Soft tissue increment scaffold material as well as preparation method and application thereof

Abstract

The invention relates to a soft tissue incremental scaffold material and a preparation method and application thereof, and belongs to the technical field of medical materials.The preparation method of the soft tissue incremental scaffold material comprises the following steps that S1, a collagen solution and bioactive glass are evenly mixed, and a mixture is obtained; the mass ratio of collagen to the bioactive glass in the collagen solution is (4-19): 1; s2, carrying out freeze drying and crosslinking on the mixed solution to obtain a crosslinked material; s3, placing the cross-linking material in a hydrogen peroxide solution for modification treatment; the mass fraction of hydrogen peroxide in the hydrogen peroxide solution is 2-6%; and S4, repeating the step S3 until the sodium content and the crosslinking agent residue in the modified material meet the requirements, thereby obtaining the soft tissue incremental scaffold material. The soft tissue increment scaffold material prepared by the invention has proper pore structure and pH, lower cytotoxicity, higher recovery rate after compression and lower thickness increase rate after water absorption, and is beneficial to regeneration of soft tissues.

Description

Technical Field

[0001] The present invention relates to the technical field of medical materials, and particularly to a soft tissue augmentation scaffold material, a preparation method thereof, and an application thereof. Background Art

[0002] Healthy periodontal soft tissue is an important prerequisite for dental implantation, restoration, and orthodontic treatment. Insufficient amount of periodontal soft tissue can cause red and white aesthetic problems of natural teeth or implants, leading to diseases such as gingivitis and peri-implantitis. When soft tissue atrophy occurs or soft tissue is lacking during dental implant surgery, traditional surgical methods repair it through autologous tissue transplantation, such as subepithelial connective tissue of the palate. However, this method will cause a secondary operation, increase the patient's pain, and increase the operation time and blood loss. Summary of the Invention

[0003] In view of one or more technical problems existing in the prior art, the present invention provides a soft tissue augmentation scaffold material, a preparation method thereof, and an application thereof. The soft tissue augmentation scaffold material prepared by the present invention has a suitable pore structure and pH, low cytotoxicity, a high compression recovery rate, and a low water absorption thickness increase rate, which is beneficial to the regeneration of soft tissue and can be used for the repair of oral soft tissue.

[0004] The present invention provides a preparation method of a soft tissue augmentation scaffold material in a first aspect, and the preparation method includes the following steps:

[0005] S1. Mix a collagen solution and bioactive glass to obtain a mixture; the mass ratio of collagen to the bioactive glass in the collagen solution is 4-19:1;

[0006] S2. Freeze-dry and crosslink the mixture to obtain a crosslinked material;

[0007] S3. Place the crosslinked material in a hydrogen peroxide solution for modification treatment; the mass fraction of hydrogen peroxide in the hydrogen peroxide solution is 2-6%;

[0008] S4. Repeat step S3 until the sodium content and the residual amount of the crosslinking agent in the modified material meet the requirements, to obtain a soft tissue augmentation scaffold material.

[0009] Preferably, in step S2, before the freeze-drying, a standing step is further included.

[0010] Preferably, the temperature of the standing is 25-35°C, and the time of the standing is 30-60 min.

[0011] Preferably, the collagen solution is obtained by dissolving collagen in an acid solution; the mass ratio of collagen to the acid solution is 4-8:100; the concentration of the acid solution is 0.05-0.5 mol / L.

[0012] Preferably, the lyophilization includes pre-freezing at a temperature below -30°C for 2 to 5 hours, then evacuating to a pressure below 5 Pa, and performing lyophilization at -3 to 3°C for 24 to 30 hours, and finally gradually heating to room temperature.

[0013] Preferably, the crosslinking is to place the lyophilized material in a crosslinking agent solution and perform vacuum impregnation.

[0014] Preferably, the crosslinking agent solution is at least one of an EDC solution and a glutaraldehyde solution; and / or

[0015] The vacuum pressure of the vacuum impregnation is 0.04 to 0.06 MPa.

[0016] Preferably, the time of the modification treatment is 48 to 72 hours.

[0017] In a second aspect, the present invention provides a soft tissue augmentation scaffold material prepared by the preparation method described in the first aspect.

[0018] In a third aspect, the present invention provides an application of the soft tissue augmentation scaffold material of the second aspect for the repair of oral soft tissues.

[0019] Compared with the prior art, the present invention has at least the following beneficial effects:

[0020] The present invention first obtains a microporous structure suitable for cell growth and having compressive strength and elasticity by controlling the mass ratio of type I collagen and bioactive glass; then uses hydrogen peroxide to regulate the surface structure and composition of the crosslinked material. The surface roughness of the modified material increases, facilitating cell attachment; the sodium content and the residual amount of the crosslinking agent in the modified material decrease, effectively reducing the cytotoxicity of the material.

[0021] The soft tissue augmentation scaffold material prepared by the present invention has a suitable pore structure and pH, low cytotoxicity, a high recovery rate after compression, and a low thickness increase rate after water absorption, which is beneficial to the regeneration of soft tissues and can be used for the repair of oral soft tissues. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the following drawings are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0023] Figure 1 The preparation flow chart of the soft tissue augmentation scaffold material provided by the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0024] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be described clearly and completely below. Apparently, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0025] The present invention provides a preparation method of a soft tissue augmentation scaffold material in a first aspect. The preparation method includes the following steps:

[0026] S1. Mix a collagen solution and bioactive glass to obtain a mixture; the mass ratio of collagen to the bioactive glass in the collagen solution is 4 to 19:1 (for example, it can be 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, or 19:1);

[0027] S2. Subject the mixture to freeze-drying and cross-linking to obtain a cross-linked material;

[0028] S3. Place the cross-linked material in a hydrogen peroxide solution for modification treatment; the mass fraction of hydrogen peroxide in the hydrogen peroxide solution is 2 to 6% (for example, it can be 2%, 3%, 4%, 5%, or 6%);

[0029] S4. Repeat step S3 until the sodium content and the residual amount of the cross-linking agent in the modified material meet the requirements, thereby obtaining the soft tissue augmentation scaffold material.

[0030] The present invention first obtains a microporous structure suitable for cell growth and having compressive strength and elasticity by controlling the mass ratio of type I collagen to bioactive glass; then uses hydrogen peroxide to regulate the surface structure and composition of the cross-linked material. The surface roughness of the modified material increases, facilitating cell attachment; the sodium content and the residual amount of the cross-linking agent in the modified material decrease, effectively reducing the cytotoxicity of the material.

[0031] The sodium content (percentage of sodium atom number) in the soft tissue augmentation scaffold material prepared by the present invention is less than 3%, and the residual amount of the cross-linking agent is less than 10 ppm.

[0032] According to some preferred embodiments, in step S2, before the freeze-drying, a step of standing is further included.

[0033] Before freeze-drying, the mixture is allowed to stand, which can form a dense film on the surface. After cross-linking, it has a good barrier effect and can play a certain role in bacteria resistance, being suitable for some application scenarios where materials are exposed.

[0034] According to some preferred embodiments, the temperature for standing is 25 - 35°C (for example, it can be 25°C, 26°C, 27°C, 28°C, 29°C or 30°C), and the time for standing is 30 - 60 min (for example, it can be 30 min, 40 min, 50 min or 60 min).

[0035] According to some preferred embodiments, the collagen solution is obtained by dissolving collagen in an acid solution; the mass ratio of the collagen to the acid solution is 4 - 8:100; the concentration of the acid solution is 0.05 - 0.5 mol / L.

[0036] According to some specific embodiments, the acid solution is placed in a stirrer, stirring is started, the stirring speed of the stirrer is set to 80 - 120 revolutions per minute, and dry collagen is added to the acid solution and stirred until it is completely swollen.

[0037] According to some specific embodiments, bioactive glass is added to the collagen solution, the stirring speed is controlled at 300 - 500 revolutions per minute, and stirring is continued until a homogeneous white viscous liquid is obtained.

[0038] According to some preferred embodiments, the freeze-drying includes pre-freezing at below -30°C for 2 - 5 h, then evacuating to below 5 Pa, freeze-drying at -3 - 3°C for 24 - 30 h, and finally gradually warming up to room temperature.

[0039] According to some preferred embodiments, the cross-linking is to place the freeze-dried material in a cross-linking agent solution and perform vacuum impregnation.

[0040] According to some preferred embodiments, after the cross-linking, it further includes the steps of soaking in water and centrifuging, and repeating more than 5 times; the soaking time is 0.5 - 2 hours, the centrifuging speed is 1000 - 2000 revolutions per minute, and the time is 5 - 10 min.

[0041] According to some preferred embodiments, the cross-linking agent solution is at least one of an EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide) solution and a glutaraldehyde solution.

[0042] According to some preferred embodiments, the vacuum pressure for the vacuum impregnation is 0.04 - 0.06 MPa.

[0043] According to some preferred embodiments, the time for the modification treatment is 48 - 72 hours.

[0044] According to some preferred embodiments, after the modification treatment, it further includes the steps of drying, packaging, and sterilization; the drying is at least one of vacuum drying and freeze drying; the vacuum degree of the vacuum drying is 0 to 2×10 4 Pa, and the time is 12 to 24 hours. The packaging is to place the sample in a PETG tray, put it into an aluminum-plastic composite bag and seal it, and the sealing temperature is 160 to 200°C. The sterilization is carried out by irradiation sterilization, and the dose of the irradiation sterilization is 15 to 25 kGr.

[0045] The present invention provides a soft tissue augmentation scaffold material in a second aspect, which is prepared by the preparation method described in the first aspect.

[0046] The soft tissue augmentation scaffold material prepared by the present invention has a suitable pore structure and pH, low cytotoxicity, a high compression recovery rate, and a low thickness increase rate after water absorption, which is beneficial to the regeneration of soft tissues and can be used for the repair of oral soft tissues. The high compression recovery rate (the thickness remains stable after 50 times of compression and pressure relief, and the recovery rate can reach more than 95%) can better maintain volume stability and is beneficial to soft tissue regeneration; the low thickness increase rate after water absorption (less than 10%) can reduce the swelling caused after clinical use.

[0047] The present invention provides an application of the soft tissue augmentation scaffold material described in the second aspect in a third aspect, which is used for the repair of oral soft tissues.

[0048] In order to more clearly illustrate the technical solutions and advantages of the present invention, the present invention will be further described below with reference to embodiments. The present invention does not specifically limit the sources of various substances used in the embodiments, and they can be directly purchased or synthesized by oneself.

[0049] Example 1

[0050] Collagen dissolution and swelling: Place a 0.05 mol / L hydrochloric acid solution in a stirrer, start stirring, add collagen to the acid solution according to the mass ratio of collagen to hydrochloric acid solution of 4:100, and stir at 80 revolutions per minute for 12 hours until the collagen is completely swollen to obtain a collagen solution.

[0051] Mixing: Add 45S5 bioactive glass to the collagen solution according to the mass ratio of 45S5 bioactive glass to collagen of 5:95, and continue to stir at a high speed of 300 revolutions per minute for 0.5 hours to obtain a uniform white viscous liquid.

[0052] Molding: Pour the obtained white viscous liquid into a mold and flatten it.

[0053] Freeze drying: Place the white viscous liquid filled in the mold into a freeze dryer for freeze drying. The specific process is as follows:

[0054]

[0055] Crosslinking: Take the freeze-dried sample and place it in a container filled with EDC solution (the concentration of EDC is 0.5 wt%, and the solvent is 75 wt% ethanol solution). Then place the container in a vacuum chamber and evacuate it to 0.06 MPa to allow the sample to fully absorb the crosslinking agent solution and sink below the liquid surface. Crosslink at room temperature for 24 hours.

[0056] Washing: Take the crosslinked sample and soak it in water for 0.5 h. Then use a centrifuge to centrifuge it. After that, use the centrifuge again with a rotation speed of 1000 rpm for 5 min. Repeat the above steps 10 times.

[0057] Modification: Take the washed sample and soak it in a 2% hydrogen peroxide solution for 24 hours. Then use a centrifuge to centrifuge it with a rotation speed of 2000 rpm for 5 min. Repeat the above steps 3 times.

[0058] Drying: Take the modified sample and place it in a vacuum drying oven for vacuum drying. The vacuum degree of the vacuum drying oven is pumped to 2×10 4 Pa for 12 hours.

[0059] Packaging: Place the sample in a PETG tray, put it into an aluminum-plastic composite bag and seal it. Use a continuous sealing machine to seal it. The sealing temperature of the aluminum-plastic composite bag is 160 - 200 °C.

[0060] Sterilization: Perform irradiation sterilization with an irradiation sterilization dose of 15 kGr.

[0061] Example 2

[0062] Collagen dissolution and swelling: Place 0.5 mol / L hydrochloric acid solution in a stirrer and start stirring. Add collagen to the acid solution at a mass ratio of collagen to hydrochloric acid solution of 8:100. Stir at 120 rpm for 24 hours until the collagen is completely swollen to obtain a collagen solution.

[0063] Mixing: Add 45S5 bioactive glass to the collagen solution at a mass ratio of 45S5 bioactive glass to collagen of 20:80 and continue to stir at 500 rpm for 2 hours to form a homogeneous white viscous liquid.

[0064] Molding: Pour the white viscous liquid into a mold and flatten it.

[0065] Freeze-drying: The same as in Example 1.

[0066] Crosslinking: Place the freeze-dried sample in a container filled with glutaraldehyde solution (the concentration of glutaraldehyde is 0.1 wt%, and the solvent is ethanol solution with a concentration of 75 wt%). Then put the container into a vacuum chamber and evacuate it to 0.04 MPa, so that the sample fully absorbs the crosslinking agent solution and sinks below the liquid surface, and crosslink at room temperature for 12 hours.

[0067] Washing: Soak the crosslinked sample in water for 2 h, then use a centrifuge and centrifuge. After that, use a centrifuge again, with a rotation speed of 2000 rpm and a time of 5 min. Repeat the above steps 10 times.

[0068] Modification: Soak the washed sample in a hydrogen peroxide solution with a mass fraction of 3% for 24 hours, then use a centrifuge, with a rotation speed of 2000 rpm and a time of 5 min. Repeat the above steps 2 times.

[0069] Drying: Place the modified sample in a vacuum drying oven and dry it under vacuum. The vacuum degree of the vacuum drying oven is pumped to 2×10 4 Pa, and the time is 24 hours to obtain the soft tissue augmentation scaffold material.

[0070] Packaging: Place the sample in a PETG tray, put it into an aluminum-plastic composite bag and seal it. Use a continuous sealing machine to seal it, and the sealing temperature of the aluminum-plastic composite bag is 200 °C.

[0071] Sterilization: Perform irradiation sterilization, and the irradiation sterilization dose is 25 kGr.

[0072] Example 3

[0073] Collagen dissolution and swelling: Place 0.5 mol / L hydrochloric acid solution in a stirrer, start stirring, and add collagen to the acid solution according to the mass ratio of collagen to hydrochloric acid solution of 6:100. After stirring at 100 rpm for 18 hours, the collagen is completely swollen to obtain a collagen solution.

[0074] Mixing: Add 45S5 bioactive glass to the collagen solution according to the mass ratio of 45S5 bioactive glass to collagen of 10:90, and continue to stir at 400 rpm for 2 hours to form a homogeneous white viscous liquid.

[0075] Molding: Pour the white viscous liquid into a mold and level it.

[0076] Freeze-drying: The same as Example 1.

[0077] Crosslinking: Place the freeze-dried sample in a container filled with glutaraldehyde solution (the concentration of glutaraldehyde is 0.1 wt%, and the solvent is ethanol solution with a concentration of 75 wt%). Then put the container into a vacuum chamber and evacuate it to 0.04 MPa, so that the product fully absorbs the crosslinking agent solution and sinks below the liquid surface, and crosslink at room temperature for 12 hours.

[0078] Washing: Take the crosslinked sample and soak it in water for 2 h, then use a centrifuge to centrifuge, and then use a centrifuge to centrifuge at a speed of 2000 revolutions / min for 5 min. Repeat the above steps 10 times.

[0079] Modification: Take the washed sample and soak it in a hydrogen peroxide solution with a mass fraction of 6% for 24 hours, then use a centrifuge to centrifuge at a speed of 2000 revolutions / min for 5 min. Repeat the above steps 2 times.

[0080] Drying: Take the modified sample and place it in a vacuum drying oven for vacuum drying. The vacuum degree of the vacuum drying oven is pumped to 2×10 4 Pa for 24 hours to obtain a soft tissue augmentation scaffold material.

[0081] Packaging: Place the sample in a PETG tray, put it into an aluminum-plastic composite bag and seal it. Use a continuous sealing machine to seal it. The sealing temperature of the aluminum-plastic composite bag is 200°C.

[0082] Sterilization: Perform irradiation sterilization with an irradiation sterilization dose of 25 kGy.

[0083] Example 4

[0084] It is basically the same as Example 2, except that: during the molding process, after filling the white viscous liquid into the mold and spreading it flat, it is left standing at 35°C for 60 min.

[0085] Comparative Example 1

[0086] It is basically the same as Example 2, except that: during the mixing process, 45S5 bioactive glass is added to the collagen solution at a mass ratio of 45S5 bioactive glass to collagen of 1:1.

[0087] Comparative Example 2

[0088] It is basically the same as Example 2, except that: during the modification process, it is soaked in a hydrogen peroxide solution with a mass fraction of 1%.

[0089] Comparative Example 3

[0090] It is basically the same as Example 2, except that: hydrogen peroxide solution is not used for modification (i.e., there is no modification step).

[0091] Comparative Example 4

[0092] It is basically the same as Example 2, except that:

[0093] Freeze-drying: Put the white viscous liquid filled in the mold into a freeze-dryer for freeze-drying. The specific process is as follows:

[0094]

[0095]

[0096] Comparative Example 5

[0097] It is basically the same as Example 2, except that: after cleaning, the cleaned sample is soaked in water for 24 hours, and then centrifuged using a centrifuge at a rotational speed of 2000 revolutions / min for 5 minutes, and the above steps are repeated 2 times; then drying, packaging, and sterilization are carried out.

[0098] The performance data of the soft tissue augmentation stent materials prepared in the examples and comparative examples of the present invention are shown in Table 1, and the test methods for each performance are as follows:

[0099] Porosity and pore size: The median pore size and porosity are measured by mercury intrusion porosimetry.

[0100] Liquid absorbency: Tested according to the method in 6.3 of YY / T 1511-2017 "Collagen Sponge".

[0101] pH value: Tested according to the method of YY / T 1511-2017. Cut 0.2 g of the sample into small pieces, put it into a suitable container, add 12 mL of water, soak it in a sealed container at 37 ± 1 °C for 24 h, then gently pour out the liquid (gently squeeze with a glass rod if necessary) and mix well, and measure the pH of the solution with a pH meter.

[0102] Thickness increase rate after water absorption: Measure the thickness of the sample before and after water absorption. The thickness increase rate after water absorption = (thickness after water absorption - thickness before water absorption) / thickness before water absorption × 100%.

[0103] Compression recovery after rehydration: The situation that it can recover to the original thickness after 50 times of 20 kPa compression and pressure relief after rehydration; Compression recovery rate = thickness after compression and pressure relief / thickness after rehydration × 100%

[0104] Cytotoxicity: Tested by the MTT method according to GBT 16886.5-2017 "Biological evaluation of medical devices - Part 5: In vitro cytotoxicity test".

[0105] Sodium content: The inorganic components of the sample after calcination at 700 °C are tested and analyzed by SED-EDS to detect the contents of sodium and silicon.

[0106] Cross-linking agent content: Detected by referring to the method for determining the residual amount of glutaraldehyde in the 2020 edition of the Chinese Pharmacopoeia 3204 or the method for determining the residual amount of carbodiimide in 3206.

[0107] Bacterial barrier property in wet state: YY / T0471.5-2017 "Test methods for contact wound dressings - Part 5: Bacterial barrier property".

[0108] Table 1. Performance data of the soft tissue augmentation scaffold materials prepared in the examples and comparative examples of the present invention

[0109]

[0110] As can be seen from Table 1, by controlling the mixing ratio of collagen and bioactive glass, the freeze-drying process and the modification treatment, the present invention can obtain a soft tissue augmentation scaffold material with a pore structure suitable for tissue ingrowth (median pore size of 80-90 μm, porosity greater than 90%) and low cytotoxicity (sodium atom percentage less than 3%, residual amount of crosslinking agent less than 10 ppm). The thickness increase rate of the soft tissue augmentation scaffold material after water absorption (less than 10%) can reduce the swelling caused after clinical use; the soft tissue augmentation scaffold material has a high recovery rate after compression (the thickness remains stable after 50 times of compression and pressure relief, and the recovery rate can reach more than 95%) and can better maintain volume stability, which is beneficial to soft tissue regeneration.

[0111] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a soft tissue augmentation scaffold material, characterized in that: The preparation method comprises the following steps: S1. Mixing the collagen solution and the bioactive glass to obtain a mixture; the mass ratio of collagen in the collagen solution to the bioactive glass is 4 to 19:1; S2. freeze-drying and cross-linking the mixture to obtain a cross-linked material; S3. placing the cross-linked material in a hydrogen peroxide solution for modification; The mass fraction of hydrogen peroxide in the hydrogen peroxide solution is 2 to 6%; S4. Repeat step S3 until the sodium content and the residual amount of the cross-linking agent in the modified material meet the requirements, thereby obtaining a soft tissue augmentation scaffold material.

2. The preparation method according to claim 1, characterized in that: In step S2, before freeze-drying, a standing step is also included.

3. The preparation method according to claim 2, characterized in that: The standing temperature is 25-35° C., and the standing time is 30-60 min.

4. The preparation method according to claim 1, characterized in that: The collagen solution is obtained by dissolving collagen in an acid solution; the mass ratio of the collagen to the acid solution is 4-8:100; and the concentration of the acid solution is 0.05-0.5 mol / L.

5. The preparation method according to claim 1, characterized in that: The freeze drying comprises pre-freezing at a temperature below -30°C for 2 to 5 hours, then vacuuming to below 5 Pa, freeze drying at -3 to 3°C for 24 to 30 hours, and finally gradually heating to room temperature.

6. The preparation method according to claim 1, characterized in that: The cross-linking is performed by placing the freeze-dried material in a cross-linking agent solution and performing vacuum impregnation.

7. The preparation method according to claim 6, characterized in that: The cross-linking agent solution is at least one of an EDC solution and a glutaraldehyde solution; and / or The vacuum pressure of the vacuum impregnation is 0.04-0.06 MPa.

8. The preparation method according to claim 1, characterized in that: The modification treatment time is 48 to 72 hours.

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

10. An application of the soft tissue augmentation scaffold material according to claim 9, characterized in that: Used for repair of oral soft tissue.

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