KR101684790B1 - A porous membrane having different specific surface double layer for hard tissue regeneration and method for preparing the same - Google Patents
A porous membrane having different specific surface double layer for hard tissue regeneration and method for preparing the same Download PDFInfo
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- KR101684790B1 KR101684790B1 KR1020150095938A KR20150095938A KR101684790B1 KR 101684790 B1 KR101684790 B1 KR 101684790B1 KR 1020150095938 A KR1020150095938 A KR 1020150095938A KR 20150095938 A KR20150095938 A KR 20150095938A KR 101684790 B1 KR101684790 B1 KR 101684790B1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/56—Porous materials, e.g. foams or sponges
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
- A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
- A61L27/222—Gelatin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
- A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
- A61L27/24—Collagen
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/40—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material
- A61L27/44—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix
Abstract
Description
TECHNICAL FIELD The present invention relates to a porous membrane for hard tissue regeneration having a double-layer structure having different specific surface areas and a method for producing the porous membrane. More particularly, the present invention relates to a microporous porous support made of a natural polymer and electrospun into collagen nanofibers Porous microporous membrane having microporous and nano-sized porous double layer structure, and a method for producing the same.
In recent years, interest in implants has been growing as implant procedures become more popular. In order to successfully perform the implant, bone growth of the tooth, which can withstand the strength of the implant to be implanted, is an important factor. However, after the tooth defect, the growth of the soft tissue is faster than the growth of the bone tissue, which interferes with bone formation, which can be a big problem in implant placement. Therefore, guided bone regeneration (GBR) has been rapidly increasing to solve the above problem, and this technique provides a space in which the hard tissue can be sufficiently regenerated by preventing the invasion of the soft tissue using the biocompatible shielding film.
Currently, shielding membranes used for bone induction regeneration prevent invasion of soft tissues and promote growth of hard tissues, but they are not suitable for biocompatibility of soft tissues. However, since the amount and quality of the soft tissue for primary suture must be ensured after the bone induction regeneration procedure, there is a need for a shielding film considering both hard tissue and soft tissue growth.
Alginic acid sodium salt is known to be biodegradable and biocompatible because alginic acid, which is mainly contained in kelp and seaweed, binds with sodium and is in the form of salt. Gelatin is a natural polymer such as alginic acid. It exists in the form of a single helix with modified triple helix structure of collagen. It is widely used in biomedical engineering due to its ease of processing and excellent biocompatibility. In addition,
In the prior art of the present invention, a multi-layered absorbent periodontal regeneration regeneration membrane is known from Korean Patent No. 10-1260757, but it relates to a regenerated periodontal regeneration membrane for preventing rapid degradation by binding a hydrogel to a biodegradable shielding membrane will be. In addition, a bone regeneration inducing membrane and a manufacturing method thereof are known in Korean Patent No. 10-0946268, but the bone regeneration inducing membrane and bone regeneration improving membrane including the inner layer made of the outer layer made of the biodegradable polymer and the bipolar polymer and the calcium phosphate mixture And a regeneration film.
Therefore, none of the above-mentioned patent documents discloses a porous membrane for hard tissue regeneration capable of uniformly regulating growth of hard tissue and soft tissues.
Therefore, an object of the present invention is to provide a biodegradable layer for hard tissue regeneration, which is characterized in that a porous support is formed using sodium alginate, a natural polymer, and a block copolymer of gelatin, and a collagen nanofiber is electrospun on the support to form a double layer having different specific surface areas. And a method for manufacturing the porous membrane.
Another object of the present invention is to provide a biodegradable porous membrane for regenerating hard tissue, which is produced by the above-described method and is biodegradable and can regulate the regeneration speed of hard tissue and soft tissue constantly during bone induction regeneration.
(A) dissolving sodium alginate and gelatin in distilled water to prepare a sodium alginate gelatin solution; (b) adding a block copolymer PPG-PEG-PPG to the solution obtained in the above step and homogenizing to prepare a porous support mixture; (c) lyophilizing the mixed solution obtained in the above step to prepare a thin film; (d) crosslinking the thin film obtained in the above step with an aqueous solution of calcium chloride anhydride and ultrasonically washing to prepare a micro-sized porous support having a specific surface area of 2.0 m 2 / g or more; (e) dissolving the
The present invention provides a biodegradable porous membrane for biodegradable hard tissue regeneration capable of regulating the regeneration rate of hard tissues and soft tissues with biodegradability in bone induction regeneration by electrospinning nanofibers on a porous support to form different double surfaces having different specific surface areas It is effective.
1 is a schematic view illustrating a method of manufacturing a porous membrane for regenerating hard tissue according to the present invention.
2 is a photograph of the surface of the porous support of Production Examples 1 to 3 prepared according to the present invention observed at 500 magnification and 3000 magnification with a scanning electron microscope.
FIG. 3 is a photograph of the surface of the collagen nanofiber prepared in Preparation Example 3 prepared according to the present invention before and after cross-linking observed with a scanning electron microscope at 5000 magnifications. FIG.
FIG. 4 is a photograph of cross-sectional photographs of a porous membrane for hard tissue regeneration according to Production Example 3 prepared according to the present invention at a magnification of 200 and a magnification of 1000 at a scanning electron microscope.
FIG. 5 is a graph showing surface analyzes of porous supports and porous membranes prepared according to the present invention using ATR-FTIR.
6 is a graph showing the proliferation rate of osteoblasts and fibroblasts in the porous membrane for hard tissue regeneration of Production Example 3 prepared according to the present invention
FIG. 7 is a photograph showing the deposition of osteoblasts and fibroblasts in a porous membrane for hard tissue regeneration according to Production Example 3 prepared according to the present invention at 500 magnifications using an injection-transfer microscope.
8 is a graph showing osteogenic activity of osteoblasts in collagen nanofibers of Preparation Example 3 prepared according to the present invention.
A dual-layer porous membrane for regenerating hard tissue of the present invention comprises: (a) dissolving sodium alginate and gelatin in distilled water to prepare a sodium alginate gelatin solution; (b) adding a block copolymer PPG-PEG-PPG to the solution obtained in the above step and homogenizing to prepare a porous support mixture; (c) lyophilizing the mixed solution obtained in the above step to prepare a thin film; (d) crosslinking the thin film obtained in the above step with an aqueous solution of calcium chloride anhydride and ultrasonically washing to prepare a micro-sized porous support having a specific surface area of 2.0 m 2 / g or more; (e) dissolving the
According to the present invention, in addition to sodium alginate, at least one of ammonium alginate, calcium alginate, potassium alginate, and sodium alginate may be used in step (a).
In the step (a), in addition to distilled water, the solvent may be selected from the group consisting of methylene chloride, chloroform, acetone, anisole, ethyl acetate, methyl acetate, N-methyl-2-pyrrolidone, hexafluoroisopropanol, tetrahydrofuran, dimethylsulfoxide, 2-pyrollidone, citric acid, It is preferable to use an organic solvent such as triethyl citrate, ethyl lactate, propylene carbonate, benzyl alcohol, benzyl benzoate, Miglyol 810, isopropanol, ethanol ethanol, supercritical carbon dioxide, and acetonitrile may be used.
The formulation of the gelatin in step (a) and the collagen in step (e) may be any one or more of a sponge, a film, a membrane, and a powder, And collagen extracted from these collagen can be used.
The solvent of the gelatin of step (a) and the collagen of step (e) may be selected from the group consisting of propanol, butanol, acetone, trifluoroethylene (TFE), tri The solvent is selected from the group consisting of trifluoroacetic acid (TFA), tetra hydrofuran (THF), dichloromethane (DCM), dimethyl formamide (DMF), dimethyl acetamide (DMA) (S), dimethyl sulfoxide, hexane, benzene, acetic acid, and formic acid may be used.
The electrospinning method in the step (f) is a method of producing very small fine nanofibers by applying a high voltage (> 10 kV) to the polymer solution, which is several hundreds of nanometers. The fibers are light, flexible and have a large specific surface area, It is a method used in engineering.
All technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art unless otherwise defined herein. Various scientific dictionaries, including the terms contained herein, are well known and available in the art. As used herein, the singular forms include a plurality of objects unless the context clearly dictates otherwise. "Or" means "and / or ", unless otherwise stated, as used herein. Furthermore, the use of the terms " comprising ", " consisting " and "consisting"
Hereinafter, the present invention will be described in detail with reference to Examples and Production Examples. The following examples are for illustrative purposes only and are not intended to limit the invention to the following examples and experimental examples.
Example 1. The present invention relates to a porous membrane for hard tissue regeneration having a micro-nano-sized double layer structure
Disclosure material
PPG having a molecular weight of 2,700 and containing 40% by weight of alginic acid sodium salt having a viscosity of 15-20 cP, gelatin extracted from a powdery fish shell, and ethylene glycol (PEG) Hexafluoro-isopropanol, 25% glutaraldehyde solution and 0.1 M glycine buffer solution were mixed with Sigma-Aldrich (Sigma-Aldrich) Aldrich Co.), calcium chloride (Cacl 2 ) was purchased from Duksan Co., and
The micro-sized porous support of the present invention
25% by weight of sodium alginate and 75% by weight of gelatin were dissolved in distilled water to prepare a sodium alginate gelatin solution. In order to form pores in the solution obtained in the above step, 25% by weight of PPG-PEG-PPG as a block copolymer was added to the sodium alginate gelatin solution, stirred at room temperature for 24 hours, homogenized at 20,000 rpm using a homogenizer To prepare a mixture of sodium alginate gelatin-PPG-PEG-PPG. The mixture obtained in the above step is freeze-dried to prepare a thin film, which is treated with 30 ml of 0.1 M aqueous solution of calcium chloride anhydride and subjected to a crosslinking reaction for 8 hours. After completion of the crosslinking reaction, distilled water and PPG-PEG-PPG were removed through ultrasonic cleaning to prepare a micro-porous support having a specific surface area of 2.0 m 2 / g or more.
The present invention relates to a porous membrane for hard tissue regeneration having a micro-nano-sized double layer structure
The first type atelocollagen was dissolved in hexafluoroisopropanol to prepare a 7 wt% collagen solution. The collagen solution obtained in the above step was placed in a syringe and electrospun on one surface of the micro-scale porous support prepared in Example 1 at a voltage of 15 kV, a spinning distance of 15 cm, and a spinning rate of 0.5 mL / h. The porous support on which the above collagen nanofibers are radiated is placed in a desiccator containing 10 mL of 25% glutaraldehyde and crosslinked at 80 ° C for 8 minutes. After completion of the crosslinking reaction, the porous support on which the collagen nanofibers were radiated was reacted with 0.1 M of glycine buffer solution for 4 hours. After the reaction, the reaction product was washed with distilled water and then washed with distilled water to obtain a microsphere (specific surface area: 2.0 m 2 / ) And a nano (specific surface area of 9.0 m 2 / g or more, outside) size of a porous membrane for hard tissue regeneration having a bilayer structure.
Example 2. Characteristic Analysis of Porous Membrane for Hard-tissue Regeneration Having Micro-and Nanoscale Double-Layered Structure
Preparation Example 1 to Preparation Example 3 in which the weight ratio of sodium alginate to gelatin was different in preparing microporous porous support among the preparation methods of Example 1 was prepared as follows.
Production Example 1. A porous support prepared by mixing sodium alginate and gelatin in a weight ratio of 3: 1
The sodium alginate-gelatin-PPG-PEG-PPG mixture was prepared by mixing 0.75 g of sodium alginate, 0.25 g of gelatin and 0.25 g of PPG-PEG-PPG in 10 mL of distilled water, Unit porous support was prepared.
Production Example 2. A porous support prepared by mixing sodium alginate and gelatin in a weight ratio of 1: 1
The sodium alginate-gelatin-PPG-PEG-PPG mixture was prepared by mixing 0.5 g of sodium alginate, 0.5 g of gelatin and 0.25 g of PPG-PEG-PPG in 10 mL of distilled water, Unit porous support was prepared.
Preparation Example 3. Preparation of micro-nano-scale porous membrane having a bilayer structure of the present invention prepared by mixing sodium alginate and gelatin at a weight ratio of 1: 3
The sodium alginate-gelatin-PPG-PEG-PPG mixture was prepared by mixing 0.25 g of sodium alginate, 0.75 g of gelatin and 0.25 g of PPG-PEG-PPG in 10 mL of distilled water, Unit porous support was prepared. 4 mL of a 7
Surface analysis of the porous support of the present invention
The surfaces of the porous supports of Production Examples 1 to 3 were confirmed by scanning electron microscopy (SEM, S-4300, 15.0 kV).
As shown in FIG. 2, in Production Example 1, a small number of pores were observed on the surface of the support, but no definite shape was observed. The size of the support was in the range of 30-100 .mu.m, and various sizes of pores were observed. In the case of Production Example 2, as in Production Example 1, a film-like support having no porosity appeared. It was confirmed that as the weight ratio of sodium alginate was decreased and the weight ratio of gelatin was increased, the formation of pores was increased, but a support having no porous type was formed. On the other hand, in the case of Production Example 3, the support had a porosity of about 3 μm to 10 μm and exhibited an inverted opal structure with constant pore size and connectivity. The inverse opal structure is a structure suitable for cell growth and is a pore shape widely used in the field of tissue engineering.
The weight ratio of sodium alginate to gelatin was preferably less than that of gelatin when preparing the porous support of the present invention. Most preferably, the weight ratio of sodium alginate to gelatin was 1: 3 as in Preparation Example 3.
Analysis of collagen nanofiber surface of the present invention
The surface of the collagen nanofibers before and after crosslinking was confirmed on a porous support of the present invention prepared by the method of Production Example 3 using a scanning electron microscope.
As shown in FIG. 3, the collagen nanofibers before crosslinking had an average diameter of 350 nm and pores of about 3 to 10 μm were observed. However, after the crosslinking, the collagen nanofibers had an average diameter of 600 nm and formed a porous network of pores of about 1 μm Respectively.
Also, as shown in FIG. 4, after cross-linking and washing with glycine, the collagen nanofibers maintained their shape well and were uniformly spread on the porous support.
Total reflection measurement Fourier transform infrared spectroscopy (ATR-FTIR)
The surfaces of the porous support and the porous membrane prepared according to Preparation Examples 1 to 3 were analyzed using ATR-FTIR (ALPHA, Bruker optics, 400-4000 nm).
As shown in Fig. 5, specific peaks of the porous support and the porous membrane prepared according to Production Examples 1 to 3 were confirmed. In the case of sodium alginate to read 1590cm -1 and 14140cm of the sodium alginate by -1 0.1M aqueous solution of anhydrous calcium chloride in the vicinity of the carboxyl groups cross-linked with the peak, and the case of gelatin, sodium alginate in the vicinity of 1590cm -1 and the peak cross-linked Amide Ⅰ peaks were observed. In addition, it was confirmed that the gelatin was contained in the porous support without filtration by confirming the peaks according to Amide IV-VI of 400 cm -1 to 900 cm -1 , and the difference in absorbance according to the weight ratio of sodium alginate and gelatin was not large. Also it confirmed the Amide Ⅰ, Ⅱ, Ⅲ peak of the gelatin and
Example 3. Identification of Biocompatibility of Porous Membrane for Hard-tissue Regeneration Having Micro-and Nano-Double Layer Structure
The biocompatibility of microporous and hard tissue regenerable porous membranes having a nanostructure of nano unit was confirmed by cell experiments using osteoblasts and fibroblasts.
Cell culture and preparation of the porous membrane of the present invention
Fibroblasts (CCD-986sk, ATCC) were cultured in 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin supplemented with α-MEM in osteoblast (MC3T3-E1, ATCC) FBS) and 1% penicillin-streptomycin in D-MEM medium at 37 ° C and 5% CO 2 .
The porous membrane of the present invention prepared in Preparation Example 3 was sterilized by UV irradiation, sterilized with 75%, 50%, 25% ethanol for 2 minutes in sterile condition, and washed five times with phosphate buffered saline (PBS) After washing, cells were used for the experiment.
Identification of cell proliferation and adhesion
The cell proliferation and adhesion of osteoblasts and fibroblasts were confirmed by MTT assay in the porous membrane of Preparation Example 3 of the present invention. The porous membrane of the present invention was brought into close contact with the bottom of a 24-well plate, and a glass ring was placed on the porous membrane, and osteoblasts or fibroblasts were dispensed to allow the cells to adhere to only the surface of the porous membrane. In this step, the osteoblasts were distributed on the collagen nanofibers outside the porous membrane, and the fibroblasts were distributed on the sodium alginate gelatin porous support inside the porous membrane. The osteoblasts and fibroblasts were seeded at 5 × 10 5 cells / well and cultured at 37 ° C and 5% CO 2 for 1 , 3, 5 and 7 days. 200 μl of MTT reagent (5 mg / ml) And the reduction reaction was induced by incubation for 4 hours. After the reaction, the medium was carefully removed and a solution of DMSO was added to dissolve the MTT formazan formed in the above step. The absorbance of each well was measured at 570 nm absorbance using an ELISA microplate reader.
As shown in FIG. 6, the cell proliferation rate of the osteoblastic cells distributed on the outer side (collagen nanofibers) of the porous membrane of the present invention and the fibroblasts distributed on the inner side (sodium alginate gelatin) coincided with the cell cultures on
Also, as shown in FIG. 7, when the osteoblasts and fibroblasts deposited on the outer side (collagen nanofiber) and the inner side (sodium alginate gelatin) of the porous membrane of the present invention were observed with a scanning electron microscope, it was confirmed that the cells were stably deposited.
Identification of osteogenic activity of osteoblasts
Alkaline phosphatase (ALP) activity experiments were carried out in order to confirm the osteogenic activity of the osteoblasts in the collagen nanofibers of the porous membrane of the present invention. In the ALP activity test, the activity of ALP was measured by measuring the amount of the hydrolysis product p-nitrophenylphosphate using the principle that ALP acts as a catalyst for hydrolysis of p-nitrophenylphosphate. Specifically, 10X10 5 osteoblasts were seeded on the outer side of the porous membrane (collagen nanofibers) and medial side (sodium alginate gelatin) under the same conditions as the MTT assay described above, and cultured for 3, 5, 7 and 14 days. After the cell culture, the medium was removed, and osteoblast cells in the porous membrane were treated with 0.1% Triton-
As shown in FIG. 8, the ALP activity of the osteoblasts in the outer side (collagen nanofiber) and the inner side (sodium alginate gelatin) of the porous membrane of the present invention showed a distinct difference from the 5th day of cell culture, appear. These results show that osteoblasts have excellent osteogenic activity in collagen nanofibers.
As described above, the present invention provides a porous membrane for regeneration of hard tissue using a biodegradable natural polymer, and a nanofiber layer formed by electrospinning to have a double-layered structure having a different specific surface area. It is a very useful invention in the biomaterial industry because it has an excellent effect of regulating the regeneration speed of the soft tissue constantly.
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| CN107296978A (en) * | 2017-08-04 | 2017-10-27 | 北京化工大学常州先进材料研究院 | A kind of spongy hemostatic material in medical use of organism |
| CN108042854A (en) * | 2017-12-16 | 2018-05-18 | 诺迈尔（苏州）医学科技有限公司 | For the production technology of the gelatin fiber guide tissue regeneration film of Dental implant surgery |
| CN108956221A (en) * | 2018-04-10 | 2018-12-07 | 上海交通大学附属第人民医院松江分院 | A method of pathology film-making is carried out using sodium alginate |
| KR101947314B1 (en) * | 2018-05-08 | 2019-02-12 | 선우윤 | Toy manufacturing method using model frame in liquid and device thereof |
| CN109481735A (en) * | 2018-12-28 | 2019-03-19 | 北京大学口腔医学院 | A kind of multi-functional Absorbable membrane production method of room temperature shaping |
| KR20190062067A (en) | 2017-11-28 | 2019-06-05 | 재단법인 대구경북첨단의료산업진흥재단 | Method for fabricating biocompatible porous structured block and biocompatible porous structured block fabricated thereby |
| CN110227356A (en) * | 2019-04-29 | 2019-09-13 | 西南石油大学 | A kind of calcium alginate compounded filter membrane, and its preparation method and application |
| CN111330083A (en) * | 2018-12-18 | 2020-06-26 | 诺一迈尔（苏州）医学科技有限公司 | Barrier membrane for guided bone regeneration and preparation method thereof |
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| CN108042854A (en) * | 2017-12-16 | 2018-05-18 | 诺迈尔（苏州）医学科技有限公司 | For the production technology of the gelatin fiber guide tissue regeneration film of Dental implant surgery |
| CN108956221A (en) * | 2018-04-10 | 2018-12-07 | 上海交通大学附属第人民医院松江分院 | A method of pathology film-making is carried out using sodium alginate |
| KR101947314B1 (en) * | 2018-05-08 | 2019-02-12 | 선우윤 | Toy manufacturing method using model frame in liquid and device thereof |
| CN111330083A (en) * | 2018-12-18 | 2020-06-26 | 诺一迈尔（苏州）医学科技有限公司 | Barrier membrane for guided bone regeneration and preparation method thereof |
| CN111330083B (en) * | 2018-12-18 | 2022-01-11 | 诺一迈尔（苏州）医学科技有限公司 | Barrier membrane for guided bone regeneration and preparation method thereof |
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