JP2022544735A - Construction method and application of nanocellulose-based cell growth factor sustained release anisotropic stent - Google Patents

Abstract

Kind Code: A1 The present invention relates to the field of biomedical material technology, and more particularly to the construction method and application of nanocellulose-based cell growth factor sustained release anisotropic stents. The method includes the steps of (1) producing pulp fibers, (2) washing and filtering the pulp fibers, and (3) using the cleanly washed pulp fibers to prepare a high-purity nanocellulose hydrogel. (4) preparing a nanocellulose film using the nanocellulose hydrogel; (5) immersing the nanocellulose film in a solution of fibroblast growth factor and complex cellulase; (6) synchronously forming an anisotropic three-dimensional stent by in situ adsorption and swelling; washing, cutting into different shapes and sizes as required, and freeze-drying to fabricate nanocellulose-based cell growth factor sustained release anisotropic stents. [Selection drawing] Fig. 1

Description

The present invention relates to the field of biomedical material technology, specifically to the field of cell growth factor sustained release stents.

Cell growth factors are active protein or peptide substances that can stimulate cell proliferation, promote cell differentiation and maturation, and regulate various activities and functions of cells. For example, basic fibroblast growth factors (bFGFs) can promote fibroblast proliferation and repair damaged cells and tissues, and platelet-derived growth factor (PDGF) plays an important role in angiogenesis. Transforming growth factors (TGFs) have biological effects that promote embryonic development, wound healing, chemotaxis and cell cycle control. Cell growth factors show enormous potential in the clinical treatment of diseases such as tissue engineering, wound healing, angiogenesis, and the nervous system. However, free cell growth factors have a short half-life of biological activity in the body, are rapidly metabolized, and are easily decomposed by enzymes. Multiple administrations and additions are required to achieve the purpose, which increases the patient's pain and economic burden. Therefore, there is a strong need in both clinical therapy and scientific research to develop products that maintain growth factor activity and that are capable of being targeted and released in a controlled manner.

Sustained release of cell growth factors can be used for scientific research and clinical treatment by complexing various biologically active cell growth factors with biocompatible materials or carriers while maintaining the activity of cell growth factors. Manufacture a product whose release rate can be controlled.

For example, patent CN105288749A mixes growth factors with biomaterials such as chitosan, polylactic acid, gelatin and freeze-dries to make biomaterial stents that release growth factors; A cell-free matrix capable of stably releasing a growth factor for a long period of time is prepared by emulsifying and freeze-drying a cell-free matrix and a degradable hydrophobic polymer such as polyglycolide lactide or polycaprolactone, and For example, patent CN101279104B discloses that epidermal growth factor and basic fibroblast growth factor are combined with collagen derived from animal skin or connective tissue, freeze-dried, and then made into a collagen sponge containing the growth factors for chronic healing. Used to promote healing of difficult wounds.

However, in the materials produced by the existing reports and related inventions, the cell factors and the matrix material are easily bound, and the matrix material is difficult to decompose or its degradability cannot be controlled, so the release rate can be precisely controlled. However, rapid or excessive release may still result in loss of activity and biological safety issues.

In addition, animal-derived matrix materials have defects and deficiencies such as immune response and batch-to-batch variation, which limit the application range and therapeutic efficacy of cell growth factors, causing many inconveniences in clinical applications and scientific research. be done.

Nanocellulose is prone to form hydrogels during fabrication, and its highly hydrated three-dimensional network structure can well simulate the extracellular matrix in human organ tissues, making it suitable for cell adhesion, growth and proliferation. provides a good three-dimensional microenvironment for Due to its excellent biocompatibility, precise controllable physical and chemical properties, and no immune allergy and rejection reaction, the development of biomedical materials based on nanocellulose is now widely used at home and abroad for artificial organs and tissues. It is more and more widely applied in clinical treatment and scientific research, such as engineering, wound repair.

The network structure and topological morphology of tissue-engineered stent materials, especially stents or matrix structures that are anisotropically aligned at the micron to nanoscale, can directly influence cell morphology and cell behavior, preserving cellular dominance. is a powerful tool for inducing directed differentiation of cells. However, at present, most of the usual nanocellulose-containing hydrogels are essentially isotropic and randomly arranged structures with their three-dimensional network scaffolds, which are microscopically similar to natural tissues such as muscle, cartilage, and heart valves. It lacks an anisotropic structure and good mechanical properties on a large scale. Matrigel network structures derived from animal basement membranes can also provide a good platform for tissue-engineered culture studies, but their batch-to-batch variability makes it difficult to control mechanical properties and network structures. Together, potentially harbored pathogens and antigens are susceptible to infection and immune responses. Conventional nanocellulose stents, such as the commercial product UPM GrowDex hydrogel, can provide an ideal three-dimensional network environment for cell growth and propagation, but the stent network is a random structure. , which lack the anisotropic structure at the microscopic scale of native tissues, cannot effectively play a role in maintaining cellular dominance and inducing directed cell differentiation.

Therefore, in order to meet the dual requirements regarding the structure and function of tissue-engineered stent materials from a biocompatibility point of view, constructing a cell growth factor sustained-release stent material with an anisotropic structural network is a biomedical application. It is an important content of materials research and development, and a difficult problem that needs to be overcome urgently at present.

The object of the present invention is to provide a cell growth factor sustained release stent with an anisotropic structural network based on nanocellulose, its construction method and application, thereby overcoming the above defects of current cell growth factor sustained release materials. and constructing anisotropic stents to dynamically provide structural and mechanical support to cells and tissues to maintain the activity of growth factors to develop products that can be targeted and controlled for release. Achieving the goal.

In the present invention, first, by combining the catalytic oxidation of TEMPO with high-pressure homogenization treatment, ultra-pure nanocellulose and its film are prepared, and the nanocellulose film adsorbs cell growth factors and cellulolytic enzymes in situ. and synchronously forming a three-dimensional three-dimensional stent material, and finally freeze-drying to fabricate a nanocellulose-based cell growth factor sustained release anisotropic stent.

Specifically, the present invention provides the following technical solutions.

A method for constructing a nanocellulose-based cell growth factor sustained release anisotropic stent, comprising the following steps.
(1) Production of pulp fiber: Disperse 1 kg of spruce dissolving pulp fiber in 30 L of sterilized water, stir to uniformly disperse, add 50 to 100 g of sodium hydroxide and 20 to 40 mL of 30% hydrogen peroxide, and homogenize. Stir and sterilize at 120° C. and high pressure for 60 minutes.
(2) Washing and filtering pulp fibers: The pulp fibers in step (1) are filtered and washed with sterilized water so that the pH of the filtrate is neutral.
(3) Preparation of high-purity nanocellulose hydrogel: Cleanly washed pulp fibers are dispersed in 50 L of sterilized water and stirred, 2,2,6,6-tetramethylpiperidine 1-oxyl 10-15 g, sodium bromide 50 Add ˜100 g, 3-5 L of 10% sodium hypochlorite, adjust and maintain pH 10.5, stir at room temperature for 3-6 hours to react, bring system to pH 7.0 with 1.0 M HCl. adjusted to 0, filtered through a 300-mesh nylon mesh, washed with sterile water until the conductivity of the filtrate was lower than 5 μS / cm, dispersed the cleanly washed pulp fibers in 30-50 L of sterile water, Stir uniformly and pass through a high-pressure homogenizer at pressures of 300 bar and 1000 bar respectively to produce high-purity nanocellulose hydrogels.
(4) Dilute the prepared nanocellulose hydrogel with sterile water to a concentration of 0.1%, put it in a vacuum filter with a PVDF filtration membrane, vacuum filter, collect the filter cake, and hot press dry. It is transferred to a machine, hot-press dried at 80° C. and a pressure of 2.3 bar for 20 minutes, and irradiated with ultraviolet rays for 30 minutes to produce a nanocellulose film.
(5) The nanocellulose film is immersed in a solution of fibroblast growth factor and complex cellulase, and swelled by in situ adsorption at 4°C to synchronously form an anisotropic three-dimensional stent.
(6) Wash the prepared nanocellulose hydrogel stent carrying fibroblast growth factor and complex cellulase with sterile water to remove fibroblast growth factor and complex cellulase remaining on the surface, and use different Cut to shape and size and freeze-dry to fabricate nanocellulose-based cell growth factor sustained release anisotropic stents.

Here, in step (4), the pore size of the PVDF filtration membrane is 0.22 microns, and the vacuum filtration time is 8-12 hours.

Here, in the above step (4), the upper surface of the filter cake is covered with a PVDF filter membrane, padded with three layers of absorbent filter paper on both sides, and then transferred to a hot press dryer.

Here, in step (5), the concentration of fibroblast growth factor is 500-1000 ng/ml and the concentration of complex cellulase is 20-100 U/ml.

Here, in step (6), the freeze-drying conditions are −50° C., a vacuum of 0.1 mbar, and the freeze-drying time is 72 hours.

The present invention also provides applications of the nanocellulose-based sustained-release anisotropic stents of the present invention in artificial organs, tissue engineering, and wound repair materials.

Compared with the prior art, the present invention has the following beneficial effects.

From the viewpoint of biomimicry of structure and biomimicry of function, the present invention is based on the fact that the binding of other cell growth factor sustained-release material matrices to the growth factor is simple, so that the release rate cannot be precisely controlled, and the release rate is rapid. or to solve the problem of loss of activity and biological safety caused by excessive release; By in situ adsorption and synchronous formation of cell growth factor sustained-release stent materials with anisotropic structural networks, the dual requirements for structure and function of tissue-engineered stent materials are met from a biocompatibility perspective. be

The nanocellulose-based sustained-release anisotropic stent material made according to the present invention has good biocompatibility, no cytotoxicity, biodegradability, and can be used in natural tissue extracellular matrix. We have simulated the tight binding of cell growth factors with anionic polysaccharides such as heparin, and the resulting stent materials stably store growth factors, release growth factors on demand, and act as stent matrix materials. Its highly hydrated three-dimensional network structure maximizes the simulation of the microenvironment and structure of the real extracellular matrix and restores it. It can provide an ideal microenvironment for growth, reproduction and differentiation.

3 is a sustained release curve of basic fibroblasts according to Example 3. FIG. FIG. 10 shows promotion of cell growth by sustained release of basic fibroblasts according to Example 3. FIG. Fig. 3 shows the distribution of fibroblasts in the growth factor sustained release anisotropic stent material according to Example 3; 1 is a photograph of a freeze-dried product of a nanocellulose-based cell growth factor sustained release anisotropic stent made in Example 1. FIG. FIG. 10 is a microscopic photograph of synchronous formation of a stent having an anisotropic structural network by in situ adsorption of a growth factor according to Example 2. FIG.

The following clearly and completely describes the technical solutions in the embodiments of the present invention in conjunction with the drawings in the embodiments of the present invention, but of course the described embodiments are merely part of the embodiments of the present invention. , but not all examples. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative efforts belong to the scope of the present invention.

Example 1
A method for constructing a nanocellulose-based cell growth factor sustained release anisotropic stent, comprising the following steps.
(1) Disperse 1 kg of spruce dissolving pulp fiber in 30 L of sterilized water, stir to uniformly disperse, add 50 to 100 g of sodium hydroxide and 20 to 40 mL of 30% hydrogen peroxide, stir uniformly, and heat to 120 ° C. , high pressure for 60 minutes.
(2) Filter and wash the pulp fibers from step (1) with sterilized water so that the pH of the filtrate is neutral.
(3) Disperse and stir the cleanly washed pulp fibers from step (2) in 50 L of sterile water, add 10-15 g of TEMPO (2,2,6,6-tetramethylpiperidine 1-oxyl), sodium bromide. Add 50-100 g, 3-5 L of 10% sodium hypochlorite, adjust and maintain pH 10.5, stir at room temperature for 3-6 hours to react, and adjust the system to pH 7 with 1.0 M HCl. .0. Filter through a 300-mesh nylon mesh and wash with sterile water until the conductivity of the filtrate is less than 5 μS/cm. The cleanly washed pulp fibers are dispersed in 30-50 L of sterilized water, stirred uniformly, and passed through a high-pressure homogenizer at pressures of 300 bar and 1000 bar, respectively, to produce high-purity nanocellulose hydrogel.
(4) Take an appropriate amount of nanocellulose prepared in step (4), dilute with sterilized water to a concentration of 0.1%, and put it into a vacuum filter with a PVDF filtration membrane with a pore size of 0.22 microns. Vacuum filter for 8-12 hours, collect the filter cake, cover the PVDF filter membrane on top, and pad 3 layers of absorbent filter paper on both sides. Transfer to a hot press dryer, hot press dry at 80° C. and 2.3 bar pressure for 20 minutes, and irradiate with ultraviolet rays for 30 minutes to produce a nanocellulose film.
(5) The nanocellulose film prepared in step (4) is immersed in 1-5 ml of a solution in which 500-1000 ng/ml of fibroblast growth factor and 20-100 U/ml of complex cellulase are dissolved at 4°C. It adsorbs in situ, swells, and synchronously forms a three-dimensional stent with an anisotropic structure.
(6) Wash the nanocellulose hydrogel stent carrying cell growth factors and cellulase prepared in step (5) with sterilized water to remove the growth factors and cellulase remaining on the surface, Cut to size and freeze-dried at −50° C. and 0.1 mbar vacuum for 72 hours to fabricate nanocellulose-based cell growth factor sustained release stents with low charge density, the product picture is shown in FIG. .

Example 2
A method for constructing a nanocellulose-based cell growth factor sustained release anisotropic stent, comprising the following steps.
(1) Disperse 1 kg of spruce dissolving pulp fiber in 30 L of sterilized water, stir to uniformly disperse, add 50 to 100 g of sodium hydroxide and 20 to 40 mL of 30% hydrogen peroxide, stir uniformly, and heat to 120 ° C. , high pressure for 60 minutes.
(2) Filter and wash the pulp fibers from step (1) with sterilized water so that the pH of the filtrate is neutral.
(3) Disperse and stir the cleanly washed pulp fibers from step (2) in 50 L of sterile water, add 10-15 g of TEMPO (2,2,6,6-tetramethylpiperidine 1-oxyl), sodium bromide. Add 50-100 g, 5-10 L of 10% sodium hypochlorite, adjust and maintain pH 10.5, stir at room temperature for 12-24 hours to react, adjust the system to pH 7 with 1.0 M HCl. .0. Filter through a 300-mesh nylon mesh and wash with sterile water until the conductivity of the filtrate is less than 5 μS/cm. The cleanly washed pulp fibers are dispersed in 30-50 L of sterilized water, stirred uniformly, and passed through a high-pressure homogenizer at pressures of 500 bar and 1500 bar, respectively, to produce high-purity nanocellulose hydrogels.
(4) Take an appropriate amount of nanocellulose prepared in step (4), dilute with sterilized water to a concentration of 0.1%, and put it into a vacuum filter with a PVDF filtration membrane with a pore size of 0.22 microns. Vacuum filter for 12-24 hours, collect the filter cake, cover the upper layer with PVDF filter membrane, and pad with 3 layers of absorbent filter paper on both sides. Transfer to a hot press dryer, hot press dry at 80° C. and 2.3 bar pressure for 20 minutes, and irradiate with ultraviolet rays for 30 minutes to produce a nanocellulose film.
(5) The nanocellulose film prepared in step (4) is immersed in 5-10 ml of a solution of 500-1000 ng/ml of fibroblast growth factor and 20-100 U/ml of complex cellulase, at 4°C. It adsorbs in situ, swells, and synchronously forms a three-dimensional stent with an anisotropic structure.
(6) Wash the nanocellulose hydrogel stent carrying cell growth factors and cellulase prepared in step (5) with sterilized water to remove the growth factors and cellulase remaining on the surface, It is cut to size and lyophilized at −50° C. and a vacuum of 0.1 mbar for 72 hours to fabricate cell growth factor sustained release stents based on nanocellulose with high charge density.

Example 3 Verification of Performance of Nanocellulose-Based Cell Growth Factor Sustained-Release Anisotropic Stent (1) To verify cell growth factor release control by the growth factor-sustained-release stents fabricated in Examples 1 and 2 , mouse b-FGF/FGF-2 ABC-ELISA enzyme-linked immunoassay kit was used to detect the release of cell growth factors in the DMEM cell culture medium of the stent material.

Take one growth factor sustained-release stent (3 mm x 3 mm x 50 μm) in Examples 1 and 2, put it in 1 ml of DMEM complete medium, and incubate it for 0.5 hours, 1.0 hours, 3.0 hours, respectively. Supernatants were taken at 0 hours, 12.0 hours, 1 day, 3 days, and 7 days to measure growth factor concentrations and draw release curves. As a result, the cell growth factor can be gradually released from the stent material, as shown in FIG. It is gradually degraded, further increasing the bioavailability of growth factors and providing space for cell growth and secretion of its own extracellular matrix.

Also, stent materials with lower nanocellulose surface charge densities can release more growth factors faster. In contrast, high charge density nanocellulose stent materials can release growth factors more loosely due to the tight association of cell growth factors with the stent network, resulting in excessive release and activation of growth factors too quickly. Effectively avoiding the loss of , it is possible to control the release rate of cell growth factors by controlling the charge density on the nanocellulose surface.

(2) In order to verify the controlled release of cell growth factors and the promotion of cell growth and proliferation by the growth factor sustained-release stents produced in Examples 1 and 2, 3D cell culture was employed to Proliferation conditions were detected.

Take one growth factor sustained release stent (3 mm x 3 mm x 50 μm) in Examples 1 and 2 and place it in a 24-well plate, inoculate with ³ x 10 mouse embryonic fibroblasts, add 1 ml of DMEM complete medium (containing 200 mM L-glutamine, 1000 IU/mL penicillin and streptomycin, and 10% inactivated fetal bovine serum) and incubated at 37° C., 5% CO ₂ , 95% humidity for 3 days. Concurrently, normal cell plating in 24-well plates and addition of free cell growth factors to cells plated in 24-well plates were controlled, respectively. Cell proliferation was analyzed by MTT cell proliferation and toxicity detection kit. Cells were stained with a live/dead fluorescent staining kit and fluorescent photographs were taken. The results are shown in Figures 2-3, respectively. The proliferation rate of fibroblasts on cell growth factor sustained release stents is significantly higher than on normal cell cultures (p<0.01 for high charge density stents***, p<0.01 for low charge density stents*). p<0.05). And the low charge density stent has a more pronounced promoting effect on cell proliferation. The growth rate of cells cultured with free cell growth factors is not significantly different when compared to normal cultures.

This proves that the cell growth factor sustained release stent of the present invention has the ability to significantly promote cell growth and proliferation while effectively avoiding denaturation and inactivation of free growth factors. . Fluorescence micrographs show a high density of viable cells distributed inside the stent, with obvious cell clusters, suggesting that the stent material produced according to the present invention is effective for cell growth and proliferation. Again proven to effectively improve speed, and also promote the generation of cell clusters as the stent matrix is gradually degraded, which is then gradually secreted to generate its own extracellular matrix can be done.

(3) In order to verify the anisotropic structure of the nanocellulose-based cell growth factor sustained release stent fabricated in Example 2, the cross-section of the stent material fabricated in Example 2 was scanned with a scanning electron microscope. I photographed and observed. As can be seen from FIG. 5, the stent material has a regular pore structure and an oriented array structure, which provides strong support for maintaining cell dominance and inducing cell directed differentiation. can be done.

While embodiments of the present invention have been shown and described, those skilled in the art can make various alterations, modifications, substitutions and alterations thereto without departing from the principles and spirit of the invention. , it is to be understood that the scope of the invention is limited by the appended claims and their equivalents.

Claims (10)

A method for constructing a nanocellulose-based cell growth factor sustained release anisotropic stent comprising:
(1) producing pulp fibers;
(2) washing and filtering the pulp fibers;
(3) making a high-purity nanocellulose hydrogel using the washed pulp fibers;
(4) The prepared nanocellulose hydrogel was diluted with sterile water to a concentration of 0.1 wt%, put into a vacuum filter with a PVDF filtration membrane, vacuum filtered, collected the filter cake, hot Transfer to a press dryer, hot press dry at 80° C., 2.3 bar pressure for 20 minutes, and irradiate with UV light for 30 minutes to produce a nanocellulose film;
(5) The nanocellulose film is immersed in a solution of a certain volume in which fibroblast growth factor and complex cellulase are dissolved, and is swollen by in situ adsorption at 4°C to produce a three-dimensional stent having an anisotropic structure. synchronously forming;
(6) The nanocellulose hydrogel stent carrying fibroblast growth factor and complex cellulase thus prepared is washed with sterile water to remove the fibroblast growth factor and complex cellulase remaining on the surface, and if necessary cutting into different shapes and sizes and freeze-drying to fabricate nanocellulose-based cell growth factor sustained release stents;
A construction method characterized by: In the step (4), the pore size of the PVDF filtration membrane is 0.22 microns, and the vacuum filtration time is 8-12 hours.
The construction method of nanocellulose-based cell growth factor sustained release anisotropic stent according to claim 1, characterized in that: In step (4), the upper surface of the filter cake is covered with the PVDF filter membrane, padded with three layers of absorbent filter paper on both sides, and then transferred to the hot press dryer.
The construction method of nanocellulose-based cell growth factor sustained release anisotropic stent according to claim 2, characterized in that: In step (5), the fibroblast growth factor has a concentration of 500-1000 ng/ml, and the complex cellulase has a concentration of 20-100 U/ml.
The construction method of nanocellulose-based cell growth factor sustained release anisotropic stent according to claim 3, characterized in that: In step (6), the freeze-drying conditions are −50° C., a vacuum of 0.1 mbar, and the freeze-drying time is 72 hours.
The construction method of nanocellulose-based cell growth factor sustained release anisotropic stent according to claim 4, characterized in that: Specifically, in the step (1), 1 kg of spruce dissolving pulp fiber is dispersed in 30 L of the sterilized water, stirred to uniformly disperse, and 50 to 100 g of sodium hydroxide and 20 to 40 mL of 30% hydrogen peroxide are added. Add, stir evenly, sterilize at 120° C., high pressure for 60 minutes,
The construction method of nanocellulose-based cell growth factor sustained release anisotropic stent according to claim 5, characterized in that: Specifically, the step (2) includes filtering and washing the pulp fibers in the step (1) with the sterilized water so that the pH of the filtrate becomes neutral.
The construction method of nanocellulose-based cell growth factor sustained release anisotropic stent according to claim 6, characterized in that: Specifically, the step (3) comprises dispersing and stirring the thoroughly washed pulp fibers in 50 L of the sterilized water, adding 10 to 15 g of 2,2,6,6-tetramethylpiperidine 1-oxyl, bromide Add 50-100 g of sodium, 3-5 L of 10% sodium hypochlorite, adjust and maintain the pH to 10.5, stir at room temperature for 3-6 hours to react, and quench the system with 1.0 M HCl. The pH is adjusted to 7.0, filtered through a 300-mesh nylon mesh, washed with the sterilized water until the conductivity of the filtrate is lower than 5 μS/cm, and the cleanly washed pulp fibers are added to the sterilized water 30%. Dispersed in ~50 L, stirred uniformly, and passed through a high pressure homogenizer at a pressure of 300 bar and 1000 bar respectively to produce the high purity nanocellulose hydrogel.
The construction method of nanocellulose-based cell growth factor sustained release anisotropic stent according to claim 7, characterized in that: A nanocellulose-based cell growth factor sustained release anisotropic stent fabricated by the construction method of any one of claims 1-8. Application of the nanocellulose-based sustained release anisotropic stent according to claim 9 in artificial organs, tissue engineering, wound repair materials.

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