minutes. During the immersion, the acid solution completely immerses the decellularized natural biological tissue. After immersion for 25 minutes, immediately continue to add an alkaline solution (specifically, a 2wt% sodium hydroxide aqueous solution) into the container to neutralize it to a neutral pH, then let it stand for 10 minutes, take out the treated natural biological tissue, and obtain a pre-treated product.

[0062] Powdering: After the pre-treated product was freeze-dried, it was impact-pulverized in liquid nitrogen to obtain a powdered product. The first powdered product was obtained at a pulverizing speed of 120 m / s and a pulverizing time of 5 min; the second powdered product was obtained at a pulverizing speed of 70 m / s and a pulverizing time of 65 s.

[0063] Surface finishing: the first powdered product and the second powdered product are respectively placed in pure water for ultrasonic treatment for 15 minutes, filtered, the filtrate is discarded, and the filter residue is vacuum dried to obtain the first natural biological particles and the second natural biological particles.

[0064] The first natural biological particles and the second natural biological particles are mixed in a mass ratio of 1:1 to obtain a natural biological particle combination.

[0065] Comparative Example 2 This example provides a natural biological particle combination, which is different from Example 1 in that the natural biological particle combination includes three sieved natural biological particles with different particle size ranges. The sieved natural biological particle combination is derived from natural biological tissues and has not been subjected to enzyme treatment and cross-linking treatment.

[0066] The sieved natural biological particle combination is obtained by decellularizing natural biological tissue, and then undergoing ion induction treatment and powderization in succession to obtain a powdered product. The powdered product is surface-treated, sieved and mixed to obtain the powdered product.

[0067] Specifically: Decellularization of natural biological tissue (specifically bladder basement membrane): The bladder basement membrane was obtained from fresh pig bladders within half an hour after death of closed-feeding pigs (weighing about 120 kg), washed with 0.15% peracetic acid aqueous solution for 3 hours, and then rinsed with PBS buffer solution for 10 minutes.

[0068] Ion induction treatment: Take a container, add an acid solution (specifically, a hydrochloric acid aqueous solution with a pH of 6.3) into the container, and immerse the decellularized natural biological tissue in the acid solution for 25 minutes. During the immersion, the acid solution completely immerses the decellularized natural biological tissue. After immersion for 25 minutes, immediately continue to add an alkaline solution (specifically, a 3wt% sodium hydroxide aqueous solution) into the container to neutralize it to a neutral pH, then let it stand for 10 minutes, take out the treated natural biological tissue, and obtain a pre-treated product.

[0069] Powdering: After the pre-treated product was freeze-dried, it was impact-pulverized in liquid nitrogen to obtain a powdered product. The first powdered product was obtained at a pulverizing speed of 150 m / s and a pulverizing time of 130 s; the second powdered product was obtained at a pulverizing speed of 90 m / s and a pulverizing time of 2 min; the third powdered product was obtained at a pulverizing speed of 70 m / s and a pulverizing time of 40 s.

[0070] Surface finishing: The first powdered product, the second powdered product and the third powdered product are respectively placed in pure water for ultrasonic treatment for 15 minutes, filtered, the filtrate is discarded, and the filter residue is vacuum dried to obtain the first natural biological particles, the second natural biological particles and the third natural biological particles.

[0071] The first natural biological particles are sieved through a 200-mesh sieve and then sieved through a 230-mesh sieve to screen out the first sieved natural biological particles of about 63-78 μm. The second natural biological particles are sieved through a 40-mesh sieve to screen out the second sieved natural biological particles of about 385-396 μm. The third natural biological particles are sieved through a 20-mesh sieve to screen out the third sieved natural biological particles of about 765-832 μm.

[0072] The first sieved natural biological particles, the second sieved natural biological particles and the third sieved natural biological particles are mixed in a mass ratio of 1.3:1:8.2 to obtain a natural biological particle combination.

[0073] Comparative Example 3 This example provides a natural biological particle combination, which is different from Example 1 in that the natural biological particle combination is derived from natural biological tissue that has been cross-linked and has not been treated with enzymes. The natural biological particle combination is obtained by decellularizing natural biological tissue, cross-linking (specifically, immersing in a 0.5% genipin aqueous solution, soaking for 72 hours, and washing with PBS buffer), and then ion-induced treatment and powderization to obtain a powdered product, which is obtained after surface finishing and mixing.

[0074] Powdering: After the pre-treated product was freeze-dried, it was impact-pulverized in liquid nitrogen to obtain a powdered product. The first powdered product was obtained at a pulverizing speed of 170 m / s and a pulverizing time of 4 min; the second powdered product was obtained at a pulverizing speed of 100 m / s and a pulverizing time of 3 min; the third powdered product was obtained at a pulverizing speed of 80 m / s and a pulverizing time of 70 s.

[0075] Comparative Example 4 This example provides a natural biological particle combination. Different from Example 1, the natural biological particle combination is obtained by decellularizing natural biological tissue, and then successively undergoing salt treatment and powderization to obtain a powdered product, and the powdered product is obtained after surface finishing and mixing.

[0076] Specifically: Decellularization of natural biological tissue (specifically bladder basement membrane): The bladder basement membrane was obtained from fresh pig bladders within half an hour after death of closed-feeding pigs (weighing about 120 kg), washed with 0.15% peracetic acid aqueous solution for 3 hours, and then rinsed with PBS buffer solution for 10 minutes.

[0077] Salt treatment: Take a container, add 25wt% sodium chloride aqueous solution into the container, soak the decellularized natural biological tissue in the sodium chloride aqueous solution for 20 minutes, during the soaking, the sodium chloride aqueous solution completely immerses the decellularized natural biological tissue, after soaking for 20 minutes, take out the treated natural biological tissue to obtain the pre-treated material.

[0078] The operation methods of powdering, surface finishing and mixing are consistent with those in Example 1.

[0079] Comparative Example 5 This example provides a natural biological particle combination. Different from Example 1, the natural biological particle combination includes three natural biological particles with different particle size ranges. Each particle size range of the natural biological particles has a certain degree of dispersion. The natural biological particle combination is derived from natural biological tissues and has not been subjected to enzyme treatment and cross-linking treatment.

[0080] The natural biological particle combination is obtained by decellularizing natural biological tissue, successively undergoing acid treatment and powderization to obtain a powdered product, and the powdered product is obtained after surface finishing and mixing.

[0081] Specifically: Decellularization of natural biological tissue (specifically bladder basement membrane): The bladder basement membrane was obtained from fresh pig bladders within half an hour after death of closed-feeding pigs (weighing about 120 kg), washed with 0.15% peracetic acid aqueous solution for 3 hours, and then rinsed with PBS buffer solution for 10 minutes.

[0082] Acid treatment: Take a container, add an acid solution (specifically, a hydrochloric acid aqueous solution with a pH of 4.5) into the container, and immerse the decellularized natural biological tissue in the acid solution for 25 minutes. During the immersion, the acid solution completely immerses the decellularized natural biological tissue. After immersion for 25 minutes, take out the treated natural biological tissue, rinse it with water, and obtain the pre-treated material.

[0083] Powdering: After the pre-treated product was freeze-dried, it was impact-pulverized in liquid nitrogen to obtain a powdered product. The first powdered product was obtained at a pulverizing speed of 130 m / s and a pulverizing time of 4 min; the second powdered product was obtained at a pulverizing speed of 100 m / s and a pulverizing time of 2 min; the third powdered product was obtained at a pulverizing speed of 60 m / s and a pulverizing time of 50 s.

[0084] Surface finishing: The first powdered product, the second powdered product and the third powdered product are respectively placed in pure water for ultrasonic treatment for 15 minutes, filtered, the filtrate is discarded, and the filter residue is vacuum dried to obtain the first natural biological particles, the second natural biological particles and the third natural biological particles.

[0085] The first natural biological particles, the second natural biological particles and the third natural biological particles are mixed in a mass ratio of 1.1:1:7.4 to obtain a natural biological particle combination.

[0086] Test Results 1. Particle size distribution characterization: The first natural biological particles, the second natural biological particles and the third natural biological particles after surface treatment in Examples 1-2 and Comparative Examples 3-5, as well as the first natural biological particles and the second natural biological particles after surface treatment in Comparative Example 1, and the three sieved natural biological particles obtained in Comparative Example 2 were tested for particle size distribution using a laser particle size tester. The results are shown in Table 1: Table 1 As can be seen from Table 1, the particle size of the first natural biological particles obtained in Examples 1-2 is in the range of 40-100 μm; the particle size of the second natural biological particles is in the range of 270-400 μm; the particle size of the third natural biological particles is in the range of 750-1000 μm, and each natural biological particle obtained in Examples 1-2 has a lower particle dispersibility. In addition, it can be observed that the PDI of the natural biological particles in Example 2 is significantly lower than that in Example 1, which indicates that the uniform performance of the particles in Example 2 is more prominent.

[0087] The natural biological particle combination of Comparative Example 1 is composed of only two natural biological particles ranging from 40 to 100 μm and 270 to 400 μm, and does not include larger natural biological particles of 750 to 1000 μm.

[0088] In Comparative Example 2, each natural biological particle was sieved, and the particle size distribution of each sieved natural biological particle was narrow.

[0089] Compared with Example 1, the particle size range of each natural biological particle in Comparative Examples 3-4 is shifted to a larger direction, and the dispersion is greater.

[0090] 2. Morphology characterization: The natural biological particles obtained in Example 1 and Comparative Examples 3-4 were characterized by SEM scanning electron microscopy. The results are as follows: Figure 1-3 It can be observed that the particles have a fluffy and rough microscopic surface structure; while the natural biological particles of Comparative Examples 3-4 have a relatively smooth and dense surface, and the particle edges are relatively sharp.

[0091] 3. Cell endotoxin content: The natural biological particle combinations obtained in Examples 1-2 and Comparative Examples 1-5 were respectively extracted with water for 2 hours, and the endotoxin content was detected by dynamic photometry. The results are shown in Table 2: Table 2 Example Endotoxin content (EU / g) Example 1 0.68 Example 2 0.73 Comparative Example 1 0.63 Comparative Example 2 0.83 Comparative Example 3 2.15 Comparative Example 4 4.74 Comparative Example 5 2.77 4. Contact angle test: The contact angle of the natural biological particle combination of Examples 1-2 and Comparative Examples 1-5 was obtained by droplet penetration method. Each sample was measured 10 times and the average value was taken. The smaller the contact angle, the higher the hydrophilicity and the higher the performance of promoting cell adhesion. During the test, it was found that for some samples, the contact angle hysteresis phenomenon was very obvious. Generally speaking, the rougher the microscopic surface of the material, the greater the amplitude of the contact angle hysteresis. The calculation of the contact angle hysteresis is the difference Δθ=θA -θR, where θA is the maximum contact angle (i.e., advancing contact angle), θR is the minimum contact angle (i.e., receding contact angle), the results are shown in Table 3, again on this basis, the experiment was repeated, the contact angles of the first natural biological particles, the second natural biological particles, and the third natural biological particles obtained in Examples 1-2 and Comparative Examples 3-5 after surface finishing were tested 10 times, respectively, and obvious contact angle hysteresis was also observed in Examples 1-2, while there was basically no contact angle hysteresis or the contact angle hysteresis was not significant in Comparative Examples 3-5, the standard deviation of the 10 contact angle hysteresis Δθ of the first natural biological particles, the second natural biological particles, and the third natural biological particles was calculated to reflect the discrete degree of the contact angle hysteresis, the results are shown in Table 4: Table 3 Example Contact angle (water, °) Δθ average value (°) Example 1 22.3 5.7 Example 2 25.5 5.4 Comparative Example 1 41.7 4.9 Comparative Example 2 27.4 4.6 Comparative Example 3 44.8 1.6 Comparative Example 4 32.6 1.2 Comparative Example 5 29.5 0.8 Table 4 It can be seen from Table 3 that the contact angle of the natural biological particle combination of Example 1-2 is small, indicating that it has a high ability to promote cell adhesion. At the same time, the contact angle hysteresis of Example 1-2 is obvious, which shows that the natural biological particle combination of Example 1-2 has a relatively rough microscopic surface, which is also consistent with the test results of the SEM scanning electron microscope, and the rough microscopic surface is further conducive to cell adhesion.

[0092] It can be seen from Table 4 that in Examples 1-2, the standard deviation of Δθ is significantly smaller than that of Comparative Examples 3-5, which indicates that the contact angle hysteresis of Examples 1-2 is relatively stable, reflecting that each natural biological particle in Examples 1-2 has a relatively uniform degree of roughness, while the roughness of each natural biological particle in Comparative Examples 3-5 is relatively uneven, which means that the product obtained in Examples 1-2 is more stable.

[0093] 5. Cell experiment: It is known that overexpression or local high concentration of TGF-β1 (transforming growth factor) will stimulate fibroblasts to transform into myofibroblasts that can synthesize and secrete α-SMA (α-smooth muscle actin), leading to scar contracture. After culturing human keloid fibroblasts (purchased from Pronocell, item number CM-H235) to 3-4 generations, 10 μL of cell suspension was aspirated and inoculated into a 24-well (8-well) plate cover glass. After 24 hours, when the cells were cultured to 80% fusion, the natural biological particle combination obtained in Example 1 was added to 2 wells, the natural biological particle combination obtained in Comparative Example 1 was added to 2 wells, the natural biological particle combination obtained in Comparative Example 2 was added to 2 wells, the aforementioned 6 wells were used as the experimental group, and the remaining 2 wells were not added with any substance as the control group. After 72 hours of continuous culture, the culture medium of the experimental group and the control group was completely aspirated, and the cells were fixed with 40% paraformaldehyde after being rinsed with PBS buffer. Then, 2 ml of BSA blocking solution was added, and the cells were incubated at 37°C in a carbon dioxide atmosphere. Rabbit anti-TGF-β1 and rabbit anti-α-SMA were added, and the cells were kept at 4°C overnight. The cells were rinsed with PBST buffer, and DAPI fluorescent dye was added to stain the nucleus for 4 hours. The cells were observed under a fluorescence microscope. The results showed that TGF-β1 and α-SMA in the control group were significantly expressed in the cytoplasm. Compared with the control group, the expression of TGF-β1 and α-SMA in the sample group of comparative example 2 in the experimental group was close to that in the control group; the expression of TGF-β1 and α-SMA in the sample group of Example 1 in the experimental group was significantly reduced, and the expression of TGF-β1 and α-SMA in the sample group of comparative example 1 in the experimental group was weakly reduced. This shows that the sample of Example 1 has a significant effect of inhibiting tissue contraction or hyperplasia and reducing scars and contractures.

Claims

1. A natural biological particle combination, characterized in that: The natural biological particle combination comprises at least three natural biological particles with different particle size ranges, wherein the particle size range of the first natural biological particle is 40-100 μm; the particle size range of the second natural biological particle is 270-400 μm; the particle size range of the third natural biological particle is 750-1000 μm; the mass ratio of the first natural biological particle, the second natural biological particle and the third natural biological particle is (0.9-1.7):1:(5.6-10.8); the natural biological particle combination is derived from natural biological tissue, and the natural biological tissue is selected from one or two of the small intestinal submucosa, pericardium, bladder basement membrane, peritoneum and dermis of mammals.

2. The natural biological particle combination according to claim 1, characterized in that: The natural bioparticle combination has not been subjected to enzyme treatment.

3. The natural biological particle combination according to claim 1, characterized in that: The natural biological particle combination is obtained by decellularizing natural biological tissue, and then undergoing ion induction treatment and powderization to obtain a powdered product. The powdered product is surface-treated and mixed to obtain the natural biological particles.

4. The natural biological particle combination according to claim 3, characterized in that: The ion induction treatment is specifically as follows: soaking the decellularized natural biological tissue in an acid solution for 15-30 minutes, immediately adding an alkaline solution to neutralize it to a neutral pH, then standing it for 10-15 minutes, taking out the treated natural biological tissue, and obtaining a pre-treated product.

5. The natural biological particle combination according to claim 4, characterized in that: The pH of the acid solution is 2.7-5.

5.

6. The natural biological particle combination according to claim 3, characterized in that: The powderization is specifically to freeze-dry the pre-treated product and then pulverize it in liquid nitrogen.

7. The natural biological particle combination according to claim 6, characterized in that: The pulverization method during pulverization in liquid nitrogen is selected from one or more of impact pulverization, shear pulverization and compression pulverization.

8. The natural biological particle combination according to claim 3, characterized in that: The surface finishing is specifically to put the powdered product into pure water, perform ultrasound for 10-20 minutes, filter, discard the filtrate, take the filter residue and dry it to obtain the natural biological particles.

9. An application of the natural biological particle combination according to claim 1 on wound surface materials, characterized in that: The wound surface materials include partial-layer skin wound repair materials and full-layer skin wound repair materials.

10. The use of the natural biological particle combination as claimed in claim 9 on wound surface materials, characterized in that: The wound surface material is in a dry state or a wet state.

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