A soft tissue repair material and its preparation method

Abstract

The present invention provides a soft tissue repair material and a preparation method thereof, comprising the following steps: soaking the extracellular matrix in a composite amino acid phosphate buffer solution containing L-arginine, L-asparagine, and L-isoleucine for protecting the structure and activity before decellularization; washing after soaking, and performing decellularization treatment with a surfactant to obtain a decellularized tissue; after washing, soaking the decellularized tissue in an MES buffer solution, and then soaking it in an MES buffer solution containing EDC and NHS for fixation, and obtaining a tissue with a fixed structure after washing; placing the tissue with a fixed structure in an NaOH solution for virus inactivation, and obtaining a soft tissue repair material after washing. The method of the present invention can maximize the retention of the structural integrity and active components of the extracellular matrix in the decellularized extracellular matrix, facilitate the influence of the extracellular matrix on cell proliferation and differentiation, and be better used for soft tissue repair.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, and particularly relates to a soft tissue repair material and a preparation method thereof. Background Art

[0002] The extracellular matrix (ECM) is secreted by cells, and its main components are collagen, elastin, fibronectin, laminin, proteoglycans (PGs), hyaluronic acid (HA), and several glycoproteins. They interact with each other to create a multi-component structural network and interact with resident cells through cell surface receptors, such as collagen, fibronectin, vitronectin, and glycosaminoglycans. As the place for cells to survive and carry out physiological activities, ECM is a storage library for various cytokines and growth factors, well simulating the in vivo microenvironment and affecting cell activities such as proliferation, migration, and differentiation through signal transduction. However, when the extracellular matrix is implanted into the human body as foreign tissue, it is necessary to remove factors such as cells and nucleic acids to avoid immune rejection reactions.

[0003] Decellularized extracellular matrix (dECM) is a scaffold that removes cells and nuclear components from ECM while maintaining the inherent ultrastructure and original biochemical composition in ECM. It allows autologous cells or multipotent progenitor cells of patients to adhere and proliferate and ultimately transform into their own specific functional tissues. In addition, the chemical composition and structure in the scaffold can increase the tissue regeneration ability of the damaged part of the patient. dECMs have been reported to successfully reconstruct different types of tissues and organs, such as skeletal muscle, blood vessels, heart valves, cornea, bladder, skin, etc.

[0004] There have been many studies on soft tissue decellularization processes, structure fixation, and removal of immunogenicity. The invention patent with the publication number CN108404212A once disclosed a preparation method of a decellularized dermal matrix material, which uses NaOH as the main decellularization reagent for decellularization treatment of dermal tissue and fixes the structure before decellularization with glutaraldehyde. Many studies have shown that glutaraldehyde has high cytotoxicity, and the control of its residual amount is a crucial factor affecting its tissue biocompatibility. Moreover, as a decellularization reagent, although NaOH has an ideal decellularization effect, it causes great damage to the tissue structure, and the cell survival rate of the prepared tissue material is also low. Although the invention patents with the publication numbers CN1569260A and CN107412868A have optimized the selection of decellularization reagents, the reagents involved are relatively complex, which is not conducive to industrial production, and cobalt-60 irradiation is used for virus inactivation of tissues. After cobalt-60 irradiation treatment, the tissues will have problems of discoloration and hardening to varying degrees.

[0005] Therefore, during the decellularization process, how to efficiently remove immunogenicity while maximizing the maintenance of the three-dimensional structural stability of the extracellular matrix and retaining bioactive components such as proteoglycans and growth factors is a current technical difficulty.

[0006] In view of this, the present invention is specifically proposed. Summary of the Invention

[0007] The object of the present invention is to provide a soft tissue repair material and a preparation method thereof, which can maximize the retention of the structural integrity and active components of the extracellular matrix by the decellularized extracellular matrix, facilitate the influence of the extracellular matrix on cell proliferation and differentiation, and be better used for soft tissue repair.

[0008] In the first aspect of the present invention, a preparation method of a soft tissue repair material is provided, including the following steps:

[0009] S1. Immerse the extracellular matrix in a composite amino acid phosphate buffer solution containing L-arginine, L-asparagine, and L-isoleucine for pre-decellularization structural and activity protection;

[0010] S2. After immersion, wash and perform decellularization treatment with a surfactant to obtain a decellularized tissue;

[0011] S3. After washing, immerse the decellularized tissue in MES buffer solution to activate the groups; then immerse it in MES buffer solution containing EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) for cross-linking and fixation, and obtain a structurally fixed tissue after washing;

[0012] S4. Immerse the structurally fixed tissue in NaOH solution for virus inactivation, and obtain a soft tissue repair material after washing.

[0013] Preferably, the mass ratio of L-arginine, L-asparagine, and L-isoleucine is (6-8):(1-3):(0.5-1.5), and more preferably 7:2:1.

[0014] Preferably, the mass concentration of the composite amino acids in the composite amino acid phosphate buffer solution is 5%-15%, and more preferably 10%.

[0015] In a specific embodiment, step S1 includes: immersing the extracellular matrix in a composite amino acid phosphate buffer solution with a pH of 7-8 containing L-arginine, L-asparagine, and L-isoleucine at room temperature for 18-24 h for pre-decellularization structural and activity protection.

[0016] Preferably, step S2 further includes: treating with DNase and / or protease inhibitor. The function of DNase is to remove antigens, and the function of protease inhibitor is to inhibit enzyme conversion.

[0017] Preferably, the protease inhibitor includes one or more of EDTA (ethylenediaminetetraacetic acid) and NEM (N-ethylmaleimide).

[0018] Preferably, step S2 further includes: treating with sodium deoxycholate and / or divalent metal cations to enhance the decellularization effect.

[0019] Preferably, the divalent metal cations include one or more of magnesium ions and calcium ions, such as MgCl2, CaCl2, etc.

[0020] Preferably, the surfactant includes one or more of non-ionic surfactants (preferably Tween80, Tween20), cationic surfactants, and zwitterionic surfactants.

[0021] In the present invention, during the decellularization process, there is no strict restriction on the addition order of DNase, protease inhibitor, sodium deoxycholate, and divalent metal cations. They can be mixed with the surfactant to perform decellularization treatment on the extracellular matrix, or can be added separately to perform decellularization treatment on the extracellular matrix.

[0022] Preferably, one or more of sodium deoxycholate and divalent metal cations are added to the surfactant.

[0023] In a specific embodiment, step S2 includes:

[0024] S21. After soaking, wash with pure water, and perform decellularization treatment with a surfactant for 24 - 72 h. Sodium deoxycholate and divalent metal cations are added to the surfactant;

[0025] S22. After washing with pure water, treat with DNase and protease inhibitor for 4 - 24 h.

[0026] In another specific embodiment, step S2 includes: after soaking, wash with pure water, and mix the surfactant added with sodium deoxycholate and divalent metal cations, DNase, and protease inhibitor for decellularization treatment.

[0027] In a specific embodiment, step S3 includes: after washing with pure water, dry the surface moisture of the tissue, soak the decellularized tissue in MES buffer for 1 h, and then continue to soak it in MES buffer containing EDC and NHS for 2 - 8 h, and wash it repeatedly with Na2HPO4, NaCl, and deionized water to obtain a tissue with a fixed structure.

[0028] Preferably, the mass concentration of the NaOH solution is 4%-8%, and more preferably 6%.

[0029] In a specific embodiment, step S4 includes: soaking the tissue with a fixed structure in the NaOH solution, treating it at room temperature for 2-8 h, then washing it with a PBS buffer solution, and then washing it with sterile physiological saline after cleaning to obtain a soft tissue repair material.

[0030] In the present invention, the extracellular matrix is derived from humans or animals, including but not limited to allogeneic dermis and allogeneic blood vessels derived from humans.

[0031] In the present invention, the above steps are all carried out in a sterile environment, and no terminal sterilization process is required.

[0032] In the present invention, the cleaning and treatment processes in the above steps can select appropriate ultrasonic oscillation methods and low-temperature treatment methods according to the extracellular matrix.

[0033] In the second aspect of the present invention, a soft tissue repair material is provided, which is prepared by the above preparation method.

[0034] Beneficial effects:

[0035] (1) The present invention uses a composite amino acid phosphate buffer solution containing L-arginine, L-asparagine, and L-isoleucine to protect the structure and activity of the extracellular matrix before decellularization, and can, on the basis of completely removing cell membranes, nucleic acids, lipids, and cytoplasmic components, retain the structural integrity and active components of the extracellular matrix to the greatest extent, which is conducive to the extracellular matrix having an impact on cell proliferation and differentiation, and further achieving the purpose of tissue repair. It can be used to replace and repair body structures and has no antigenicity to the body.

[0036] (2) The present invention uses a surfactant for decellularization treatment, which has little damage to the tissue structure, and the prepared tissue material has a high cell survival rate.

[0037] (3) The present invention uses an MES buffer solution to activate the groups, and then uses an MES buffer solution containing EDC and NHS for cross-linking and fixation, which has low cytotoxicity to cells and better tissue biocompatibility.

[0038] (4) The present invention uses an NaOH solution for virus inactivation, which can effectively avoid the problem of excessive cytotoxicity of traditional virus inactivation methods and has no destructive effect on the tissue structure. Description of the Drawings

[0039] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0040] Figure 1 HE staining map of allogeneic dermis before decellularization provided in Example 1 of the present invention;

[0041] Figure 2 HE staining map of the tissue without structural fixation after decellularization obtained from the control group provided in Example 1 of the present invention;

[0042] Figure 3 HE staining map of the tissue without structural fixation after decellularization obtained from Experimental Group 1 provided in Example 1 of the present invention;

[0043] Figure 4 HE staining map of the tissue without structural fixation after decellularization obtained from Experimental Group 2 provided in Example 1 of the present invention;

[0044] Figure 5 HE staining map of the tissue without structural fixation after decellularization obtained from Experimental Group 3 provided in Example 1 of the present invention;

[0045] Figure 6 HE staining map of the tissue after decellularization, structural fixation and virus inactivation obtained from Experimental Group 3 provided in Example 1 of the present invention;

[0046] Figure 7 HE staining map of allogeneic blood vessel before decellularization provided in Example 2 of the present invention;

[0047] Figure 8 HE staining map of allogeneic blood vessel after decellularization provided in Example 2 of the present invention;

[0048] Figure 9 Normal fibroblast map provided in Example 3 of the present invention;

[0049] Figure 10 Co - culture result map of decellularized allogeneic dermal matrix and fibroblasts obtained by the preparation method with the publication number CN108404212A provided in Example 3 of the present invention;

[0050] Figure 11 Co - culture result map of decellularized allogeneic dermal matrix and fibroblasts obtained from Experimental Group 3 in Example 1 provided in Example 3 of the present invention;

[0051] Figure 12The figure of normal vascular endothelial cells provided in Example 4 of the present invention;

[0052] Figure 13 The figure of the co - culture result of decellularized blood vessels and endothelial cells provided in Example 4 of the present invention;

[0053] Figure 14 The figure of the cytotoxicity results of three virus inactivation methods provided in Example 5 of the present invention. Detailed implementation manners

[0054] It should be noted that the following detailed description is exemplary and is intended to provide further illustration of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present application belongs.

[0055] It should be noted that the terms used herein are only for describing specific implementation manners and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular forms also include the plural forms. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0056] The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of them. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts belong to the scope of protection of the present invention.

[0057] Example 1

[0058] (1) Protection of the extracellular matrix structure before decellularization

[0059] Allogeneic dermis was placed in the following buffers with a pH of 7.4, with an addition ratio of 1:5 (g:ml), soaked at 25 °C for 24 h, and placed in a constant - temperature shaker at 80 r / min.

[0060] Control group: Phosphate buffer solution;

[0061] Experimental group 1: 10% L - arginine phosphate buffer solution;

[0062] Experimental group 2: 8% L - arginine + 2% L - asparagine phosphate buffer solution;

[0063] Experimental group 3: 7% L - arginine + 2% L - asparagine + 1% L - isoleucine phosphate buffer solution.

[0064] (2) Decellularization process

[0065] A. Wash the processed tissue in (1) three times with pure water for 15 min each time, and then perform decellularization treatment with 0.05% Triton X-100, 0.1% Tween 80, 0.2% sodium deoxycholate, and 20 mM MgCl2 at 4°C for 24 h.

[0066] B. Ultrasonically wash with pure water five times for 5 min each time, and then treat with a mixed solution of DNase (1 U / ml) containing 20 mM EDTA and 1% SDS at room temperature for 8 h.

[0067] The HE staining image of allogeneic dermis before decellularization is shown in Figure 1 , after being protected by different buffer solutions and then undergoing the decellularization process, the HE staining image of the tissue without structural fixation is shown in Figures 2 - 5 , it can be seen from the figure that compared with the control group ( Figure 2 ) that uses phosphate buffer without added amino acids for structural and activity protection, experimental groups 1 to 3 can all play a certain role in protecting the dermal matrix with different amino acid phosphate buffers ( Figures 3 - 5 ), but there is a certain degree of damage to the tissue structure in experimental groups 1 and 2, while the tissue structure in experimental group 3 is not damaged, and compared with the tissue without decellularization treatment ( Figure 1 ), the tissue structure is basically the same, indicating that the composite amino acid phosphate buffer in experimental group 3 has a more significant protective effect on the tissue structure.

[0068] (3) Structural fixation

[0069] Wash five times with pure water for 30 min each time. After washing, dry the surface moisture of the tissue. Immerse the tissue in 0.05 M MES (pH 5.6) buffer solution for 60 min, and then immediately put it into MES buffer solution containing 5% EDC and 3% NHS and continue to soak for 4 h. Then wash three times repeatedly with 0.1 mol / L Na2HPO4, 4 mol / L NaCl, and deionized water.

[0070] (4) Virus inactivation process

[0071] Immerse in 6.0% NaOH solution and treat at room temperature for 4 h. Then wash three times with PBS buffer for 20 min each time. After washing, wash 2 - 3 times with sterile normal saline for 10 min each time.

[0072] The HE staining image of the tissue in experimental group 3 after structural fixation and virus inactivation is shown in Figure 6 , it can be seen from the figure that after structural fixation, using NaOH solution for virus inactivation has no destructive effect on the tissue structure.

[0073] Example 2

[0074] (1) Protection of the extracellular matrix structure before decellularization

[0075] Place the allogeneic blood vessel in a composite amino acid phosphate buffer with a pH of 7.4 containing L-arginine, L-asparagine, and L-isoleucine. The addition ratio is 1:8 (g:ml), and the mass ratio of L-arginine, L-asparagine, and L-isoleucine is 7:2:1, with a concentration of 10%. Immerse it at 25°C for 24 hours.

[0076] (2) Decellularization process

[0077] A. Ultrasonically clean the tissue treated in (1) three times with pure water for 5 minutes each time, and then perform decellularization treatment with 0.1% SDS, 0.1% Tween20, 20 mM CaCl2 at 4°C for 48 hours;

[0078] B. Ultrasonically clean with pure water three times for 5 minutes each time, and then treat with a mixed solution of 1% TritonX-100 and DNase (1 U / ml) at room temperature for 12 hours.

[0079] (3) Structure fixation

[0080] Wash with pure water three times, 15 minutes each time. Immerse the tissue in 0.05 mol / L MES buffer for 30 minutes, and then immediately place it in a MES buffer containing EDC and NHS. Gently shake and react at 37°C for 4 hours. After the reaction, wash successively with 0.1 mol / L Na2HPO4 (2 hours), 4 mol / L NaCl (4 times, 6 hours each time), and pure water repeatedly (3 times, 3 hours each time).

[0081] (4) Virus inactivation process

[0082] Immerse in 4.0% NaOH solution and treat at room temperature for 2 hours. Then wash with PBS buffer three times, 20 minutes each time. After washing, wash with sterile normal saline 2 - 3 times, 10 minutes each time.

[0083] The blood vessel is a tissue with a relatively soft texture and a thin wall. To ensure the integrity of its structure, the requirements for the decellularization process are also relatively high. The structure of the allogeneic blood vessel after structure protection and decellularization treatment by this method ( Figure 8 ) is basically the same as that of the non-decellularized blood vessel structure ( Figure 7 ).

[0084] Example 3

[0085] Normal fibroblasts are shown in Figure 9, the acellular allogeneic dermal matrix obtained by the preparation method with the publication number CN108404212A was co-cultured with fibroblasts, and the culture results are shown in Figure 10 , the acellular allogeneic dermal matrix obtained in experimental group 3 of Example 1 was co-cultured with fibroblasts, and the culture results are shown in Figure 11 , it can be seen from the figure that the tissue material obtained in experimental group 3 of Example 1 has better activity, and its cell viscosity and cell proliferation effect are more significant.

[0086] Example 4

[0087] Normal vascular endothelial cells are shown in Figure 12 , the acellular blood vessel was co-cultured with endothelial cells, and the culture results are shown in Figure 13 , it can be seen from the figure that no contact or growth inhibition of endothelial cells was found around the acellular blood vessel material. The endothelial cells around the material were in normal morphology, and a large number of endothelial cells were seen to grow in, indicating that the acellular blood vessel matrix prepared by this method has no cytotoxicity, good biocompatibility, high activity level, and is easy for cell growth and later proliferation.

[0088] Example 5

[0089] In this example, the method of experimental group 3 of Example 1 was adopted. After the structure was fixed, 3 samples were taken and immersed in 5 ml of indicator virus solution (PRV, PPV) respectively, mixed well and placed at room temperature for 60 min. Then, the peracetic acid-ethanol inactivation method, hydrogen peroxide inactivation method, and 6% NaOH inactivation were used respectively. The medicinal liquid was added for immersion treatment, and placed on an air bath constant temperature oscillating shaker. The set temperature was controlled at about 24 °C, and the oscillation was stopped after 4 h, and then continued to immerse until 20 h ended. (The ratio of the sample to the medicinal liquid is: 0.25 cm 2 : 1 ml).

[0090] Samples were taken at 0.5 h, 1 h, 2 h, 4 h, 8 h, and 20 h respectively. After sampling, 9 times the volume of neutralizer (2% glycine) was added. The samples were cut into pieces to make suspensions, centrifuged at 4000 r / min for 15 min, the supernatant was aspirated, and sterilized by filtration to detect the inactivation effect of PRV (pseudorabies virus) and PPV (porcine parvovirus) of the treated samples. The results are shown in Table 1-2, and the lesion results of the virus-inactivated samples during cell passage are shown in Table 3. Among them, the negative control was the sample after structure fixation, and the positive control was the sample immersed in the virus solution after structure fixation.

[0091] Table 1 Inactivation effect of PRV by different reagents at different times

[0092]

[0093] Note: "-" indicates that the virus titer could not be detected

[0094] Table 2 Inactivation Effect of Different Reagents on PPV at Different Times

[0095]

[0096] Note: "-" indicates that the virus titer could not be detected

[0097] Table 3 Lesion Results of Virus-Inactivated Samples during Cell Passage

[0098]

[0099]

[0100] As can be seen from Table 1, Table 2 and Table 3, the 6% NaOH solution can achieve the same virus inactivation effect as peracetic acid - ethanol and hydrogen peroxide. No detectable virus was found in all three groups, and the titers of PRV and PPV can be decreased by more than 4 Log. For samples with undetectable virus titers, the supernatant of the lowest dilution well was taken and inoculated into sensitive cells, and blind passage was carried out for 3 generations. No specific cytopathic effect was observed in the results of all three groups, indicating that 6% NaOH is effective in inactivating viruses. The cytotoxicity results of the three virus inactivation methods are shown in Figure 14 , and as can be seen from the figure, regarding the effects of the peracetic acid - ethanol inactivation method, hydrogen peroxide inactivation method, and 6% NaOH inactivation method on cell proliferation rate, both the peracetic acid - ethanol inactivation method and the hydrogen peroxide inactivation method have an impact on cell proliferation, and 6% NaOH inactivation can avoid the problem of excessive cytotoxicity caused by the residues of peracetic acid and hydrogen peroxide.

[0101] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a soft tissue repair material, characterized in that: The steps include: S1. Immersing the extracellular matrix in a composite amino acid phosphate buffer containing L-arginine, L-asparagine, and L-isoleucine to protect the structure and activity before decellularization; the mass ratio of L-arginine, L-asparagine, and L-isoleucine is (6-8):(1-3):(0.5-1.5); the mass concentration of the composite amino acids in the composite amino acid phosphate buffer is 5%-15%; S2, washing after soaking, and performing decellularization treatment with a surfactant to obtain a decellularized tissue; S3, after washing, the decellularized tissue is immersed in MES buffer, and then immersed in MES buffer containing EDC and NHS for fixation, and a tissue with a fixed structure is obtained after washing; S4. Place the structurally fixed tissue in a NaOH solution to inactivate the virus, and obtain the soft tissue repair material after washing.

2. The preparation method according to claim 1, characterized in that: Step S2 also includes: treating with DNase and / or protease inhibitors.

3. The preparation method according to claim 2, characterized in that: The protease inhibitors include: one or more of EDTA and NEM.

4. The preparation method according to claim 1, characterized in that: Step S2 also includes: treating with sodium deoxycholate and / or divalent metal cations.

5. The preparation method according to claim 4, characterized in that: The divalent metal cations include one or more of magnesium ions and calcium ions.

6. The preparation method according to claim 1, characterized in that: The surfactant includes: one or more of a nonionic surfactant, a cationic surfactant, and a zwitterionic surfactant.

7. The preparation method according to claim 1, characterized in that: The mass concentration of the NaOH solution is 4%-8%.

8. A soft tissue repair material, characterized in that: The method is prepared by the preparation method according to any one of claims 1 to 7.

Images