Preparation method of double-layer guided tissue regeneration membrane, double-layer guided tissue regeneration membrane and application of double-layer guided tissue regeneration membrane

Abstract

The invention provides a preparation method of a double-layer guided tissue regeneration membrane, the double-layer guided tissue regeneration membrane and application of the double-layer guided tissue regeneration membrane, and relates to the field of biomedical materials. The preparation method comprises the following steps: carrying out enzymolysis treatment on pretreated fish skin by combining an acid method with a restrictive enzymolysis method to obtain telopeptide-removed collagen; carrying out electrostatic spinning on the blended spinning solution of the telopeptide-removed collagen and the polylactic acid to form a loose layer, and carrying out electrostatic spinning on the loose layer by adopting the polylactic acid spinning solution to form a compact layer, so as to obtain a double-layer composite membrane; and crosslinking the double-layer composite membrane to obtain the double-layer guided tissue regeneration membrane. The double-layer guided tissue regeneration membrane has low immunogenicity, good mechanical properties and excellent histocompatibility, the compact layer can play a barrier role and prevent fibroblasts from occupying an osteogenic space, the loose surface can promote cell adhesion and growth and accelerate bone tissue repair, and the interface of the double-layer structure is tightly combined.

Description

Technical Field

[0001] The present invention relates to the field of biomedical materials, and in particular to a preparation method of a double-layer guided tissue regeneration membrane, a double-layer guided tissue regeneration membrane and applications thereof. Background Art

[0002] Collagen is widely used in biomedical materials because of its low immunogenicity and biocompatibility. At present, collagen used in biomedical materials mainly comes from mammals. Mammalian collagen has restrictions on use and risks of disease transmission such as mad cow disease and foot-and-mouth disease, while marine collagen does not have these problems. In addition, my country's aquatic product processing produces a large number of by-products such as fish skin and fish scales, which can be used as raw materials for extracting collagen, which not only reduces the waste of aquatic resources, but also realizes the high-value utilization of aquatic products. Therefore, it is very important to extract collagen from marine sources as a substitute for mammalian collagen.

[0003] Marine collagen has broad development prospects in the field of medical materials, and its safety, especially the control of immunogenicity, remains the focus. Although it is generally believed that collagen has low immunogenicity, collagen implantation still causes inflammation, allergies and other reactions in clinical practice. Collagen is a triple helix structure composed of three peptide chains. There is a non-helical region at both ends of the triple helix structure called the telopeptide. The telopeptide is the main antigen binding site of collagen and the main source of collagen immunogenicity. The removal of the telopeptide by pepsin hydrolysis to obtain de-telopeptide collagen can effectively reduce the immunogenicity of collagen.

[0004] Guided tissue regeneration membranes can play an important role in guided bone regeneration of dental implants. On the one hand, they guide bone regeneration in the tooth defect, and on the other hand, they block the growth of soft tissue around the tooth defect into the bone defect area to leave enough space for bone regeneration. Therefore, most of the guided tissue regeneration membranes reported in literature and patents are designed as double-layer or even multi-layer structures, generally including a dense layer and a loose layer. The dense layer plays a blocking role, and the loose layer plays a role in guiding bone regeneration.

[0005] Guided tissue regeneration membranes can be divided into absorbable membranes and non-absorbable membranes according to whether the membrane material is absorbed and degraded in the body. At present, the absorbable membranes that are widely used at home and abroad are mainly polymer membranes and collagen membranes. At present, the polymer membranes that have achieved good results include and The collagen membranes that have achieved good results include Extend, etc. Collagen membranes have good biocompatibility and functional activity, but their poor mechanical properties make their degradation time short. Although polymer membranes have good mechanical properties, they lack biological activity. Therefore, it is necessary to develop a new guided tissue regeneration membrane with low immunogenic collagen and polymer as raw materials that takes into account both biological activity and good mechanical properties.

[0006] CN110141687A discloses a gradient material for guiding the regeneration of periodontal hard and soft tissues and a preparation method thereof, comprising the following steps: S1. dispersing nano-hydroxyapatite in a solvent, ultrasonically dispersing for 1-2 hours, then adding fish collagen and polylactic acid-glycolic acid, shaking and evenly dispersing for 1.5-3 hours, and then ultrasonically dispersing for 0.5-1 hour to obtain a spinning solution; S2. stirring the spinning solution obtained in step S1 to volatilize the solvent to obtain a 3D printing ink; S3. preparing an electrospun fiber membrane layer from the spinning solution obtained in step S1 by an electrospinning method; S4. placing the electrospun fiber membrane layer obtained in step S3 on a biological 3D printer platform, and using the 3D printing ink obtained in step S2 to print on the electrospun fiber membrane layer through a biological 3D printer to construct a gradient material composited with an electrospun fiber membrane layer and a 3D printed scaffold layer.

[0007] It should be noted that in the subsequent use of the material, the drug can be slowly released from the fiber by diffusion and other means, while the drug loading and release of the 3D printed scaffold layer mainly depends on its designed pore structure and the possible addition of drug carriers, and the flexibility of controlling drug release is relatively poor. And for tissue engineering applications that require long-term slow release of drugs, the density of the 3D printed scaffold layer is poor. In addition, the 3D printed scaffold layer may require more complex designs and material combinations to achieve sustained release effects over days or even weeks.

[0008] In view of this, the present invention is proposed. Summary of the invention

[0009] The purpose of the present invention is to provide a preparation method of a double-layer guided tissue regeneration membrane, a double-layer guided tissue regeneration membrane and its application. The double-layer guided tissue regeneration membrane prepared by the method of the present invention has low immunogenicity, good mechanical properties and excellent tissue compatibility, the dense layer can act as a barrier to prevent fibroblasts from occupying the osteogenic space, the loose surface can promote cell adhesion and growth, accelerate bone tissue repair, and the double-layer structure interface is tightly combined and not easy to delaminate.

[0010] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0011] In a first aspect, the present invention provides a method for preparing a double-layer guided tissue regeneration membrane, the preparation method comprising the following steps:

[0012] The pre-treated fish skin is enzymatically treated by an acid method combined with a restricted enzymatic method to obtain atelocollagen;

[0013] A polylactic acid spinning solution and a blended spinning solution of atelocollagen and polylactic acid are respectively prepared; the blended spinning solution of atelocollagen and polylactic acid is subjected to electrostatic spinning to form a loose layer, and the polylactic acid spinning solution is used to perform electrostatic spinning on the loose layer to form a dense layer, thereby obtaining a double-layer composite membrane;

[0014] The double-layer composite membrane is cross-linked to obtain the double-layer guided tissue regeneration membrane.

[0015] Preferably, the pre-treatment includes defatting, removing impurity proteins, decolorizing and virus inactivation.

[0016] Preferably, the fish skin comprises any one of cod skin, tilapia skin, basa fish skin or black fish skin, or a combination of at least two thereof.

[0017] Preferably, the pre-treatment comprises the following steps:

[0018] The fish skin is washed and cut into pieces, and then added to a mixture of hydrogen peroxide and sodium hydroxide, and stirred at low temperature for the first time until the fish skin turns white; residual hydrogen peroxide is washed off with physiological saline, and the solution is filtered off; sodium hydroxide solution is added again, and stirred at low temperature for the second time, the solution is filtered off, and the mixture is washed with physiological saline until it is neutral.

[0019] Preferably, in the mixed solution of hydrogen peroxide and sodium hydroxide, the mass percentage of the hydrogen peroxide solution is 0.05-1%, and the concentration of the sodium hydroxide is 0.05-0.1 mol / L.

[0020] Preferably, the temperature of the first low-temperature stirring is 2 to 8° C., and the time of the first low-temperature stirring is 1 to 2 hours.

[0021] Preferably, the concentration of the sodium hydroxide solution is 0.05-0.1 mol / L.

[0022] Preferably, the temperature of the second low-temperature stirring is 2-8° C., and the time of the second low-temperature stirring is 6-8 h.

[0023] Preferably, the enzymatic treatment step comprises:

[0024] The pre-treated fish skin is mixed with acid, and protease is added, and the telopeptide is removed by stirring at low temperature to obtain a crude collagen extract; the crude collagen extract is sequentially subjected to salting out, standing and centrifugation to obtain a precipitate; the precipitate is redissolved and dialyzed to obtain a collagen solution, and the collagen solution is freeze-dried to obtain de-telopeptide collagen.

[0025] Preferably, the acid comprises any one of hydrochloric acid, acetic acid, sulfuric acid or phosphoric acid, or a combination of at least two thereof, preferably acetic acid.

[0026] Preferably, the mass volume ratio of the fish skin to the acid is 1:(40-60), and the concentration of the acid is 0.1-0.5 mol / L.

[0027] Preferably, the added amount of the protease is 400-1200 U / g fish skin.

[0028] Preferably, the temperature of the low-temperature stirring is 2 to 8° C., and the time of the low-temperature stirring is 24 to 72 hours.

[0029] Preferably, the salting out comprises: adding sodium chloride to the crude collagen extract for salting out.

[0030] Preferably, the final concentration of sodium chloride is 0.5-1.0 mol / L.

[0031] Preferably, the standing time is 6 to 18 hours.

[0032] Preferably, the centrifugal speed is 9000-10000 rpm, and the centrifugal time is 25-30 min.

[0033] Preferably, the re-dissolving step comprises: adding water at 5-20 g / mL according to the weight of the precipitate, or re-dissolving with 0.05-0.1 mol / L acetic acid according to the weight of the precipitate to obtain a re-dissolved collagen solution.

[0034] Preferably, the dialysis step comprises: transferring the reconstituted collagen solution into a dialysis bag with a molecular weight cutoff of 9000 to 60000 kDa, the dialysate being a 0.1 to 0.3 mol / L sodium dihydrogen phosphate solution, and when the pH of the dialysate is greater than 8, dialyzing with 0.1 to 0.5 mol / L acetic acid for 12 to 24 hours, replacing deionized water for dialysis desalination, and replacing the dialysate every 6 to 10 hours until the conductivity of the dialysate is less than 50 μs / cm, thereby obtaining a de-telopeptide collagen solution.

[0035] Preferably, the freeze-drying is carried out by gradient cooling freeze-drying, and the procedure of the gradient cooling freeze-drying includes: first freeze-drying at -25 to -15°C for 8 to 12 hours, then freeze-drying at -85 to -75°C for 8 to 12 hours, and finally vacuum drying at -90 to -50°C for 48 to 72 hours.

[0036] Preferably, the pore size of the loose layer is 11.71±4.41 μm, and the porosity of the loose layer is 48.54±1.33%.

[0037] Preferably, the pore size of the dense layer is 2.43±1.31 μm, and the porosity of the dense layer is 29.86±2.89%.

[0038] Preferably, the polylactic acid spinning solution comprises, by mass percentage, 3-10% polylactic acid and the remainder hexafluoroisopropanol.

[0039] Preferably, the blended spinning solution of the atelocollagen and polylactic acid comprises, by mass percentage, 2-6% polylactic acid, 2-6% atelocollagen, and the balance being hexafluoroisopropanol.

[0040] Preferably, the process parameters of the electrospinning of the loose layer include: positive voltage 6-10 kV; negative voltage 2-5 kV; receiving distance 15-20 cm; 2.5 mL syringe injection speed 1-1.5 mm / min.

[0041] Preferably, the process parameters of the electrospinning of the dense layer include: positive voltage 8 to 15 kV; negative voltage 1 to 4 kV; receiving distance 8 to 15 cm; and 2.5 mL syringe injection speed 1 to 2 mm / min.

[0042] Preferably, the cross-linking method comprises any one of glutaraldehyde vapor cross-linking, glutaraldehyde solution cross-linking or EDC / NHS cross-linking.

[0043] Preferably, the cross-linking step comprises:

[0044] The double-layer composite film is placed in a dryer with glutaraldehyde solution poured at the bottom, and steam cross-linking is performed by natural volatilization.

[0045] Preferably, the mass percentage of the glutaraldehyde solution is 5-10%.

[0046] Preferably, the steam cross-linking time is 4 to 12 hours.

[0047] Preferably, after the cross-linking is completed, a cleaning step is also included: the cross-linked double-layer composite membrane is cleaned in a 0.01-0.05 mol / L glycine solution for 20-40 min, then cleaned in a 0.05-0.1 mol / L disodium hydrogen phosphate solution for 6-18 h, and then cleaned in pure water for 6-18 h.

[0048] In a second aspect, the present invention provides a double-layer guided tissue regeneration membrane, wherein the double-layer guided tissue regeneration membrane is prepared by the preparation method of the double-layer guided tissue regeneration membrane as described in the first aspect.

[0049] In a third aspect, the present invention provides a use of the double-layer guided tissue regeneration membrane as described in the second aspect in preparing a guided bone regeneration material for dental implants.

[0050] Compared with the prior art, the present invention has the following beneficial effects:

[0051] (1) The raw materials used in the guided tissue regeneration membrane prepared by the present invention are poly-L-lactic acid (PLLA) and atelopeptide aquatic type I collagen, wherein PLLA is approved by the U.S. Food and Drug Administration (FDA) for clinical use, has good biocompatibility and its degradation products are non-toxic. Type I collagen is the main component of the extracellular matrix and has good biocompatibility itself, and marine collagen does not have the use restriction of the risk of disease transmission.

[0052] (2) The telopeptide is the main antigen binding site of collagen and the main source of collagen immunogenicity. Removing the telopeptide without destroying the structure of collagen itself effectively reduces the immunogenicity of collagen, retains the biological activity of collagen, improves the biocompatibility of the material, and reduces immunogenicity.

[0053] (3) The guided tissue regeneration membrane prepared by the present invention is an electrospun double-layer structure, consisting of a loose layer and a dense layer. The material obtained by electrospinning has a micro-nano structure, which is conducive to cell proliferation and adhesion. The double-layer fibers have different diameters and different densities. The dense layer fibers have a smaller pore size and low porosity, which act as a barrier to non-osteoblasts. The loose layer fibers have a larger pore size and high porosity, which allow osteoblasts to grow in, which is conducive to cell proliferation and diffusion, and is suitable for the guided bone regeneration scenario of dental implants.

[0054] (4) The guided tissue regeneration membrane prepared by the present invention and Erigen Compared with absorbable biorepair membranes, it has a better ability to promote cell proliferation, and because the electrospun material has a higher specific surface area, it is beneficial for cells to diffuse into the material, accelerate tissue repair, have good adhesion, and have better operability.

[0055] (5) The dense layer and the loose layer of the guided tissue regeneration membrane prepared by the present invention are tightly combined, which solves the problem of easy delamination of the electrospinning double-layer membrane. The dense layer and the loose layer have common components, and the voltage, receiving distance, environmental humidity and injection speed during the electrospinning of the two layers are optimized and adjusted to ensure that the double-layer membrane has a strong bonding force. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0057] Figure 1This is the mass spectrum of the atelopepeptide marine collagen extracted in Example 1.

[0058] Figure 2 The surface image and scanning electron microscope image of the double-layer guided tissue regeneration membrane; wherein, the surface image (A), the dense layer (B), the loose layer (C), and the cross section (D).

[0059] Figure 3 These are the results of the cytotoxicity test of the double-layer guided tissue regeneration membrane.

[0060] Figure 4 These are the test results for evaluating the effect of the double-layer guided tissue regeneration membrane on cells related to bone regeneration. DETAILED DESCRIPTION

[0061] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear, however, in the case of any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. In this application, unless otherwise stated, the use of "or" means "and / or". In addition, the use of the term "including" and other forms is non-limiting.

[0062] It should be noted that specific details are described in the following description to facilitate a full understanding of the present invention. However, the present invention can be implemented in a variety of other ways different from those described herein, and those skilled in the art can make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0063] The technical solution 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 the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0064] In a first aspect, the present invention provides a method for preparing a double-layer guided tissue regeneration membrane, the preparation method comprising the following steps:

[0065] The pre-treated fish skin is enzymatically treated by an acid method combined with a restricted enzymatic method to obtain atelocollagen;

[0066] A polylactic acid spinning solution and a blended spinning solution of atelocollagen and polylactic acid are respectively prepared; the blended spinning solution of atelocollagen and polylactic acid is subjected to electrostatic spinning to form a loose layer, and the polylactic acid spinning solution is used to perform electrostatic spinning on the loose layer to form a dense layer, thereby obtaining a double-layer composite membrane;

[0067] The double-layer composite membrane is cross-linked to obtain the double-layer guided tissue regeneration membrane.

[0068] In the present invention, the fish skin after pretreatment is first subjected to an acid method combined with a restricted enzymatic hydrolysis method to obtain low immunogenicity atelocollagen, which is then mixed with polylactic acid for electrospinning to obtain a loose layer, and the polylactic acid dense layer is obtained by continuing to spin on the loose layer by adjusting the solution and electrospinning parameters, and then modified by cross-linking to prepare the double-layer guided tissue regeneration membrane. The double-layer guided tissue regeneration membrane in the present invention has low immunogenicity, good mechanical properties and excellent tissue compatibility, the dense layer can act as a barrier to prevent fibroblasts from occupying the osteogenic space, the loose surface can promote cell adhesion and growth, accelerate bone tissue repair, and the interface of the double-layer structure is tightly combined and not easy to delaminate.

[0069] As an optional embodiment, the pre-treatment includes defatting, removing impurity proteins, decolorizing and virus inactivation.

[0070] In the present invention, the fish skin is firstly washed and chopped, and then placed in a mixture of hydrogen peroxide and sodium hydroxide for a first low-temperature stirring to remove glycoprotein and endotoxin on the surface of the fish skin; then the residual hydrogen peroxide is washed with physiological saline; and then the fish skin is placed in a sodium hydroxide solution for a second low-temperature stirring to remove fat, myofibrillar protein and other protein impurities, thereby completing the steps of defatting, removing impurity proteins, decolorizing and inactivating viruses.

[0071] As an optional embodiment, the fish skin includes any one of cod skin, tilapia skin, basa fish skin or black fish skin, or a combination of at least two of them.

[0072] As an optional implementation, the pre-processing includes the following steps:

[0073] The fish skin is washed and cut into pieces, and then added to a mixture of hydrogen peroxide and sodium hydroxide, and stirred at low temperature for the first time until the fish skin turns white; residual hydrogen peroxide is washed off with physiological saline, and the solution is filtered off; sodium hydroxide solution is added again, and stirred at low temperature for the second time, the solution is filtered off, and the mixture is washed with physiological saline until it is neutral.

[0074] As an optional embodiment, in the mixed solution of hydrogen peroxide and sodium hydroxide, the mass percentage of the hydrogen peroxide solution is 0.05-1%, for example, it can be 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, etc., and the concentration of sodium hydroxide is 0.05-0.1 mol / L, for example, it can be 0.05mol / L, 0.06mol / L, 0.07mol / L, 0.08mol / L, 0.09mol / L, 0.1mol / L, etc.

[0075] As an optional embodiment, the temperature of the first low-temperature stirring is 2-8°C, for example, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, etc., and the time of the first low-temperature stirring is 1-2h, for example, 1h, 1.2h, 1.4h, 1.5h, 1.6h, 1.8h, 2h, etc.

[0076] As an optional embodiment, the concentration of the sodium hydroxide solution is 0.05-0.1 mol / L, for example, it can be 0.05 mol / L, 0.06 mol / L, 0.07 mol / L, 0.08 mol / L, 0.09 mol / L, 0.1 mol / L, etc.

[0077] As an optional embodiment, the temperature of the second low-temperature stirring is 2-8°C, for example, it can be 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, etc., and the time of the second low-temperature stirring is 6-8h, for example, it can be 6h, 6.2h, 6.4h, 6.5h, 6.6h, 6.8h, 7h, 7.2h, 7.4h, 7.5h, 7.6h, 7.8h, 8h, etc.

[0078] As an optional implementation, the pre-processing specifically includes the following steps:

[0079] (a) Decolorization, glycoprotein and endotoxin removal: adding 0.5-2% hydrogen peroxide solution to the fish skin, and adding sodium hydroxide to a concentration of 0.05-0.1 mol / L, stirring continuously at a low temperature of 2-8° C. for 1-2 hours, filtering the solution to remove glycoprotein and endotoxin on the surface of the fish skin;

[0080] (b) removing residual hydrogen peroxide: washing the fish skin obtained by the treatment in step (a) with physiological saline to remove the residual hydrogen peroxide, and filtering the solution;

[0081] (c) defatting and deproteinizing: adding a 0.05-0.1 mol / L sodium hydroxide solution precooled at 2-8° C. to the fish skin obtained by the treatment in step (b), stirring continuously at low temperature of 2-8° C. for 6-8 h to remove fat, myofibrillar protein and other protein impurities, filtering the solution, and then washing the fish skin with physiological saline until neutral to obtain a pre-treated fish skin.

[0082] As an optional embodiment, the enzymatic treatment step includes:

[0083] The pre-treated fish skin is mixed with acid, and protease is added, and the telopeptide is removed by stirring at low temperature to obtain a crude collagen extract; the crude collagen extract is sequentially subjected to salting out, standing and centrifugation to obtain a precipitate; the precipitate is redissolved and dialyzed to obtain a collagen solution, and the collagen solution is freeze-dried to obtain de-telopeptide collagen.

[0084] As an optional embodiment, the acid includes any one of hydrochloric acid, acetic acid, sulfuric acid or phosphoric acid, or a combination of at least two thereof, preferably acetic acid.

[0085] As an optional embodiment, the mass volume ratio of the fish skin and the acid is 1:(40-60), for example, it can be 1:40, 1:42, 1:44, 1:45, 1:46, 1:48, 1:50, 1:52, 1:54, 1:55, 1:56, 1:58, 1:60, etc.

[0086] As an optional embodiment, the concentration of the acid is 0.1-0.5 mol / L, for example, it can be 0.1 mol / L, 0.15 mol / L, 0.2 mol / L, 0.25 mol / L, 0.3 mol / L, 0.35 mol / L, 0.4 mol / L, 0.45 mol / L, 0.5 mol / L, etc.

[0087] As an optional embodiment, the added amount of the protease is 400-1200U / g fish skin, for example, it can be 400U / g, 500U / g, 600U / g, 700U / g, 800U / g, 900U / g, 1000U / g, 1100U / g, 1200U / g, etc.

[0088] As an optional embodiment, the temperature of the low-temperature stirring is 2 to 8°C, for example, it can be 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, etc., and the time of the low-temperature stirring is 24 to 72h, for example, it can be 24h, 30h, 36h, 42h, 46h, 48h, 50h, 52h, 54h, 60h, 66h, 72h, etc.

[0089] As an optional embodiment, the enzymatic treatment step specifically includes:

[0090] 0.1-0.5 mol / L of acid is added to the fish skin after the previous treatment, and 400-1200 U / g of enzyme is added according to the weight of the fish skin. The terminal peptide is removed by stirring at a low temperature of 2-8° C., and after hydrolysis for 24-72 hours, a crude collagen extract is obtained.

[0091] As an optional embodiment, the salting-out includes: adding sodium chloride to the crude collagen extract for salting-out.

[0092] As an optional embodiment, the final concentration of sodium chloride is 0.5-1.0 mol / L, for example, it can be 0.5 mol / L, 0.6 mol / L, 0.7 mol / L, 0.8 mol / L, 0.9 mol / L, 1.0 mol / L, etc.

[0093] As an optional embodiment, the standing time is 6 to 18 hours, for example, it can be 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, etc.

[0094] As an optional embodiment, the centrifugal rotation speed is 9000-10000rpm, for example, it can be 9000rpm, 9100rpm, 9200rpm, 9300rpm, 9400rpm, 9500rpm, 9600rpm, 9700rpm, 9800rpm, 9900rpm, 10000rpm, etc., and the centrifugal time is 25-30min, for example, it can be 25min, 26min, 27min, 28min, 29min, 30min, etc.

[0095] As an optional embodiment, the precipitate is obtained by the following steps:

[0096] Slowly add finely ground NaCl to the crude collagen extract until the final concentration of NaCl is 0.5-1.0 mol / L. After salting out, let stand for 6-18 hours, collect the precipitate by centrifugation, and weigh it.

[0097] As an optional embodiment, the re-dissolving step comprises:

[0098] Add water at 5-20 g / mL according to the weight of the precipitate (for example, 5 g / mL, 6 g / mL, 8 g / mL, 10 g / mL, 12 g / mL, 14 g / mL, 15 g / mL, 16 g / mL, 18 g / mL, 20 g / mL, etc.) to obtain a reconstituted collagen solution.

[0099] As an optional embodiment, the re-dissolving step comprises:

[0100] The precipitate is re-dissolved with 0.05-0.1 mol / L acetic acid (for example, 0.05 mol / L, 0.06 mol / L, 0.07 mol / L, 0.08 mol / L, 0.09 mol / L, 0.1 mol / L, etc.) according to the weight of the precipitate to obtain a reconstituted collagen solution.

[0101] Preferably, the dialysis step comprises:

[0102] The reconstituted collagen solution was transferred into a dialysis bag with a molecular weight cutoff of 9000-60000 kDa. The dialysate was 0.1-0.3 mol / L disodium hydrogen phosphate solution. When the pH of the dialysate was greater than 8, it was dialyzed with 0.1-0.5 mol / L acetic acid for 12-24 hours. The deionized water was replaced for dialysis desalination. The dialysate was replaced every 6-10 hours until the conductivity of the dialysate was less than 50 μs / cm to obtain a de-telopeptide collagen solution.

[0103] In the present invention, 0.1-0.3 mol / L disodium hydrogen phosphate solution is first used for dialysis, and enzymatic hydrolysis is stopped by changing the pH. After the pH of the atelocollagen in the dialysis bag is greater than 8, 0.1-0.5 mol / L acetic acid and deionized water are used for dialysis desalination until the conductivity is below 50 μs / cm, thereby obtaining a high-purity (purity of 96-98%) atelocollagen solution.

[0104] As an optional embodiment, during the dialysis process, the retention molecular weight of the dialysis bag is 9000-60000 kDa, for example, it can be 9000 kDa, 10000 kDa, 12000 kDa, 15000 kDa, 18000 kDa, 20000 kDa, 22000 kDa, 25000 kDa, 28000 kDa, 30000 kDa, 32000 kDa, 35000 kDa, 38000 kDa, 40000 kDa, 42000 kDa, 45000 kDa, 48000 kDa, 50000 kDa, 52000 kDa, 55000 kDa, 60000 kDa, etc.

[0105] As an optional embodiment, during the dialysis process, the concentration of the dialysate disodium hydrogen phosphate solution is 0.1-0.3 mol / L, for example, it can be 0.1 mol / L, 0.12 mol / L, 0.14 mol / L, 0.15 mol / L, 0.16 mol / L, 0.18 mol / L, 0.2 mol / L, 0.22 mol / L, 0.24 mol / L, 0.25 mol / L, 0.26 mol / L, 0.28 mol / L, 0.3 mol / L, etc.

[0106] As an optional embodiment, during the dialysis process, the concentration of acetic acid in the dialysate is 0.1-0.5 mol / L, for example, it can be 0.1 mol / L, 0.15 mol / L, 0.2 mol / L, 0.25 mol / L, 0.3 mol / L, 0.35 mol / L, 0.4 mol / L, 0.45 mol / L, 0.5 mol / L, etc.

[0107] As an optional embodiment, during the dialysis process, the acetic acid dialysis time is 12 to 36 hours, for example, it can be 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 26 hours, 28 hours, 30 hours, 32 hours, 34 hours, 36 hours, etc.

[0108] As an optional embodiment, during the dialysis process, the dialysate in the desalination is replaced every 6 to 10 hours, for example, 6 hours, 6.5 hours, 7 hours, 7.5 hours, 8 hours, 8.5 hours, 9 hours, 9.5 hours, 10 hours, etc.

[0109] As an optional embodiment, the freeze-drying is performed by gradient cooling freeze-drying.

[0110] As an optional embodiment, the gradient cooling freeze-drying procedure includes: first freeze-drying at -25 to -15°C (for example, -25°C, -24°C, -22°C, -20°C, -18°C, -16°C, -15°C, etc.) for 8 to 12 hours (for example, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, etc.), and then cooling at -85 to -75°C (for example, -85°C, -84°C, -82°C, -80°C, -78°C, -76°C, -75°C, etc.) Freeze-dry for 8 to 12 hours (for example, it can be 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, etc.), and finally vacuum dry at -90 to -50°C (for example, it can be -90°C, -85°C, -80°C, -75°C, -70°C, -65°C, -60°C, -55°C, -50°C, etc.) for 48 to 72 hours (for example, it can be 48 hours, 50 hours, 52 hours, 54 hours, 56 hours, 58 hours, 60 hours, 62 hours, 64 hours, 66 hours, 68 hours, 70 hours, 72 hours, etc.).

[0111] As an optional implementation, the pore size of the loose layer is 11.71±4.41 μm.

[0112] As an optional embodiment, the porosity of the loose layer is 48.54±1.33%.

[0113] As an optional embodiment, the pore size of the dense layer is 2.43±1.31 μm.

[0114] As an optional embodiment, the porosity of the dense layer is 29.86±2.89%.

[0115] In the present invention, spinning on the basis of the loose layer to obtain a dense layer has the following advantages: ① Microstructure advantage: The fibers can be interwoven with each other, and the fibers can better combine with the loose layer because it is a continuous fiber deposition process, so the combination between the loose layer and the dense layer will be tighter. ② Mechanical property advantage: Due to the interweaving and stacking of the fibers, better flexibility and tensile strength can be provided. The 3D printed dense layer and the electrospun loose layer will have different mechanical properties due to their different structures, which will affect the mechanical properties of the overall material. ③ Cost and efficiency: The dense layer can be quickly prepared by adjusting the spinning parameters, with low time cost, and the 3D printing cost is higher than that of electrospinning. ④ Potential drug loading capacity: The dense layer of polylactic acid obtained by electrospinning has good drug loading capacity. Due to its porous fiber structure, drugs can be loaded in the fiber by various methods such as physical adsorption and embedding.

[0116] As an optional implementation, the polylactic acid spinning solution comprises, by mass percentage, 3-10% polylactic acid and the remainder hexafluoroisopropanol.

[0117] As an optional embodiment, based on the total mass of the polylactic acid spinning solution as 100%, the content of the polylactic acid is 3-10%, for example, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, etc.

[0118] As an optional embodiment, the step of configuring the polylactic acid spinning solution includes:

[0119] Polylactic acid is dissolved in hexafluoroisopropanol to prepare a polylactic acid spinning solution.

[0120] As an optional embodiment, the blended spinning solution of the atelocollagen and polylactic acid comprises, by mass percentage, 2-6% polylactic acid, 2-6% atelocollagen, and the balance being hexafluoroisopropanol.

[0121] As an optional embodiment, based on the total mass of the blended spinning solution of the de-terminated collagen and polylactic acid as 100%, the content of the polylactic acid is 2-6%, for example, it can be 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, etc.

[0122] As an optional embodiment, based on the total mass of the blended spinning solution of the atelocollagen and polylactic acid as 100%, the content of the atelocollagen is 2-6%, for example, it can be 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, etc.

[0123] As an optional embodiment, the steps of preparing the blended spinning solution of atelocollagen and polylactic acid include:

[0124] The polylactic acid and the atelocollagen are dissolved in hexafluoroisopropanol to prepare a blended spinning solution of the atelocollagen and the polylactic acid.

[0125] As an optional embodiment, the process parameters for electrospinning of the loose layer include: positive voltage 6 to 10 kV (for example, 6 kV, 7 kV, 8 kV, 9 kV, 10 kV, etc.); negative voltage 2 to 5 kV (for example, 2 kV, 2.5 kV, 3 kV, 3.5 kV, 4 kV, 4.5 kV, 5 kV, etc.); receiving distance 15 to 20 cm (for example, 15 cm, 16 cm, 17 cm, 18 cm, 19 cm, 20 cm, etc.); 2.5 mL syringe injection speed 1 to 1.5 mm / min (for example, 1 mm / min, 1.1 mm / min, 1.2 mm / min, 1.3 mm / min, 1.4 mm / min, 1.5 mm / min, etc.).

[0126] As an optional embodiment, the steps of preparing the loose layer specifically include:

[0127] Use a syringe to absorb the blended spinning solution of the atelocollagen and polylactic acid, fix it on a 2.5mL injection pusher, use a No. 23 needle, and connect high-voltage direct current to the needle. The injection speed is 1-1.5mm / min. Take tin foil and fix it on a flat receiver. Set the receiving distance between it and the syringe needle to 15-20cm, set the positive voltage to 6-10kV, and the negative voltage to 2-5kV, and perform electrostatic spinning to obtain the loose layer.

[0128] As an optional embodiment, the process parameters of the electrospinning of the dense layer include: positive voltage 8-15kV (for example, 8kV, 9kV, 10kV, 11kV, 12kV, 13kV, 14kV, 15kV, etc.); negative voltage 1-4kV (for example, 1kV, 1.5kV, 2kV, 2.5kV, 3kV, 3.5kV, 4kV, etc.); receiving distance 8-15cm (for example, 8cm, 9cm, 10cm, 11cm, 12cm, 13cm, 14cm, 15cm, etc.); 2.5mL syringe push speed 1-2mm / min (for example, 1mm / min, 1.2mm / min, 1.4mm / min, 1.5mm / min, 1.6mm / min, 1.8mm / min, 2mm / min, etc.).

[0129] As an optional embodiment, the steps of preparing the dense layer specifically include:

[0130] The polylactic acid electrospinning liquid was absorbed by a syringe and fixed on a 2.5 mL injection pusher. A No. 23 needle was selected and a high voltage direct current was connected to the needle. The specific parameters were positive voltage 8-15 kV, negative voltage 1-4 kV, receiving distance 8-15 cm, and injection speed 1-2 mm / min. The dense layer was directly received on the loose layer.

[0131] As an optional embodiment, the cross-linking method includes any one of glutaraldehyde vapor cross-linking, glutaraldehyde solution cross-linking or EDC / NHS cross-linking.

[0132] As an optional embodiment, the cross-linking step includes:

[0133] The double-layer composite film is placed in a dryer with glutaraldehyde solution poured at the bottom, and steam cross-linking is performed by natural volatilization.

[0134] As an optional embodiment, the mass percentage of the glutaraldehyde solution is 5-10%, for example, it can be 5%, 6%, 7%, 8%, 9%, 10%, etc.

[0135] As an optional embodiment, the steam cross-linking time is 4 to 12 hours, for example, it can be 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, etc.

[0136] As an optional embodiment, after the cross-linking is completed, a cleaning step is also included:

[0137] The cross-linked double-layer composite membrane is washed with 0.01-0.05 mol / L glycine solution for 20-40 min, then washed with 0.05-0.1 mol / L disodium hydrogen phosphate solution for 6-18 h, and then washed with pure water for 6-18 h.

[0138] As an optional embodiment, in the cleaning step, the concentration of the glycine solution is 0.01-0.05 mol / L, for example, it can be 0.01 mol / L, 0.02 mol / L, 0.03 mol / L, 0.04 mol / L, 0.05 mol / L, etc.

[0139] As an optional embodiment, in the cleaning step, the cleaning time using the glycine solution is 20 to 40 minutes, for example, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, etc.

[0140] As an optional embodiment, in the cleaning step, the concentration of the disodium hydrogen phosphate solution is 0.05-0.1 mol / L, for example, it can be 0.05 mol / L, 0.06 mol / L, 0.07 mol / L, 0.08 mol / L, 0.09 mol / L, 0.1 mol / L, etc.

[0141] As an optional embodiment, in the cleaning step, the cleaning time with disodium hydrogen phosphate solution is 6 to 18 hours, for example, it can be 6h, 7h, 8h, 9h, 10h, 11h, 12h, 13h, 14h, 15h, 16h, 17h, 18h, 19h, 20h, etc.

[0142] As an optional embodiment, in the cleaning step, the time for cleaning with pure water is 6 to 18 hours, for example, it can be 6h, 7h, 8h, 9h, 10h, 11h, 12h, 13h, 14h, 15h, 16h, 17h, 18h, 19h, 20h, etc.

[0143] In a second aspect, the present invention provides a double-layer guided tissue regeneration membrane, wherein the double-layer guided tissue regeneration membrane is prepared by the preparation method of the double-layer guided tissue regeneration membrane as described in the first aspect.

[0144] In a third aspect, the present invention provides a use of the double-layer guided tissue regeneration membrane as described in the second aspect in preparing a guided bone regeneration material for dental implants.

[0145] The present invention is further described below by way of examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or directly purchased from the market.

[0146] Example 1

[0147] This embodiment provides a method for extracting atelocollagen, the extraction method comprising the following steps:

[0148] S1. Pre-treatment:

[0149] The tilapia skin is washed and cut into pieces, a 0.5 wt% hydrogen peroxide solution (1:25, w / v) precooled at 4°C is added to the fish skin, and sodium hydroxide is added to a concentration of 0.08 mol / L, and the mixture is continuously stirred at 4°C for 2 hours, and the solution is filtered out; then, residual hydrogen peroxide is washed out with physiological saline, and the solution is filtered out; then, a 0.1 mol / L sodium hydroxide solution (1:25, w / v) precooled at 4°C is added, and the mixture is continuously stirred at 4°C for 6 hours, and the solution is filtered out, and the fish skin is washed with physiological saline until it is neutral, so as to obtain the pre-treated fish skin.

[0150] S2, Enzymatic Hydrolysis:

[0151] 0.5 mol / L acetic acid (1:50, w / v) was added to the fish skin after the forward treatment, and pepsin was added at 1000 U / g according to the weight of the fish skin. The fish skin was enzymatically hydrolyzed and the terminal peptide was removed at 4°C with stirring. After hydrolysis at 4°C for 48 hours, a crude collagen extract was obtained.

[0152] S3. After salting out, re-dissolve with acid and perform dialysis:

[0153] Slowly add finely ground NaCl to the crude collagen extract until the final concentration of NaCl is 0.9 mol / L. After salting out, stand at 4°C for 12 hours, centrifuge at 9500 rpm for 30 minutes, collect and weigh the precipitate; add pure water at 10 g / mL to the precipitate obtained after salting out, and stir at 4°C for redissolution; the redissolved collagen solution is transferred into a dialysis bag with a molecular weight cutoff of 50000 kDa, and the dialysate is 0.2 mol / L disodium hydrogen phosphate solution. When the pH of the dialysate is greater than 8, dialyze with 0.1 mol / L acetic acid for 24 hours, replace with deionized water for dialysis desalination, and replace the dialysate every 8 hours until the conductivity of the dialysate is less than 50 μs / cm, thereby obtaining a high-purity medical collagen solution with de-telopeptide;

[0154] S4. Freeze drying:

[0155] The high-purity atelocollagen medical collagen solution is first frozen at -20°C for 12 hours, then frozen at -80°C for 12 hours, and after gradient low-temperature freezing, is freeze-dried at -60°C in a vacuum for 48 hours to obtain the atelocollagen.

[0156] Example 2

[0157] This embodiment provides a method for extracting atelocollagen, the extraction method comprising the following steps:

[0158] S1. Pre-treatment:

[0159] Wash the tilapia skin and cut it into pieces. Add 1wt% hydrogen peroxide solution (1:25, w / v) precooled at 4°C to the skin, add sodium hydroxide to a concentration of 0.1mol / L, stir continuously at 4°C for 2h, filter out the solution, then wash the residual hydrogen peroxide with saline, and filter out the solution. Add 0.05mol / L sodium hydroxide solution (1:25, w / v) precooled at 4°C to the skin, stir continuously at 4°C for 8h, change the solution once, and then wash with saline until neutral.

[0160] S2, Enzymatic Hydrolysis:

[0161] 0.5 mol / L acetic acid (1:50, w / v) was added to the fish skin after the forward treatment, and pepsin was added at 1200 U / g according to the weight of the fish skin. The fish skin was enzymatically hydrolyzed and the terminal peptide was removed at 4°C with stirring. After hydrolysis at 4°C for 48 hours, a crude collagen extract was obtained.

[0162] S3. After salting out, re-dissolve with acid and perform dialysis:

[0163] Slowly add finely ground NaCl to the crude collagen extract until the final concentration of NaCl is 0.9 mol / L. After salting out, stand at 4°C for 12 hours, centrifuge at 9500 rpm for 30 minutes, collect and weigh the precipitate; add 0.05 mol / L acetic acid at 10 g / mL to the precipitate obtained after salting out, and stir at 4°C for redissolution; the redissolved collagen solution is transferred into a dialysis bag with a molecular weight cutoff of 50000 kDa, and the dialysate is 0.2 mol / L disodium hydrogen phosphate solution. When the pH of the dialysate is greater than 8, dialyze with 0.1 mol / L acetic acid for 24 hours, replace with deionized water for dialysis desalination, and replace the dialysate every 8 hours until the conductivity of the dialysate is less than 50 μs / cm, thereby obtaining a high-purity medical collagen solution with de-telopeptide;

[0164] S4. Freeze drying:

[0165] The high-purity atelocollagen medical collagen solution was first frozen at -20°C for 12 hours, then frozen at -80°C for 12 hours, and after gradient low-temperature freezing, it was freeze-dried at -60°C in a vacuum for 48 hours to obtain atelocollagen.

[0166] Example 3

[0167] This embodiment provides a method for extracting atelocollagen, which is different from Embodiment 1 only in that fish skin is replaced by cod skin, and other steps are exactly the same as Embodiment 1.

[0168] Example 4

[0169] This embodiment provides a method for extracting atelocollagen, which is different from Embodiment 1 only in that the acetic acid used in S2 enzymatic hydrolysis is replaced by hydrochloric acid, and the enzyme used in enzymatic hydrolysis is replaced by trypsin, and the other steps are completely consistent with Embodiment 1.

[0170] Example 5

[0171] This embodiment provides a method for extracting atelocollagen, which is different from Embodiment 1 only in that the acetic acid used in S2 enzymatic hydrolysis is replaced by phosphoric acid, and the enzyme used in enzymatic hydrolysis is replaced by bromelain, and the other steps are completely consistent with Embodiment 1.

[0172] Example 6

[0173] This embodiment provides a method for extracting atelocollagen, which is different from the embodiment 1 only in that the molecular weight cutoff of the S3 dialysis bag is replaced with 9000 kDa, and the other steps are completely consistent with the embodiment 1.

[0174] Example 7

[0175] This embodiment provides a method for extracting atelocollagen, which is different from Embodiment 1 only in that the molecular weight cutoff of the S3 dialysis bag is replaced with 60000 kDa, and the other steps are completely consistent with Embodiment 1.

[0176] Test Example 1

[0177] Atelocollagen Detection

[0178] Test sample: the atelocollagen provided in Examples 1 to 7.

[0179] Test method: The atelocollagen prepared in Example 1 was enzymatically hydrolyzed, and the peptide segments after enzymatic hydrolysis were compared with the database to test the effect of atelocollagen. The specific method was as follows: After pre-treatment of the atelocollagen sample, the peptide chain coverage was analyzed by nano-liquid chromatography tandem mass spectrometry (nano-HPLC-MS / MS, purchased from Thermo Fisher, USA) equipped with an online nanospray ion source.

[0180] Test results:

[0181] like Figure 1 As shown, the telopeptide collagen provided in Example 1 has no peptide coverage at both ends of the peptide chain, indicating that the telopeptides at both ends are completely removed. Further, after testing the telopeptide collagen provided in Examples 2 to 7, it was found that the samples provided in Examples 2 to 7 also had no peptide coverage at both ends of the peptide chain, indicating that the telopeptides at both ends were completely removed.

[0182] Example 8

[0183] This embodiment provides a method for preparing a double-layer guided tissue regeneration membrane, the preparation method comprising the following steps:

[0184] S1. Configuration of spinning solution:

[0185] Poly-L-lactic acid was dissolved in hexafluoroisopropanol to obtain a 5% poly-L-lactic acid electrospinning solution; the atelocollagen and poly-L-lactic acid provided in Example 1 were mixed and dissolved in hexafluoroisopropanol to obtain a blended spinning solution of 5% atelocollagen and 5% poly-lactic acid.

[0186] S2. Preparation of loose layer:

[0187] Use a 2.5mL syringe to absorb the blended spinning solution prepared by S1, fix it on the injection pusher, use a No. 23 needle, and connect high-voltage direct current to the needle. The injection speed is 1.5mm / min. Take tin foil and fix it on the flat receiver. Set the receiving distance between it and the syringe needle to 15cm, set the positive voltage to 10kV, and the negative voltage to 2kV, and perform electrospinning to obtain the loose layer.

[0188] S3. Preparation of dense layer:

[0189] Use a 2.5mL syringe to absorb the poly-L-lactic acid electrospinning solution prepared by S1, fix it on the injection pusher, use a 23-gauge needle, and connect high-voltage direct current to the needle. The injection speed is 1mm / min. Take the dense layer prepared by S2 and fix it on the flat receiver. Set the receiving distance from the syringe needle to 10cm, set the positive voltage to 8kV, and the negative voltage to 2kV. Perform electrospinning to obtain a dense layer to obtain a double-layer composite membrane.

[0190] S4, cross-linking:

[0191] The double-layer composite membrane obtained in S3 was cross-linked with glutaraldehyde vapor for 12 h, washed in 0.02 mol / L glycine solution for 30 min, then washed with 0.05 mol / L disodium hydrogen phosphate solution for 12 h, then washed with pure water for 12 h, and then vacuum dried for 72 h to obtain the double-layer guided tissue regeneration membrane.

[0192] Example 9

[0193] This embodiment provides a method for preparing a double-layer guided tissue regeneration membrane, the preparation method comprising the following steps:

[0194] S1. Configuration of spinning solution:

[0195] Poly-L-lactic acid was dissolved in hexafluoroisopropanol to obtain a 5% poly-L-lactic acid electrospinning solution; the atelocollagen and poly-L-lactic acid provided in Example 2 were mixed and dissolved in hexafluoroisopropanol to obtain a blended spinning solution of 5% atelocollagen and 5% poly-lactic acid.

[0196] S2. Preparation of loose layer:

[0197] Use a 2.5mL syringe to absorb the blended spinning solution prepared by S1, fix it on the injection pusher, use a No. 23 needle, and connect high-voltage direct current to the needle. The injection speed is 1.5mm / min. Take tin foil and fix it on the flat receiver. Set the receiving distance between it and the syringe needle to 15cm, set the positive voltage to 10kV, and the negative voltage to 2kV, and perform electrospinning to obtain the loose layer.

[0198] S3. Preparation of dense layer:

[0199] Use a 2.5mL syringe to absorb the poly-L-lactic acid electrospinning solution prepared by S1, fix it on the injection pusher, use a 23-gauge needle, and connect high-voltage direct current to the needle. The injection speed is 1mm / min. Take the dense layer prepared by S2 and fix it on the flat receiver. Set the receiving distance from the syringe needle to 10cm, set the positive voltage to 8kV, and the negative voltage to 2kV. Perform electrospinning to obtain a dense layer to obtain a double-layer composite membrane.

[0200] S4, cross-linking:

[0201] The double-layer composite membrane obtained in S3 was cross-linked with glutaraldehyde vapor for 12 h, washed in 0.02 mol / L glycine solution for 30 min, then washed with 0.05 mol / L disodium hydrogen phosphate solution for 12 h, then washed with pure water for 12 h, and then vacuum dried for 72 h to obtain the double-layer guided tissue regeneration membrane.

[0202] Example 10

[0203] This embodiment provides a method for preparing a double-layer guided tissue regeneration membrane, the preparation method comprising the following steps:

[0204] S1. Configuration of spinning solution:

[0205] Poly-L-lactic acid was dissolved in hexafluoroisopropanol to obtain a 5% poly-L-lactic acid electrospinning solution; the atelocollagen and poly-L-lactic acid provided in Example 2 were mixed and dissolved in hexafluoroisopropanol to obtain a blended spinning solution of 9% atelocollagen and 5% poly-lactic acid.

[0206] S2. Preparation of loose layer:

[0207] Use a 2.5mL syringe to absorb the blended spinning solution prepared by S1, fix it on the injection pusher, use a No. 23 needle, and connect high-voltage direct current to the needle. The injection speed is 1.5mm / min. Take tin foil and fix it on the flat receiver. Set the receiving distance between it and the syringe needle to 15cm, set the positive voltage to 10kV, and the negative voltage to 2kV, and perform electrospinning to obtain the loose layer.

[0208] S3. Preparation of dense layer:

[0209] Use a 2.5mL syringe to absorb the poly-L-lactic acid electrospinning solution prepared by S1, fix it on the injection pusher, use a 23-gauge needle, and connect high-voltage direct current to the needle. The injection speed is 1mm / min. Take the dense layer prepared by S2 and fix it on the flat receiver. Set the receiving distance from the syringe needle to 10cm, set the positive voltage to 8kV, and the negative voltage to 2kV. Perform electrospinning to obtain a dense layer to obtain a double-layer composite membrane.

[0210] S4, cross-linking:

[0211] The double-layer composite membrane obtained in S3 was cross-linked with glutaraldehyde vapor for 12 h, washed in 0.02 mol / L glycine solution for 30 min, then washed with 0.05 mol / L disodium hydrogen phosphate solution for 12 h, then washed with pure water for 12 h, and then vacuum dried for 72 h to obtain the double-layer guided tissue regeneration membrane.

[0212] Embodiment 11

[0213] This embodiment provides a method for preparing a double-layer guided tissue regeneration membrane, which is different from Example 8 only in that the atelocollagen provided in Example 1 is replaced with an equal mass of atelocollagen provided in Example 3, and the other steps are consistent with Example 8.

[0214] Example 12

[0215] This embodiment provides a method for preparing a double-layer guided tissue regeneration membrane, which is different from Example 8 only in that the atelocollagen provided in Example 1 is replaced with an equal mass of atelocollagen provided in Example 4, and the other steps are consistent with Example 8.

[0216] Embodiment 13

[0217] This embodiment provides a method for preparing a double-layer guided tissue regeneration membrane, which is different from Example 8 only in that the atelocollagen provided in Example 1 is replaced with an equal mass of atelocollagen provided in Example 5, and the other steps are consistent with Example 8.

[0218] Embodiment 14

[0219] This embodiment provides a method for preparing a double-layer guided tissue regeneration membrane, which is different from Example 8 only in that the atelocollagen provided in Example 1 is replaced with an equal mass of atelocollagen provided in Example 6, and the other steps are consistent with Example 8.

[0220] Embodiment 15

[0221] This embodiment provides a method for preparing a double-layer guided tissue regeneration membrane, which is different from Example 8 only in that the atelocollagen provided in Example 1 is replaced with an equal mass of atelocollagen provided in Example 7, and the other steps are consistent with Example 8.

[0222] Comparative Example 1

[0223] This comparative example provides a method for preparing a guided tissue regeneration membrane, which differs from Example 8 only in that the atelocollagen provided in Example 1 is replaced with fish collagen of equal mass, and the other steps are consistent with Example 8.

[0224] Comparative Example 2

[0225] This comparative example provides a method for preparing a guided tissue regeneration membrane, which is different from Example 8 only in that the S2 loose layer is not prepared, and the other steps are consistent with Example 8.

[0226] Comparative Example 3

[0227] This comparative example provides a method for preparing a guided tissue regeneration membrane, which is different from Example 8 only in that the S3 dense layer is not prepared, and the other steps are consistent with Example 8.

[0228] Comparative Example 4

[0229] This comparative example provides a method for preparing a guided tissue regeneration membrane, which is different from Example 8 only in that the S3 dense layer is prepared first, and then the S2 loose layer is prepared. The other steps are consistent with Example 8.

[0230] Comparative Example 5

[0231] This comparative example provides a method for preparing a guided tissue regeneration membrane, which is different from Example 8 only in that the S4 cross-linking step is not performed, and the other steps are consistent with Example 8.

[0232] Test Example 2

[0233] Test sample: the double-layer guided tissue regeneration membrane provided in Example 8.

[0234] Test method: Observe on a JSM-7500 scanning electron microscope produced by JEOL, Japan.

[0235] like Figure 2 As shown, the double-layer guided tissue regeneration membrane provided in Example 8 of the present invention is a white membrane ( Figure 2 A), with certain strength and toughness. It can be cut into different shapes and sizes according to requirements. The product was observed on a JSM-7500 scanning electron microscope produced by Japan JEOL Company, and it can be seen that the product's dense layer is relatively dense ( Figure 2 B), the loose layer is relatively loose ( Figure 2 C), the cross-sectional photo shows that the two layers are tightly bonded ( Figure 2 D).

[0236] Test Example 3

[0237] Mechanical properties testing

[0238] Test samples: the double-layer guided tissue regeneration membranes provided in Examples 8 to 15 and the guided tissue regeneration membranes provided in Comparative Examples 1 to 5.

[0239] Test method: The mechanical properties of the material were measured according to the national standard "GBT1040.4-2006 Determination of tensile properties of plastics Part 4: Test conditions for isotropic and orthotropic fiber reinforced composite materials". The specific method is: take the double-layer guided tissue regeneration membrane before and after cross-linking in Example 4, prepare it into a dumbbell shape of 5.0 cm × 0.5 cm according to the standard, soak it in physiological saline for 4 hours to completely wet it, and then use an electronic universal testing machine (WDW-1, purchased from Jinan Youchuang Testing Machine Co., Ltd.) to stretch it at a speed of 20 mm / min.

[0240] The specific test results are shown in Table 1:

[0241] Table 1

[0242]

[0243]

[0244] As shown in Table 1, the tensile strength of the double-layer guided tissue regeneration membrane of the present invention can reach above 1.5 MPa, cross-linking has a significant effect on improving the tensile strength of the double-layer guided tissue regeneration membrane, and the obtained double-layer guided tissue regeneration membrane has good mechanical properties.

[0245] Test Example 4

[0246] Cytotoxicity evaluation and hemolysis rate determination

[0247] Test samples: the double-layer guided tissue regeneration membranes provided in Examples 8 to 15 and the guided tissue regeneration membranes provided in Comparative Examples 1 to 5.

[0248] Test method: The cytotoxicity of the double-layer guided tissue regeneration membrane was determined according to the national standard "GBT16886.5-2017 Biological Evaluation of Medical Devices Part 5: In vitro Cytotoxicity Test". The hemolysis rate of the prepared double-layer guided tissue regeneration membrane was determined according to "GB-T14233.2-2005 Inspection Methods for Medical Infusion, Transfusion and Injection Equipment".

[0249] Test results such as Figure 3 As shown in Table 2:

[0250] Table 2

[0251]

[0252] As shown in Table 2, the cytotoxicity of the double-layer guided tissue regeneration membrane of the present invention is 77.828% to 100.710%, and the hemolysis test result is 1.491 to 4.307%. This shows that the double-layer guided tissue regeneration membrane of the present invention has low cytotoxicity and can be used for cell adhesion, growth and proliferation.

[0253] Test Example 5

[0254] Evaluation of cell growth

[0255] Test sample: the double-layer guided tissue regeneration membrane provided in Example 8.

[0256] Test method: The membrane was cut into discs with the same size as the pore size of the 48-well plate, and randomly divided into two groups with the dense layer facing up and the loose layer facing up. L929 fibroblasts and MC3T3 osteoblasts were inoculated respectively, and 300 μL complete culture medium was added to each well. As a product control, the treatment method was the same. The culture medium was replaced every day. On the third day, the culture medium was discarded, and each well was filled with formaldehyde fixative. Frozen sections were made to observe the growth of cells in the cross-section of the double-layer guided tissue regeneration membrane.

[0257] Test results such as Figure 4 shown.

[0258] like Figure 4 As shown in the figure, the specific growth of cells is as follows: the cells in the dense layer grow closely on the membrane and do not show any migration tendency, which is consistent with the Erigen Compared with other products, the number of cells growing is larger; the cells in the loose layer migrate within the membrane and grow within the membrane, which is different from Erigen. Compared with other products, the number of cells is larger and the degree of migration is higher.

[0259] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a double-layer guided tissue regeneration membrane, characterized in that: The preparation method comprises the following steps: The pre-treated fish skin is enzymatically treated by an acid method combined with a restricted enzymatic method to obtain atelocollagen; A polylactic acid spinning solution and a blended spinning solution of atelocollagen and polylactic acid are respectively prepared; the blended spinning solution of atelocollagen and polylactic acid is subjected to electrostatic spinning to form a loose layer, and the polylactic acid spinning solution is used to perform electrostatic spinning on the loose layer to form a dense layer, thereby obtaining a double-layer composite membrane; The double-layer composite membrane is cross-linked to obtain the double-layer guided tissue regeneration membrane.

2. The method for preparing a double-layer guided tissue regeneration membrane according to claim 1, characterized in that: The pre-treatment includes defatting, removing impurity proteins, decolorizing and inactivating viruses; Preferably, the fish skin comprises any one of cod skin, tilapia skin, basa fish skin or black fish skin, or a combination of at least two thereof; Preferably, the pre-treatment comprises the following steps: After cleaning the fish skin and chopping it up, add it to a mixed solution of hydrogen peroxide and sodium hydroxide, and stir at low temperature for the first time until the fish skin becomes white; wash the residual hydrogen peroxide with normal saline, and filter the solution; then add sodium hydroxide solution, stir at low temperature for the second time, filter the solution, and wash it to neutrality with normal saline; Preferably, in the mixture of hydrogen peroxide and sodium hydroxide, the mass percentage of hydrogen peroxide solution is 0.05-1%, and the concentration of sodium hydroxide is 0.05-0.1 mol / L; Preferably, the temperature of the first low-temperature stirring is 2 to 8° C., and the time of the first low-temperature stirring is 1 to 2 hours; Preferably, the concentration of the sodium hydroxide solution is 0.05-0.1 mol / L; Preferably, the temperature of the second low-temperature stirring is 2 to 8° C., and the time of the second low-temperature stirring is 6 to 8 hours.

3. The method for preparing a double-layer guided tissue regeneration membrane according to claim 1, characterized in that: The enzymatic treatment step comprises: The pre-treated fish skin is mixed with acid, and protease is added, and the telopeptide is removed by stirring at low temperature to obtain a crude collagen extract; the crude collagen extract is sequentially subjected to salting out, standing and centrifugation to obtain a precipitate; the precipitate is redissolved and dialyzed to obtain a collagen solution, and the collagen solution is freeze-dried to obtain de-telopeptide collagen; Preferably, the acid comprises any one of hydrochloric acid, acetic acid, sulfuric acid or phosphoric acid or a combination of at least two thereof, preferably acetic acid; Preferably, the mass volume ratio of the fish skin to the acid is 1:(40-60), and the concentration of the acid is 0.1-0.5 mol / L; Preferably, the protease comprises any one of acidic protease, trypsin, bromelain or pepsin or a combination of at least two thereof, preferably pepsin; Preferably, the added amount of the protease is 400-1200 U / g fish skin; Preferably, the low temperature stirring temperature is 2 to 8°C, and the low temperature stirring time is 24 to 72 hours; Preferably, the salting out comprises: adding sodium chloride to the crude collagen extract for salting out; Preferably, the final concentration of sodium chloride is 0.5-1.0 mol / L; Preferably, the standing time is 6 to 18 hours; Preferably, the centrifugal speed is 9000-10000 rpm, and the centrifugal time is 25-30 min; Preferably, the re-dissolving step comprises: Adding water at 5 to 20 g / mL according to the weight of the precipitate, or redissolving with 0.05 to 0.1 mol / L acetic acid according to the weight of the precipitate to obtain a redissolved collagen solution; Preferably, the dialysis step comprises: The reconstituted collagen solution was transferred into a dialysis bag with a molecular weight cutoff of 9000-60000 kDa, and the dialysate was 0.1-0.3 mol / L disodium hydrogen phosphate solution. When the pH of the dialysate was greater than 8, it was dialyzed with 0.1-0.5 mol / L acetic acid for 12-24 hours, and deionized water was replaced for dialysis desalination. The dialysate was replaced every 6-10 hours until the conductivity of the dialysate was less than 50 μs / cm, thereby obtaining a de-telopeptide collagen solution. Preferably, the freeze-drying is carried out by gradient cooling freeze-drying, and the procedure of the gradient cooling freeze-drying includes: first freeze-drying at -25 to -15°C for 8 to 12 hours, then freeze-drying at -85 to -75°C for 8 to 12 hours, and finally vacuum drying at -90 to -50°C for 48 to 72 hours.

4. The method for preparing a double-layer guided tissue regeneration membrane according to claim 1, characterized in that: The pore size of the loose layer is 11.71±4.41 μm, and the porosity of the loose layer is 48.54±1.33%; And / or, the pore size of the dense layer is 2.43±1.31 μm, and the porosity of the dense layer is 29.86±2.89%.

5. The method for preparing a double-layer guided tissue regeneration membrane according to claim 1, characterized in that: The polylactic acid spinning solution comprises, by weight percentage, 3-10% polylactic acid and the remainder hexafluoroisopropanol; And / or, the blended spinning solution of the atelocollagen and polylactic acid comprises, by mass percentage, 2-6% polylactic acid, 2-6% atelocollagen, and the remainder being hexafluoroisopropanol.

6. The method for preparing a double-layer guided tissue regeneration membrane according to claim 1, characterized in that: The process parameters of the electrospinning of the loose layer include: positive voltage 6 to 10 kV; negative voltage 2 to 5 kV; receiving distance 15 to 20 cm; 2.5 mL syringe injection speed 1 to 1.5 mm / min; And / or, the process parameters of the electrospinning of the dense layer include: positive voltage 8 to 15 kV; negative voltage 1 to 4 kV; receiving distance 8 to 15 cm; 2.5 mL syringe injection speed 1 to 2 mm / min.

7. The method for preparing a double-layer guided tissue regeneration membrane according to claim 1, characterized in that: The cross-linking method includes any one of glutaraldehyde vapor cross-linking, glutaraldehyde solution cross-linking or EDC / NHS cross-linking.

8. The method for preparing a double-layer guided tissue regeneration membrane according to claim 1 or 7, characterized in that: The cross-linking step comprises: The double-layer composite film is placed in a dryer with glutaraldehyde solution poured at the bottom, and steam cross-linked by natural volatilization; Preferably, the mass percentage of the glutaraldehyde solution is 5-10%; Preferably, the steam cross-linking time is 4 to 12 hours; Preferably, after the cross-linking is completed, a cleaning step is also included: the cross-linked double-layer composite membrane is cleaned in a 0.01-0.05 mol / L glycine solution for 20-40 min, then cleaned in a 0.05-0.1 mol / L disodium hydrogen phosphate solution for 6-18 h, and then cleaned in pure water for 6-18 h.

9. A double-layer guided tissue regeneration membrane, characterized in that: The double-layer guided tissue regeneration membrane is prepared by the preparation method of the double-layer guided tissue regeneration membrane according to any one of claims 1 to 8.

10. Use of the double-layer guided tissue regeneration membrane according to claim 9 in preparing guided bone regeneration materials for dental implants.

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