Acellular dermal matrix particle and preparation method thereof

Abstract

The invention provides a decellularized dermal matrix particle and a preparation method thereof, the preparation method comprises the steps of pretreatment, degreasing, virus inactivation, decellularization, grinding, crosslinking, freeze-drying and sterilization subpackaging, and solves the problems that the existing decellularized particle product is non-uniform in particle size, incomplete in decellularization, poor in injection effect and incapable of meeting the requirements of tissue repair and regeneration. The invention develops a product which is short in production cycle, relatively concentrated in particle size distribution, thorough in cell removal and complete in matrix function preservation, can effectively remove fat and viruses in animal skin, and has the advantages of being thorough in decellularization, short in time, high in efficiency, light in damage to dermal matrix, capable of removing antigens, capable of retaining a natural structure and capable of effectively guiding tissue regeneration. When the temperature of the freeze-dried particle dispersion liquid is reduced, the water binding capacity of the particles is reduced, free water in the prepared solution is increased, the particles naturally float and gather (dense at the upper part and sparse at the lower part), and the free water is in the lower layer and still has good dispersity after being sterilized.

Description

Technical Field

[0001] The present invention relates to the technical field of acellular dermal matrix, and particularly to an acellular dermal matrix particle and a preparation method thereof. Background Art

[0002] As a representative material for bio-tissue engineering scaffolds, acellular dermal matrix (ADM) has attracted attention in the field of plastic surgery due to its good clinical application prospects. ADM is prepared by removing the epidermal layer in xenogeneic full-thickness skin and the cellular components in the dermal tissue, and only preserving the dermal extracellular matrix (ECM) with a complete fiber structure. Since this material removes the main cells and epidermis with immunogenicity, it can guide host cells to grow inwards and tissue regeneration to promote the repair of the body. It can be derived from human, bovine or porcine skin tissues, and there are reports of clinical applications. Currently, ADM has been widely used in soft tissue filling and lifting, including rhinoplasty, eyelid repair, breast reconstruction, reproductive plastic surgery, etc.

[0003] Existing acellular particle products have problems such as uneven particle size, incomplete decellularization that is likely to cause an immunogenic reaction, poor injection effect, and do not meet the requirements of tissue repair and regeneration. Therefore, an acellular dermal matrix particle and a preparation method thereof are proposed. Summary of the Invention

[0004] The purpose of the present invention is to provide a product and method with a short production cycle, relatively concentrated particle size distribution of particles, thorough cell removal, and complete preservation of matrix functions.

[0005] To achieve the above object, the present invention proposes the following technical solutions:

[0006] A preparation method of an acellular dermal matrix particle, comprising the following steps:

[0007] Step 1: Pretreatment: Use a skin harvesting machine to peel the body surface skin of a mammal, and remove the attached subcutaneous part to obtain an acellular matrix material;

[0008] Step 2, degreasing: Mix and oscillate the acellular matrix material in Step 1 with a degreasing agent;

[0009] Step 3, virus inactivation: Mix and oscillate the acellular matrix material processed in Step 2 with a disinfectant;

[0010] Step 4, decellularization: Mix and oscillate the acellular matrix material processed in Step 3 with SDS;

[0011] Step 5, grinding: Mix the acellular matrix material processed in Step 4 with purified water. When grinding, set the centrifugal speed of the colloid mill to 8000 rpm - 10000 rpm, and grind while injecting water.

[0012] Step 6, crosslinking: Mix the acellular matrix material processed in Step 5 with a crosslinking agent.

[0013] Step 7, freeze-drying: Centrifuge the crosslinked solution processed in Step 6 and then put it into a freeze-dryer for freeze-drying.

[0014] Step 8, sterilization and packaging: Sterilize to obtain the acellular biological matrix material. The sterilization method is one of Co60, electron beam, or ethylene oxide sterilization.

[0015] In Step 2, the weight ratio of the acellular matrix material to the degreasing agent is 1:1 - 1:10, and the oscillation time is 10 - 20 h. The degreasing reagent is selected from at least one of 0.1 - 1 wt% sodium hydroxide solution, 1 - 20 wt% sodium carbonate solution, 1 - 20 wt% isopropyl alcohol solution, 20 - 80 wt% n-hexane solution, and 40 - 80 wt% ethanol mixed solution.

[0016] The mammal is selected from one of pig, cattle, and sheep.

[0017] In Step 1, the parts attached to the subcutaneous layer include the fat, muscle, and hair components attached to the subcutaneous layer of the mammal, and the mammalian skin is made into a split skin graft with a thickness of about 0.1 - 1 mm.

[0018] In Step 3, the weight ratio of the acellular matrix material to the disinfectant is 1:1 - 1:10, and the oscillation time is 4 - 8 h. The disinfectant is selected from at least one of 0.02 - 0.2 wt% peracetic acid and 5 - 40% ethanol solution.

[0019] In Step 4, the weight ratio of the acellular matrix material to SDS is 1:1 - 1:10, and the oscillation time is 1 - 4 h. The concentration of SDS is 0.01 - 2 g / L.

[0020] In Step 5, the shear rotation speed of the colloid mill is 8000 rpm - 14000 rpm.

[0021] In Step 6, the acellular matrix material is mixed with a crosslinking agent. The concentration of the crosslinking agent is 0.0001% - 0.01%. The crosslinking agent is selected from at least one of glutaraldehyde, formaldehyde, genipin, and diphenylphosphoryl azide.

[0022] Centrifuge the crosslinked solution processed in Step 7 and then put it into a freeze-dryer for freeze-drying. The centrifugation conditions are 5000 - 10000 rpm, 10 - 20 min; the freeze-drying time is 12 - 20 h.

[0023] Use one of the Co60 or electron beam or ethylene oxide sterilization methods for the acellular biological matrix material in Step Eight.

[0024] An acellular dermal matrix microparticle is prepared according to the described method.

[0025] The present invention develops a product with a short production cycle, a relatively concentrated particle size distribution of microparticles, thorough cell removal, and intact preservation of matrix functions. It can effectively remove fat and viruses in animal skin, has the advantages of thorough acellularization, short time, high efficiency, slight damage to the dermal matrix, antigen removal, retention of its natural structure, and effective guidance of tissue regeneration. When the freeze-dried microparticle dispersion cools down, the ability of the microparticles to bind water decreases, the free water in the prepared solution increases, and the microparticles naturally float and aggregate (dense on top and sparse at the bottom), with the free water at the bottom layer. After sterilization, it still has good dispersibility. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is a schematic diagram of the acellular dermal matrix microparticle product of the present invention.

[0027] Figure 2 It is an analysis chart of the particle size of the acellular dermal matrix microparticle of the present invention.

[0028] Figure 3 It is an analysis chart of the particle size of the acellular dermal matrix microparticle of the present invention.

[0029] Figure 4 It is an SDS-PAGE chart of the acellular dermal matrix microparticle of the present invention.

[0030] Figure 5 In it, a is a HE staining (500μm) chart of the acellular dermal matrix microparticle of the present invention before acellular treatment, Figure 5 In it, b is a HE staining (500μm) chart after acellular treatment;

[0031] Figure 5 In it, c is a HE staining (100μm) chart of the acellular dermal matrix microparticle of the present invention before acellular treatment, Figure 5 In it, c is a HE staining (100μm) chart after acellular treatment.

[0032] Figure 6 It is an in vitro enzymatic hydrolysis experiment chart of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0033] To better understand the technical content of the present invention, specific embodiments are hereby given and described in conjunction with the accompanying drawings. In the present disclosure, aspects of the present invention are described with reference to the drawings, in which a number of illustrative embodiments are shown. The embodiments of the present disclosure do not necessarily define all aspects of the present invention. It should be understood that the various concepts and embodiments introduced above, as well as those described in more detail below, can be implemented in any of many ways, because the concepts and embodiments disclosed in the present invention are not limited to any implementation manner. In addition, some aspects of the present invention can be used alone, or in any suitable combination with other aspects of the present invention.

[0034] Example 1: Preparation and performance detection of acellular dermal matrix microparticles

[0035] The following steps are adopted in this example to prepare acellular dermal matrix microparticles:

[0036] (A) Pretreatment: Fresh pigskin is collected from a freshly slaughtered pig. After mechanical and manual hair removal, the attached subcutaneous part is removed with a slicer, and the dermis about 0.5 mm thick is cut.

[0037] (B) Degreasing: The cut skin pieces and the degreasing agent are in a weight ratio of 1:4, and the degreasing agent is 0.4% NaOH. The degreasing treatment is carried out by shaking for 10 h.

[0038] (C) Virus inactivation: The skin pieces treated in (B) and the disinfectant are in a weight ratio of 1:6, and the disinfectant is 0.5% peracetic acid for virus removal. The treatment is carried out by shaking for 3 h.

[0039] (D) Decellularization: The skin pieces treated in (C) and SDS are in a weight ratio of 1:5, and the decellularization is carried out by shaking with 0.2 g / L SDS for 1 h.

[0040] (E) Grinding: The colloid mill is centrifugally set at 10,000 rpm, and grinding is carried out while injecting water.

[0041] (F) Crosslinking: The microparticles treated in (E) and the crosslinking agent are in a weight ratio of 1:150, and the crosslinking agent is 0.0005% glutaraldehyde for treatment for 6 h.

[0042] (G) Freeze-drying: The crosslinked microparticles in (F) are placed in a centrifuge at 15,000 rpm for 10 min. After centrifugation, the precipitate is spread out flat on a freeze-drying tray for freeze-drying.

[0043] (H) Sub-packaging and sterilization: After freeze-drying, the samples are sub-packaged as shown in Figure 1 , and sterilized by ethylene oxide.

[0044] Performance detection:

[0045] The measurement shows that the particle size D50 of the material is (209.85 ± 10) μm, and the span is (1.325 ± 0.1) (N > 5). For specific reference Figure 2 ; Differential scanning calorimetry analysis shows that the initial denaturation temperature of the acellular matrix microparticles is (57 ± 0.4) °C (N > 5), and the enthalpy change value is (60 ± 5) J / g (N > 5). Compared with the raw materials in the natural state, the entire process greatly retains the biological characteristics of the acellular matrix. The degree of crosslinking is 40% - 60%, which are 36.6%, 42.0%, 38.7%, 35.6%, and 40.8% respectively (N > 5).

[0046] According to "YY / T 1561-2017 Residues of Animal-derived Scaffold Materials for Tissue Engineering Medical Devices" for α-Gal antigen detection, the test results show that the clearance rate of the original skin / skin after decellularization is greater than 90%. The α-Gal antigen test results are 94.55%, 92.37%, and 94.06% respectively.

[0047] According to "YYT 0606.25-2014 Tissue Engineering Medical Products Part 25 Determination Method for DNA Residue in Animal-derived Biomaterials" for DNA residue detection, the test results show that the acellular dermal matrix microparticles of the present invention are less than 50 ng / mg, which are 16.2 ng / mg, 20.0 ng / mg, and 15.7 ng / mg respectively.

[0048] Example 2:

[0049] Using the acellular dermal matrix microparticles prepared in Example 1, the HE section staining results before and after decellularization during the process are shown in Figure 5 .

[0050] Example 3:

[0051] Using the acellular dermal matrix microparticles prepared in Example 1, according to "YYT 1805.2-2021 Tissue Engineering Medical Devices Products Collagen Part 2: Detection of Molecular Weight of Type I Collagen - Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis Method", the SDS-PAGE test results are shown in Figure 4 .

[0052] Example 4: (Degradation experiment)

[0053] Using the acellular dermal matrix microparticles prepared in Example 1 and uncrosslinked acellular dermal matrix microparticles, according to the ratio of adding 10 mL of 5 Units / mL enzyme degradation solution to every 10 mg of freeze-dried sample, at 37 °C ± 1 °C, record the degradation rate of the samples at 0, 1, 2, 3, 4, 5, and 6 h respectively, as shown in Figure 6 .

[0054] Although the present invention has been disclosed above in preferred embodiments, it is not intended to limit the present invention. Those of ordinary skill in the technical field to which the present invention pertains can make various modifications and refinements without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention shall be determined by the scope defined in the claims.

Claims

1. A method for preparing acellular dermal matrix microparticles, characterized in that: It includes the following steps: Step 1: Pretreatment: Use a skin harvester to strip the body surface skin of a mammal, and remove the attached subcutaneous part to obtain a decellularized matrix material; Step 2, Degreasing: Mix and oscillate the decellularized matrix material in Step 1 with a degreasing agent; Step 3, Virus inactivation: Mix and oscillate the decellularized matrix material after the treatment in Step 2 with a disinfectant; Step 4, Decellularization: Mix and oscillate the decellularized matrix material after the treatment in Step 3 with SDS; Step 5, Grinding: Mix the decellularized matrix material after the treatment in Step 4 with purified water. When grinding, set the centrifugal of the colloid mill to 8000 rpm - 10000 rpm, and grind while injecting water; Step 6, Crosslinking: Mix the decellularized matrix material after the treatment in Step 5 with a crosslinking agent; Step 7, Freeze-drying: Centrifuge the crosslinked solution after the treatment in Step 6 and then put it into a freeze-dryer for freeze-drying; Step 8, Sterilization and packaging: Sterilize to obtain the decellularized biological matrix material. The sterilization uses one of Co60 or electron beam or ethylene oxide sterilization; In the said Step 2, the weight ratio of the decellularized matrix material to the degreasing agent is 1:1 - 1:10, and the oscillation time is 10 - 20 h. Among them, the degreasing reagent is selected from at least one of 0.1 - 1 wt% sodium hydroxide solution, 1 - 20 wt% sodium carbonate solution, 1 - 20 wt% isopropanol solution, 20 - 80 wt% n-hexane solution, and 40 - 80 wt% ethanol mixed solution.

2. The preparation method of an acellular dermal matrix particle according to claim 1, wherein: The said mammal is selected from one of pig, cattle, and sheep.

3. The preparation method of an acellular dermal matrix particle according to claim 1, characterized in that: In Step 1, the attached subcutaneous part includes the attached fat, muscle, and hair components under the mammal's skin, and the mammal's skin is made into a split skin graft with a thickness of about 0.1 - 1 mm.

4. The preparation method of an acellular dermal matrix particle according to claim 1, characterized in that: In the said Step 3, the weight ratio of the decellularized matrix material to the disinfectant is 1:1 - 1:10, and the oscillation time is 4 - 8 h. Among them, the disinfectant is selected from at least one of 0.02 - 0.2 wt% peracetic acid and 5 - 40% ethanol solution.

5. A method for preparing acellular dermal matrix microparticles according to claim 1, characterized in that: In the said Step 4, the weight ratio of the decellularized matrix material to SDS is 1:1 - 1:10, and the oscillation time is 1 - 4 h. Among them, the concentration of SDS is 0.01 - 2 g / L.

6. The preparation method of an acellular dermal matrix particle according to claim 1, wherein: In the said Step 5, the shear rotation speed of the colloid mill is 8000 rpm - 14000 rpm.

7. A method for preparing acellular dermal matrix microparticles according to claim 1, characterized in that: In the said Step 6, the decellularized matrix material is mixed with a crosslinking agent, and the concentration of the crosslinking agent is 0.0001% - 0.01%. Among them, the crosslinking agent is selected from one of glutaraldehyde, formaldehyde, genipin, and diphenylphosphoryl azide.

8. The preparation method of an acellular dermal matrix particle according to claim 1, characterized in that: Centrifuge the crosslinked solution after the treatment in Step 7 and then put it into a freeze-dryer for freeze-drying. The centrifugation conditions are 5000 - 10000 rpm, 10 - 20 min; the freeze-drying time is 12 - 20 h.

9. A decellularized dermal matrix particle, prepared by the method according to any one of claims 1 - 8.

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