Method for preparing collagen membranes

Abstract

The application provides a collagen drying film forming method, which comprises the following steps: a preparation step, a dilution step, a defoaming step, a coating step, a pressing step, a drying step, a heat cross-linking step, a water covering step and a freeze drying step. The collagen drying film forming method provided by the application can improve the drying efficiency and product yield while retaining the integrity of the triple helix of collagen, and the dried collagen film has good mechanical properties and good flatness.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of collagen processing, in particular to a collagen membrane preparation method, and more particularly to a collagen membrane suitable for periodontal regeneration and dura repair and a preparation method thereof. BACKGROUND

[0002] Collagen is a natural fibrous protein mainly present in the skin, tendons and bones of animals. It is also the most abundant functional protein in mammals, accounting for 25-30% of the total protein. Collagen is widely used in the food industry, cosmetics, medical materials and other fields.

[0003] Connective tissue in the body is mainly composed of collagen, so collagen is often used as a membrane to replace skin, blood vessels, ligaments and other connective tissues, and has quite extensive applications in the field of medical materials.

[0004] Currently, the method for preparing collagen membranes is usually oven drying, which involves pouring collagen solution into a sterilized stainless steel tray and drying the tray in an oven. However, the collagen membranes prepared by this method often have uneven thickness, uneven surface and insufficient tensile strength, which affects the quality of the product.

[0005] Therefore, it is necessary to improve the uneven thickness and uneven surface of collagen membranes, and to enhance the tensile strength to make the collagen membranes more applicable. SUMMARY

[0006] In view of the above problems of the prior art, one of the purposes of the present application is to provide a collagen membrane preparation method that can improve the uneven thickness and uneven surface of collagen membranes, and can enhance the tensile strength of collagen membranes.

[0007] After the inventors' research, it was found that if double-layer and double-direction coating is performed by a dispensing machine, the vertical and horizontal tensile strength of the collagen membrane will be improved. By using a layer plate containing water-absorbing material and a pressing frame, the problems of uneven thickness and uneven surface of the membrane can be improved, and the defects in the preparation of traditional collagen membranes are solved. The collagen membrane prepared by the present application maintains the integrity of collagen stock helix, has high tensile strength, uniform thickness and smooth surface, and can be used to separate soft tissue and hard tissue and accelerate tissue healing.

[0008] The present application provides a collagen membrane preparation method, which comprises the following steps:

[0009] S4, coating the defoamed collagen raw material into the layer plate by a dispensing machine; and

[0010] S5, placing a pressing frame on the defoamed collagen raw material after step S4.

[0011] In some embodiments, the defoamed collagen raw material is obtained by diluting and defoaming a collagen raw material.

[0012] In some embodiments, the collagen raw material is at least one selected from the group consisting of collagen extracted from animal tissue and recombinant collagen.

[0013] In some embodiments, the collagen raw material can be in a solid state, a liquid state, or a combination of a solid state and a liquid state.

[0014] In some embodiments, the method further comprises S6: heat pump drying the defoamed collagen raw material to obtain a collagen dry film.

[0015] In some embodiments, the method further comprises S7: heat crosslinking the collagen dry film to obtain a collagen semi-finished film.

[0016] In some embodiments, the method further comprises S8: soaking the collagen semi-finished film in water to obtain a collagen flat film.

[0017] In some embodiments, the method further comprises S9: freeze-drying the collagen flat film to obtain a collagen film.

[0018] In some embodiments, the collagen raw material is at least one selected from the group consisting of collagen extracted from animal tissue and recombinant collagen.

[0019] In some embodiments, the collagen raw material can be in a solid state, a liquid state, or a combination of a solid state and a liquid state.

[0020] In some embodiments, the solvent used to dilute the collagen raw material is at least one selected from the group consisting of an organic solvent and an inorganic solvent.

[0021] In some embodiments, the solvent is an alcohol, such as a C1-C5 alcohol solvent. In some embodiments, the solvent is isopropyl alcohol. In some embodiments, the solvent is water.

[0022] In some embodiments, the concentration of the diluted collagen raw material is 10-50 mg / g.

[0023] In some embodiments, the coating step is a double-layer and double-direction coating using a dispensing machine. In some embodiments, the reason why the double-layer and double-direction coating using a dispensing machine is more desirable is as follows.

[0024] If the collagen raw material is merely poured into the layer plate and dried, the fiber direction of the collagen is randomly arranged, and thus the mechanical strength of the collagen membrane formed is poor. If the double-layer double-direction coating is performed using the dispensing machine as in the present application, the collagen fibers can be arranged in a specific direction, and thus the mechanical strength of the collagen membrane can be improved.

[0025] In some embodiments, the so-called double-layer double-direction coating refers to performing the first layer coating in the left-right direction using the dispensing machine, and performing the second layer coating in the up-down direction using the dispensing machine, i.e., perpendicular to the first layer coating direction.

[0026] In some embodiments, it is more ideal that the bottom of the layer plate contains water-absorbing materials. Furthermore, it is more ideal that the water-absorbing materials are porous inorganic materials or organic polymer materials. Furthermore, the inventors of the present application speculate that the reasons are as follows.

[0027] If there is no water-absorbing material at the bottom of the layer plate during the drying process of the collagen raw material, the water will flow during the drying process, and thus the thickness of the collagen membrane formed is not uniform.

[0028] If the water-absorbing material is placed at the bottom of the layer plate during the drying process of the collagen raw material, part of the water can be moved into the water-absorbing material, which can effectively reduce the flowing water, and thus the thickness of the collagen membrane formed in this way is more uniform.

[0029] In some embodiments, it is more ideal that the contact surface of the pressing frame and the collagen raw material is provided with a net. Furthermore, it is more ideal that the net is made of nylon material. The inventors of the present application speculate that the reasons are as follows.

[0030] During the drying process of the collagen raw material, the pressing frame and the net can apply a weight to the collagen raw material, which can effectively reduce the excessive movement of the collagen and water, and the nylon net is breathable and does not affect the evaporation of water vapor during the drying process, and thus the thickness of the collagen membrane formed is more uniform.

[0031] In some embodiments, it is more ideal that the temperature of the heat pump drying is controlled at 40-75°C.

[0032] In some embodiments, it is more ideal that the drying time of the heat pump drying is 5-30 hours.

[0033] In some embodiments, it is more ideal that the temperature of the heat crosslinking is controlled at 60-90°C.

[0034] In some embodiments, it is more ideal that the crosslinking time of the heat crosslinking is 8-20 hours. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 is a flow chart showing the method according to the embodiments of the present application.

[0036] Figure 2 Appearance of the collagen membrane prepared in Example 1 of the present application is shown.

[0037] Figure 3 Appearance of the collagen membrane prepared in Comparative Example 2 of the present application is shown. DETAILED DESCRIPTION

[0038] The following describes embodiments of the present application.

[0039] Referring to FIG. 1, which is a flow chart of a method for preparing a collagen membrane according to an embodiment of the present application. As shown, the method for preparing a collagen membrane of the present application comprises the following steps: providing a collagen raw material (S1); diluting the collagen (S2); defoaming the collagen raw material (S3); applying the collagen raw material to a tray by a dispenser (S4); placing a press frame on the collagen raw material (S5); heat pump drying the collagen raw material (S6); heat cross-linking the collagen membrane (S7); soaking the collagen membrane (S8); and drying the collagen membrane (S9). Figure 1

[0040] In step (S1), the collagen raw material provided can be collagen extracted from animal tissue or recombinant collagen. For example, collagen extracted from cowhide or cow tendon, or recombinant collagen produced using yeast as a genetic engineering carrier.

[0041] In an embodiment, the collagen raw material is in a liquid state, but can also be in a solid state or a solid-liquid mixed state. For example, it can be collagen solution in a solvent (liquid state), lyophilized sponge (solid state), or fibrous collagen paste (solid-liquid mixed state).

[0042] In step (S2), the solvent used to dilute the collagen raw material includes but is not limited to organic solvents, inorganic solvents, and combinations thereof. For example, isopropyl alcohol, water, and the like.

[0043] If water is used, the collagen raw material can be uniformly dispersed, which is desirable. If isopropyl alcohol is used, the collagen raw material can also be uniformly dispersed, and isopropyl alcohol has antibacterial function, which is even more desirable.

[0044] ​The concentration of the diluted collagen raw material, i.e. the collagen raw material is 10-50 mg / g per 1 g of solvent, is ideal. It is more ideal to be 15-45 mg / g, even more ideal to be 20-40 mg / g, and particularly ideal to be 25-30 mg / g. Since the collagen has a viscosity in the solvent, if the concentration is more than 50 mg / g, the viscosity is too high, which makes the operability poor. If the concentration is less than 10 mg / g, the mechanical strength of the film is insufficient due to the low concentration of collagen.

[0045] In step (S3), the conventional method can be used for defoaming, including but not limited to centrifugation, vacuum, or a combination thereof. It is more ideal to use a centrifugal vacuum defoaming machine to defoam, and the obtained raw material is uniform and bubble-free.

[0046] In step (S4), if the conventional method is used to pour the raw material into the layer plate for coating, a multi-layer structure cannot be achieved, and the tensile strength of the collagen film is poor. In contrast, if the dispensing machine is used for coating, multi-layer and multi-directional coating can be achieved, and the tensile strength of the collagen film is higher.

[0047] In addition, the dispensing machine of Pinxu Automation Equipment Co., Ltd. is used in the present application. The collagen raw material is poured into the cylinder, the glue gun is positioned at the origin, and the coating path is input into the controller to start the program.

[0048] If a double-layer and double-direction coating is used, the tensile strength is more ideal because the collagen fibers are vertically arranged and have a double-layer structure.

[0049] In step (S4), a layer plate containing water-absorbing material is used for coating, and diatomite plate is more ideal as the water-absorbing material. The diatomite plate has a large number of small pores, which can achieve rapid water absorption.

[0050] In step (S5), a pressing frame is placed above the collagen raw material after coating, and nylon mesh is more ideal on the pressing frame. The nylon mesh can fix the shape of the collagen raw material during drying, reduce the deformation after drying, and improve the problems of uneven film thickness and uneven surface.

[0051] In addition, the pressing frame of the present application is made of stainless steel, and its size is the same as that of the layer plate. It is placed above the collagen raw material after the coating is completed.

[0052] In step (S6), the temperature of the heat pump drying is preferably 40-75°C, more preferably 45-70°C, even more preferably 50-65°C, and particularly preferably 55-60°C. If the temperature is too low, the drying efficiency is poor. On the other hand, if the temperature is too high, there is a concern that the collagen protein will be denatured. The drying time is preferably 5-30 hours, more preferably 7-27 hours, even more preferably 10-25 hours, and particularly preferably 15-20 hours. If the time is too short, the collagen protein raw material cannot be completely dried. On the other hand, if the time is too long, only the burden of time cost is increased.

[0053] In step (S7), the temperature of the heat crosslinking is preferably 60-90°C, more preferably 63-87°C, even more preferably 66-84°C, and particularly preferably 73-77°C. If the temperature is too low, the heat crosslinking effect is not good. On the other hand, if the temperature is too high, there is a concern that the collagen protein will be denatured. The crosslinking time is preferably 8-20 hours, more preferably 10-18 hours, and even more preferably 13-15 hours. If the crosslinking time is too short, the collagen protein cannot be sufficiently crosslinked, and the excellent effect of the present application cannot be obtained. On the other hand, if the crosslinking time is too long, only the burden of time cost is increased.

[0054] Further, in the heat crosslinking of the present application, the collagen protein raw material is placed in a heat pump drying oven, and the heat pump drying oven is adjusted to the target temperature and the target time, and then left to stand.

[0055] In step (S8), water for injection is used as the solvent for the water coating step. The water coating step of step (S8) of the present application means that the collagen protein film is soaked in water. This step can reduce the wrinkles on the surface of the collagen protein film and increase the flatness of the collagen protein film.

[0056] In step (S9), the drying method can be heat pump drying, vacuum drying, or freeze drying. Among them, freeze drying is relatively ideal because it can preserve the shape of the collagen protein film while drying, and the process is performed at a relatively low temperature.

[0057] The present application will be specifically described below by way of examples, but these examples are only illustrative and do not mean that the present application is limited to these examples.

[0058] [Example 1]

[0059] A collagen protein drying film formation method, the specific steps are as follows:

[0060] 1) The water and fibrous collagen protein are uniformly mixed, and the concentration is 24 mg / g.

[0061] 2) Defoaming is performed by a centrifugal defoaming machine at a rotation speed of 2000 rpm and a vacuum degree of -90 kPa for 5 minutes.

[0062] 3) The defoamed collagen raw material is coated into a layer tray by a dispensing machine, and the bottom of the layer tray is provided with diatom mud board as water absorption material. A total of 2 layers are coated, and the directions are perpendicular to each other.

[0063] 4) A pressing frame is placed on the defoamed collagen raw material, and a nylon net is arranged on the contact surface between the pressing frame and the defoamed collagen raw material.

[0064] 6) The collagen dry film is subjected to heat crosslinking in the above-mentioned manner, wherein the temperature is controlled at 70°C, and the time is 16 hours, to obtain a collagen semi-finished film.

[0065] 7) The collagen semi-finished film is soaked in water for injection to perform water covering, so as to smooth the film surface.

[0066] 8) The collagen smooth film after water covering is placed in a freeze dryer to perform freeze drying, to obtain a collagen film.

[0067] [Example 2]

[0068] A collagen dry film forming method, and the specific steps are as follows:

[0069] 1) Water and fibrous collagen are uniformly mixed, and the concentration is 35 mg / g.

[0070] 2) Defoaming is performed by a centrifugal defoaming machine, and the defoaming is performed at a rotation speed of 2000 rpm and a vacuum degree of -90 kPa for 5 minutes.

[0071] 3) The defoamed collagen raw material is coated into a layer tray by a dispensing machine, and the bottom of the layer tray is provided with gypsum board as water absorption material. A total of 2 layers are coated, and the directions are perpendicular to each other.

[0072] 4) A pressing frame is placed on the defoamed collagen raw material, and a nylon net is arranged on the contact surface between the pressing frame and the defoamed collagen raw material.

[0073] 5) Heat pump drying is performed, and the temperature is controlled at 60°C, and the time is 12 hours. After this step is completed, the collagen raw material has become a film.

[0074] 6) The collagen dry film is subjected to heat crosslinking in the above-mentioned manner, wherein the temperature is controlled at 80°C, and the time is 12 hours, to obtain a collagen semi-finished film.

[0075] 7) The collagen film is soaked in water for injection to perform water covering, so as to smooth the film surface.

[0076] 8) The collagen membrane coated with water was placed in a freeze dryer to be freeze-dried, and a collagen membrane was obtained.

[0077] [Comparative Example 1]

[0078] A collagen membrane was prepared in the same manner as in Example 1, except that the collagen material was not coated using a dispenser, but was directly poured into the layer plate.

[0079] [Comparative Example 2]

[0080] A collagen membrane was prepared in the same manner as in Example 1, except that the layer plate did not use a water-absorbing material as a base plate, and no pressure frame was placed above the collagen material.

[0081] [Comparative Example 3]

[0082] A collagen membrane was prepared in the same manner as in Example 1, except that the temperature during heat pump drying was 80°C.

[0083] [Comparative Example 4]

[0084] A collagen membrane was prepared in the same manner as in Example 1, except that the dispenser coated in a single layer in one direction.

[0085] The average thickness, thickness standard deviation, participation helix structure, and tensile strength of the collagen membranes obtained in Example 1, Example 2, and Comparative Examples 1 to 4 were evaluated in the following manner, and the results are shown in Table 1 below.

[0086] <Average Thickness>

[0087] The average thickness was measured by measuring the thickness of 20 points on the collagen membrane using a thickness gauge and calculating the average value.

[0088] <Thickness Standard Deviation>

[0089] The thickness standard deviation was measured by measuring the thickness of 20 points on the collagen membrane using a thickness gauge and calculating the standard deviation.

[0090] <Participation Helix Structure>

[0091] The participation helix structure was determined by separating proteins using electrophoresis, and determining whether the participation helix structure was complete using α1 and α2 bands.

[0092] <Tensile Strength>

[0093] Tensile strength, is above Shanghai cooperation strength instrument manufacturing limited company's microcomputer control electronic universal material testing machine, cut into 5*15mm size of collagen membrane, with 1mm / min clamp moving rate to test, the maximum load is divided by the sample cross-sectional area to obtain the tensile strength.

[0094] Table 1

[0095]

[0096]

[0097] In addition, in order to more clearly understand the difference in flatness of the films of example 1 and comparative example 2, their appearance results are also shown in Figure 2 、 Figure 3 .

[0098] <Comparison with commercially available products>

[0099] The tensile strength of example 1 and commercially available products was determined in the above-mentioned manner, and the results are shown in table 2.

[0100] Among them, commercially available product 1 is Remaix manufactured by Matricel company, commercially available product 2 is Bio-Gide manufactured by Geistlich Biomaterials company, and commercially available product 3 is Ossix Plus manufactured by Datum Dental Biotech company.

[0101] Table 2

[0102]

[0103] From the results in table 1, it can be found that the collagen membrane prepared by meeting the process conditions of the present application has a lower thickness standard deviation than comparative example 2 which does not meet the process conditions of the present application, that is, it has better film flatness.

[0104] From the results in table 1, it can also be found that the collagen membrane prepared by meeting the process conditions of the present application has a complete collagen stock spiral structure compared with comparative example 3 which does not meet the process conditions of the present application. From the results in table 1 and table 2, it can be found that the collagen membrane prepared by meeting the process conditions of the present application has more sufficient physical strength compared with comparative example 1 which does not meet the process conditions of the present application, or compared with the conventional commercially available products.

[0105] In summary, the collagen membrane prepared according to the present application has the following advantages:

[0106] (1) Collagen is a heat-sensitive material, and if the temperature during the drying process is too high, the collagen helix structure will be destroyed, and the original effect will be lost. The drying temperature in the application is controlled within a specific range, which can avoid the destruction of the collagen helix structure and retain the integrity of the collagen.

[0107] (2) According to the collagen film prepared by the application, due to the action of the pressing frame and the water absorption material, the thickness of the dried collagen film is relatively uniform, and the surface unevenness and wrinkle phenomenon are reduced.

[0108] (3) According to the collagen film prepared by the application, the tensile strength of the collagen film is effectively improved by the action of the dispensing machine.

[0109]

Symbol Description

[0110] S1-S9: Step.

Claims

1. A method for preparing a collagen membrane, comprising the following steps: S4, applying the defoamed collagen raw material to the layer tray using a dispensing machine; S5, placing a pressing frame on the defoamed collagen raw material after step S4; S6: subjecting the defoamed collagen raw material to heat pump drying to obtain a collagen dry film; S7: thermally cross-linking the dried collagen film to obtain a semi-finished collagen film; S8: soaking the semi-finished collagen film in water to obtain a smooth collagen film; S9: freeze-drying the collagen flat film to obtain a collagen film; Wherein, the coating step is implemented by using a dispensing machine to perform double-layer bidirectional coating; The bottom of the layer tray contains a water-absorbing material; A net is stretched on the contact surface between the pressing frame and the defoamed collagen raw material; The heat pump drying temperature is 40~75 °C， described Drying time is 5 to 30 hours; described Thermal crosslinking temperature 60~90 °C， described Cross-linking time is 8~20 hours .

2. The preparation method according to claim 1, wherein The defoamed collagen raw material is obtained by diluting and degassing the collagen raw material.

3. The preparation method according to claim 2, wherein The collagen raw material is at least one selected from the group consisting of collagen extracted from animal tissues and recombinant collagen.

4. The preparation method according to claim 2, wherein The collagen raw material is in a solid state, a liquid state, or a combination of solid and liquid states.

5. The preparation method according to claim 2, wherein The solvent used in the dilution step is at least one selected from the group consisting of organic solvents and inorganic solvents; and / or the concentration of the diluted collagen raw material is 10-50 mg / g.

6. The preparation method according to claim 5, wherein The organic solvent is a C1-C5 alcohol solvent; the inorganic solvent is water.

7. The preparation method according to claim 5, wherein The organic solvent is isopropyl alcohol.

8. The preparation method according to claim 1, wherein The water-absorbing material is at least one selected from the group consisting of porous inorganic materials and organic polymer materials.

9. The preparation method according to claim 1, wherein The net is made of nylon material.

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