TWI515024B - Composition of blended chitosan-biodegradable polymer and use thereof - Google Patents

Description

Chitosan-biodegradable polymer blending composition and use thereof

The present invention relates to a tissue engineering material. In particular, the present invention utilizes a method of mixing chitosan and a biodegradable polymer to form a new tissue engineering material suitable for in vitro culture of corneal cells and corneal replacement. The invention also relates to the use and process of the tissue engineering material.

Tissue engineering uses a matrix of suitable biomedical materials to signal, attach, grow, differentiate, and even perform normal functions. Some people call it "regenerative medicine." As described above, the three elements constituting tissue engineering are cells, artificial extracellular matrices (ie, tissue growth scaffolds), and growth information molecules. The tissues and organs of the human body are basically composed of the extracellular matrix of cells and supporting cells. The extracellular matrix is the basic framework and metabolic site of cell attachment, and its morphology and function directly affect the tissue morphology. and function. The ideal extracellular matrix should have the following characteristics: good biocompatibility, absorbability and plasticity, surface chemical properties and surface structure for cell adhesion and growth, and degradation rate can be adjusted according to the tissue regrowth of different cells. The substrate in tissue engineering basically functions as an extracellular matrix, so the aforementioned characteristics are also the goals pursued in the current research field.

Corneal blindness caused by corneal lesions is one of the main causes of blindness in the world, so the development of artificial cornea has become one of the hot topics. The artificial cornea currently used clinically is based on cell-free biomedical materials made of collagen fibers (Griffth et al., Invest Ophthalmol Vis Sci 47(5): 1869-75, 2006; Griffth et al., Invest Ophthalmol Vis Sci 49(9): 3887-94, 2008), the principle is mainly to make collagen fibers a corneal substitute by cross-linking, although it can be used in emergency situations, but Because it can not be introduced into normal corneal cells for growth, it can only be used as a temporary substitute. Finally, it is still necessary to wait for the normal cornea for corneal transplantation.

Like other organ transplants, corneal transplants also face a shortage of organ sources. The current trend is to establish an in vitro culture system with corneal cells to solve the diseases of simple corneal cells with specific cell therapy. However, the above method has a property of being difficult to reproduce, and a cell denaturation phenomenon is apt to occur in a general tissue culture dish, and once denaturation occurs, the cell loses its original function. These problems make the research of cultured corneal cells in vitro still to be explored and broken.

A number of biodegradable materials for cultivating tissue have been disclosed in the prior art. For example, the Chinese Patent Application No. 200505514 discloses a biocompatible stent for implantable animals, comprising a biocompatible, Polymer matrix and fibrous mat for pores; European Patent No. 2,385,105 provides a matrix of a plurality of polymers consisting of a plurality of polymers for maintaining cell growth, coating and regeneration; and U.S. Patent No. The publication application No. 2003/0211130 discloses a method of cultivating alternative tissues comprising providing a substrate for cell attachment culture, and a multilayered fiber composed of an artificial polymer and a natural polymer. However, all of the above materials are opaque and porous, and are not suitable for culturing corneal cells and for corneal replacement.

In order to improve the deficiencies of the prior art, the inventors mixed two kinds of biomedical materials: chitosan and polyester biodegradable polymers to prepare a new blend, which can be used as a cell culture substrate. The blended product of the present invention utilizes the difference in the attachment ability of corneal cells to chitosan and polyester biodegradable polymers to control the shape and growth of cells. In addition, the admixture of the present invention has the characteristics of no pores, is suitable for culturing corneal cells, and forms corneal cells to form a cell layer such as a human corneal structure; and the mixed material of the present invention has a film shape and good light transmittance. It is suitable for corneal replacement, so that patients can receive light before the mixture is completely decomposed and absorbed, without visual obstruction.

The main advantages of the invention are:

First, the method of culturing corneal cells using a biologically derived substrate may be discarded, which may cause side effects of infection and pathogens, and the pathogen of infected cells is not created in the process of mixing.

Second, the method of culturing corneal cells using a chemically functional matrix may be discarded, which may cause cytotoxic side effects, and the cells do not produce toxic cells during the mixing process.

Third, the new material created by the mixed material, the material property changed is the change of the whole material, not limited to the surface of the material, while retaining the characteristics of biocompatibility and cleavability, so that the corneal cells can be cultured and applied. Replacement of the cornea.

Fourth, the process of mixing the chitosan with the polyester biodegradable polymer to form a mixed material, not only saves time and saves cost.

As used herein, the term "blend" refers to a method of mixing two polymers to develop a novel biological material that has properties that are not unique to either of the two polymers described above. As used herein, the terms "blend," "mixed material," or "mixed composition" refer to a product made by a blending process. The blends formed from synthetic polymers and natural polymers have the biochemical properties of synthetic polymers, as well as the biocompatibility and bio-interaction properties of natural polymers. As used herein, the term "cell culture medium" or "mixed cell culture medium" refers to a state in which the blend of the present invention is used to culture cells, which may be in vivo or in vitro, and the cells may be any mammalian cells, For example but not limited to: human corneal cells.

As used herein, the term "Corneal cells" includes corneal epithelial cells, corneal interstitial cells, and corneal endothelial cells.

Herein, the term "polyester biodegradable polymer" means a polyester-based polymer material which can be completely degraded into a low molecular substance by the action of microorganisms or enzymes in nature. Polyester polymers account for a large proportion of biodegradable polymer materials, mainly because their ester chains can easily break bonds through hydrolysis, produce lactic acid that can be absorbed by organisms, and then undergo metabolic transformation in living organisms. Excreted from carbon dioxide and water molecules. Therefore, polyester-based polymers are widely used in biomedical materials. Polyester biodegradable polymers include, but are not limited to, polylactic acid (PLA), polyglycolic acid (PGA), polylactic acid-glycolic acid (PLGA), polyhydroxybutyrate (PHB), polyhydroxybutyl ester Amyl ester (PHBV), polycaprolactone (PCL), polyhydroxyalkanoate (PHA), and polyhydroxysuccinic acid (PMA).

In this paper, the term "non-porous" means that the completely non-porous hole or hole is smaller than the limit that can be observed by current microscopy, rather than an absolute property. Figure 9 and Figure 10 show the electron microscopy of three sample blends. Like, no obvious holes were observed in the figure. However, the limits of hole measurement may vary with the evolution of the technology, so the "non-porous" blends described in this specification may detect the presence of holes in the future. There is a close relationship between the non-porous nature of the blend and its transparency. When there is no hole at all or the hole is less than 100 nm, the transmittance of the blend is as high as 80~99.99%; when the hole size is between 100~500 nm, the larger the hole, the light transmittance of the blend The degree has also declined.

As used herein, the terms "PCL 25", "PCL 50", "PCL 75", and "PCL 100" represent the overall proportion of polycaprolactone in the blended composition. PCL 25 represents 25% of polycaprolactone in the blended composition; similarly, PCL 50 and PCL 75 represent 50% and 75% of polycaprolactone in the blended composition, respectively; PCL 100 represents the composition It consists entirely of polycaprolactone.

In this paper, the term "transmittance" refers to the ratio of brightness before and after the light penetrates the object, and can be used as an indicator of the transparency of the object. The higher the transmittance, the better the transparency.

As used herein, the term "corneal replacement" or "keratoplasty" means removing the cornea of a patient's lesion and suturing the donated human cornea, synthetic substitute or cell culture medium containing the cornea to the patient's eye. The function of the original cornea.

As used herein, the term "plurality" is used to describe the number of elements or units of the invention. This term should be understood to mean two or more unless expressly stated otherwise.

The articles "a" or "an" are used herein to describe the elements and compositions of the invention. This terminology is only for convenience of description and the basic idea of the invention. This description is to be construed as inclusive of the singular

The term "or" in this document means "and/or".

The present invention provides a blended composition comprising a chitosan and a polyester biodegradable polymer; wherein the blended composition has the characteristics of no voids. The non-porous nature allows the corneal cells to grow into a layer of cells that mimic the natural cornea, and this non-porous nature is not affected by the type of biodegradable polymer used (Figures 9 through 10). In a specific embodiment, the blended composition further has a transmittance of more than 30% and a thickness of no greater than 2000 microns.

Chitosan is obtained from crustaceans via deacetylation and has been used in many medical fields (eg, drug delivery and wound dressings). For in vitro cell culture, chitosan can cause cells to be difficult to attach, which in turn leads to rounded cells; however, previous studies have indicated that chitosan has the ability to promote the secretion of extracellular matrices by cells (Howling et al., Biomaterials) 22(22): 2959-2966, 2001).

In a specific embodiment, the blended composition has a transmittance of greater than 50%. In another embodiment, the blended composition has a light transmission between 70% and 99.99%. In a specific embodiment, the blended composition has a thickness of no greater than 1000 microns. In another embodiment, the blended composition has a thickness between 5 microns and 250 microns.

In a specific embodiment, the polyester biodegradable polymer constituting the blended composition accounts for 25% to 100% of the total proportion. In another embodiment, the polyester biodegradable polymer constituting the blended composition accounts for from 25% to 75% of the total proportion.

In a specific embodiment, the polyester biodegradable polymer constituting the blended composition is selected from the group consisting of polycaprolactone, polylactic acid, and polyhydroxybutyrate. Among them, polycaprolactone is a synthetic aliphatic acid polyester, which is formed by ring-opening polymerization of a cyclic monomer caprolactone (ε-caprolactone, ε-CL). Polycaprolactone has good mechanical strength, biocompatibility, degradability and permeable properties. It has been widely used in various fields of biomedicine and has been approved by the US Food and Drug Administration (FDA) for human body use. It is a tissue engineering substrate material with great potential. In addition, when polycaprolactone is used for cell culture or tissue engineering, it is easy to attach cells to its surface, so cells tend to have a flat shape on the surface of polycaprolactone (Baker et al., Biomaterials 30(7): 1321-1328, 2009; Kweon et al., Biomaterials 24(5): 801-808, 2003).

The blended composition of the present invention is suitable for culturing corneal cells and corneal replacement based on the above-described non-pores and characteristics such as good light transmittance. Further, the blend composition of the present invention is also suitable as a wound dressing, a tissue repair patch or a drug release carrier.

The blended composition of the present invention is prepared by a blending process. In detail, the chitosan and polyester biodegradable polymers are separately dissolved in an acidic solution (for example, aqueous acetic acid or glacial acetic acid) to prepare a chitosan solution and a biodegradable polyester. Molecular solution. Then, different volumes of the polyester biodegradable polymer solution are slowly added to the chitosan solution according to the ratio, and the volume is supplemented with the above acidic solution to finally obtain the polyester biodegradable polymer having the desired concentration. Mixed solution. The cell culture substrate (i.e., the blend) is then prepared from the blended solution. First, the mixed solution obtained in the foregoing step is coated on a tissue culture dish, and after standing at room temperature for a suitable period of time, the excess solution is removed and the culture dish is dried. Before the cell culture, the cell culture medium is subjected to an acid solution of an acidic solution, an alkaline solution (for example, sodium hydroxide), a washing, and ultraviolet irradiation sterilization.

The present invention also provides a use of a blended composition as a corneal substitute, wherein the blended composition comprises chitosan, a polyester biodegradable polymer, and a plurality of corneal cells cultured thereon; The blended composition has the characteristics of no pores, allowing the corneal cells to grow into a single layer of cells, and the non-porous properties are not affected by the type of biodegradable polymer used (Figs. 9 to 10). In a specific embodiment, the blended composition further has a transmittance of more than 30% and a thickness of no greater than 2000 microns.

In a specific embodiment, the blended composition has a transmittance of greater than 50%. In another embodiment, the blended composition has a light transmission between 70% and 99.99%. In a specific embodiment, the blended composition has a thickness of no greater than 1000 microns. In another embodiment, the blended composition has a thickness between 5 microns and 250 microns.

In a specific embodiment, the polyester biodegradable polymer constituting the blended composition accounts for 25% to 100% of the total proportion. In another embodiment, the polyester biodegradable polymer constituting the blended composition accounts for from 25% to 75% of the total proportion.

In a specific embodiment, the polyester biodegradable polymer constituting the blended composition is selected from the group consisting of polycaprolactone, polylactic acid, and polyhydroxybutyrate.

The following examples provide some illustrative specific embodiments of the invention.

The invention may be embodied in different forms and is not limited to the examples mentioned below. The following examples are merely representative of the various aspects and features of the present invention.

Embodiment 1 Preparation and qualitative analysis of mixed admixture

The biodegradable polymer in this embodiment is exemplified by polycaprolactone.

The preparation method of chitosan and polycaprolactone is as follows: chitosan (purchased from Sigma-Aldrich, the degree of deacetylation is 85%) is dissolved in 0.5 M acetic acid to make 1 a wt% solution of chitosan; dissolving polycaprolactone (PCL) in glacial acetic acid to make a 10 wt% polycaprolactone solution; then, proportionally different volumes of 10 wt% The ester solution and glacial acetic acid were slowly added to 3 mL of 1 wt% chitosan solution to finally obtain a chitosan/polycaprolactone mixed solution having a concentration of polycaprolactone of 25, 50 and 75 wt%.

The cell culture substrate was prepared by directly coating the chitosan/polycaprolactone blend solution on a polystyrene culture dish for tissue culture (TCPS plate, available from Coster, 60 mm in diameter). After standing at room temperature for 30 minutes, the remaining solution was removed and the TCPS dish was placed in a 60 ° C convection oven for 24 hours. The above cell culture medium must be soaked in a 0.5 N NaOH aqueous solution for 24 hours to neutralize the acidity of the acetic acid, followed by thorough washing with deionized water. The cell culture medium is sterilized by ultraviolet irradiation overnight before being used for cell culture.

The prepared cell culture substrate was placed on a visual acuity meter together with a TCPS culture dish, and photographed using a digital camera to determine the transparency of the cell culture substrate. In addition, a self-made instrument is also used to quantify the transparency. The process is briefly described as follows: First, the digital illuminance meter (MLM-1010, Minipa) Measuring the brightness emitted by a white light source; then, inserting a blank TCPS culture dish between the white light source and the digital illuminometer, recording the brightness of the transmitted light, and setting the blank TCPS dish to 100% as Benchmark for subsequent measurements. When measuring, the cell culture medium made of PCL 25 (ie, a polybutyrolactone concentration of 25 wt% of chitosan/polycaprolactone mixed solution, the following representations have the same meaning), PCL 50. A PCL 75 blend and a TCPS petri dish of PCL100 were inserted between the white light source and the digital illuminometer, and the brightness of the transmitted light was recorded one by one to convert the transparency. The transmittance is defined as the ratio of the transmitted light luminance value recorded by the blend to be tested and the transmitted light luminance value recorded by the blank TCPS petri dish.

As can be seen from Figure A, the number on the visual force meter can be clearly observed through the blank TCPS culture dish, and replaced with a TCPS culture dish coated with chitosan, PCL 25, PCL50 and PCL75 blends. The numbers are still legible. However, as the proportion of PCL in the blend is higher, the transparency of the blend decreases. If you pass pure PCL (PCL 100), you cannot clearly observe the number on the power gauge. If expressed in a quantitative manner, it can be seen that the penetration brightness of PCL 100 has a significant decrease (Fig. 1B).

Embodiment 2 Isolation and culture of Bovine Corneal Emtothelial Cells (BCECs)

The following animal experiments were performed in accordance with the animal experiment plan approved by the Taipei Medical University Review Committee. Fresh bull's eye was taken from a regional slaughterhouse and immersed in an iodine solution for 3 minutes, removed and placed in phosphate buffered saline (PBS). Subsequent experimental procedures are referenced and modified from previous research literature (YT Zhu et al., Invest. Ophthalmol. Vis. Sci. 49 (2008) 3879-3886; and W. Li et al., nvest. Ophthalmol. Vis. Sci. 48 (2007) 614-620): Incubation with fresh bovine eyes at 37 ° C for 30-60 minutes using trypsin to strip the corneal endothelial cell layer from the underlying tissue. Next, the supernatant was centrifuged at 1,500 rpm for 5 minutes to collect endothelial cells. The obtained cells were cultured in a supplemented hormonal epithelial medium consisting mainly of an equal volume of HEPES buffered DMEM and Ham F12 (Invitrogen), and the remaining components were: 5% FBS, 0.5% dimethyl hydrazine. (Sigma-Aldrich), 2 ng/mL hEGF (Sigma-Aldrich), 5 μg/mL insulin, 5 μg/mL transferrin, 5 ng/mL selenium (Invitrogen), 1 nM cholera toxin (Sigma-Aldrich) 50 μg/mL Jiantaimycin (Invitrogen) and 1.25 μg/mL Amphotericin B (Invitrogen). The cell culture environment was 37 ° C, 95% air and 5% CO ₂ . When cell aggregation was reached after 14-21 days, the cells were washed with PBS, the cells were detached from the culture dish by trypsin, finally collected by centrifugation, and then the cells were suspended in SHEM. Next, about 10,000 cells were implanted in each dish and maintained at 37 ° C in an environment of 5% CO ₂ humid air. The medium was changed every 2 to 3 days, and the cell type was observed by a phase contrast microscope (Leica) up to 7 days later.

Embodiment 3 Cell adhesion test

The cells were cultured in a 24-well culture plate coated with chitosan/polycaprolactone blend, and the attached cells were weighed 4 hours later to determine the adhesion of the cells to various blends. Specifically, 1 mL of cell culture medium (90% DMEM + 10% FBS) suspended in about 10,000 cells was added to each well of a 24-well culture plate, and the plate was incubated at 37 ° C with 5% CO ₂ damp. Cultivate in an air environment. Unattached cells were collected 4 hours later, quantified using a hemocytometer, and the number of attached cells was estimated accordingly.

After adhering the cells for 4 hours, the number of cells on the cell culture substrate was as follows: 5.56±0.6×10 ³ for the chitosan group, 7.56±0.3×10 ³ for the PCL 25 group, and 8.11±0.2× for the PCL50 group. 10 ³ , PCL 75 group is 8.31 ± 0.1 × 10 ³ . If the results are expressed as the ratio of the number of adherent cells in each chitosan/PCL blended group and the chitosan group, the results are as follows: 136.0±4.8% in the PCL 25 group and 145.8±4.2% in the PCL 50 group. The PCL 75 group was 149.4 ± 1.8%. The number of cells attached to pure chitosan cell culture medium was significantly lower than the number of cells attached to the mixed cell culture medium of any PCL ratio ( p < 0.01) (Fig. 2).

Optical microscopy further confirmed the above phenomenon. It is difficult for BCEC cells to adhere to the surface coated with pure chitosan. As the proportion of PCL in the blend increases, the coverage of BCEC cells is getting better and better. It can be seen under the microscope that most of the cells are spherical on the surface coated with pure chitosan, whereas on the surface of the coated blend, the cells are spread out.

Embodiment 4 Cell proliferation test

In order to test the cell proliferation on each blend, 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl was used on days 1, 3 and 7 of the culture, respectively. The number of cells was determined by the tetrazolium bromide (MTT, Sigma-Aldrich) assay. The principle of MTT assay is that living cells can reduce the water-soluble yellow dye to an insoluble purple formazan product, so the MTT value obtained by the reaction is positively correlated with cell viability.

First, 100 μL of MTT solution was added to each well. After incubating for 3 hours at 37 ° C, 200 μL of dimethyl hydrazine (DMSO) was added to dissolve formazan crystals. The mixed solution was placed on an shaker and shaken evenly for 15 minutes. The transmission density of the formazan solution at a wavelength of 570 nm was measured using an enzyme immunoassay (ELISA) disc analyzer (ELx800, BIO-TEK). All experimental data are four replicates.

As can be seen from Fig. 3, the number of BCEC cells increased with the proportion of PCL in the blend at each measurement time point, indicating that the cells proliferated in the chitosan/PCL blend. This result is consistent with the observations of the phase difference microscope.

Embodiment 5 Type measurement and fluorescence microscope observation

The type analysis was carried out at the 4th hour of cell culture by observing the distribution of actin fibers using an immunofluorescence microscope. In detail, the cells were cultured on a mixed cell culture medium, and after 4 hours, they were fixed with 2% paraformaldehyde for 1 hour. Prior to staining, cells were permeabilized with 0.1% polyethylene glycol octylphenyl ether (Triton X-100) for 10 minutes and then incubated with 10% bovine serum albumin/PBS for 20 minutes to remove non-specific staining binding. . Use Alexa- respectively The actin cytoskeleton and nuclear DNA were stained with phalloidin 546 and 300 nM DAPI (Invitrogen). After the dyeing was completed, measurement was performed with Image J software (Universal Imaging). The shape factor is defined as σ = 4π A / P ² , where A is the surface area of the cell and P is the cell perimeter. A perfect round cell has a shape factor of 1, while an extended cell has a shape factor value close to zero. The standard deviation (SEM) of the measurements and the statistical analysis of the shape factor and cell range were calculated using Graphpad Prism (Graphpad Software) software and plotted.

The shape factor of BCEC cells on chitosan at the 4th hour was 0.92 ± 0.03, indicating that most of the BCEC cells attached to chitosan were spherical. When the PCL content in the blend is increased, the shape factor of the cells is also reduced (PCL 25: 0.81 ± 0.10; PCL 50: 0.72 ± 0.12; and PCL 75: 0.67 ± 0.12), representing the cell type in the circle Between spherical and elongated. Among them, the PCL 75 group had the lowest cell shape factor value, which confirmed that the cell group had the best adhesion.

The cytoskeleton is extremely important for cell type, cell attachment, and gene expression. Figure 5 and Annex 1 show the effect of chitosan/PCL blend on the cytoskeletal structure of BCEC cells at 4 hours and 1 day after culture. From the immunofluorescence images stained with rosin-labeled phalloidin, it can be seen that the actin fiber elongation on the blend is very significant. In contrast, cells cultured on pure chitosan have a structure in which actin is aggregated into small pieces. In the case of blends, as the proportion of PCL increases, the extent of the actin network also becomes wider.

Fig. 6 is a photograph showing the BCEC cell type after 7 days of culture observation by a phase contrast microscope. Although BCEC cells can attach to chitosan, the cells appear to be more dispersed. As the proportion of PCL content in the blend increases, the BCEC cells expand and the cell morphology becomes nearly hexagonal. On the 7th day of culture, the cells in the PCL50 and PCL75 groups occupied the confluence of the plane, indicating that the cell-adhesive state was changed by the regulation of cell attachment, and the chitosan/PCL mixture also changed the cell type. However, limited to the opaque nature of pure PCL (PCL 100), the cell type cultured thereon could not be observed.

Embodiment 6 Immunofluorescence analysis

For immunofluorescence staining, BCEC cells were fixed with 4% paraformaldehyde (pH 7.4) for 30 minutes at room temperature at the indicated time points. Cells were cultured in 10% bovine serum albumin containing 0.5% polyethylene glycol octylphenyl ether at room temperature prior to staining to block non-specific antibody responses while increasing cell membrane penetration of antibodies. . Next, the cells were co-cultured with the following primary antibody at 4 ° C overnight: anti-ZO-1 rabbit polyclonal antibody (1:200; Millipore) and anti-calcium adhesion protein (N-cadherin) antibody (1:400, BD) ). The cells were washed twice in PBS every other day for 15 minutes, then labeled with Goat anti-rabbit IgG (1:100; Invitrogen) of 568 was incubated for 1 hour at room temperature. All groups of cells were stained with DAPI (1:5000; Invitrogen) for 5 minutes at room temperature to label DNA in the nucleus. Finally, several washes were performed, and the cells were fixed on a slide with a fluorescent fixative (VectA Mount; Vector Laboratories). After the slides were dried, the cell images were taken with a digital imaging system (Leica, Germany).

ZO-1 is a protein related to tight junction, which is usually expressed in a single layer of hexagonal retinal endothelial cells. N-cadherin is a differentiation marker and is expressed in the retina during eye development. The junction of endothelial cells, both used to confirm the cell appearance of BCEC cells on the substrate of chitosan, PCL 25, PCL 50 and PCL 75 cells on day 7. As can be seen from Figure VII and Annex II, only BCEC cells cultured on PCL 25, PCL 50 and PCL75 cell culture media showed ZO-1 and calcium-adhesive proteins. The higher the PCL content in the blend, the more apparent the form of ZO-1 and calcium-adhesive proteins. In contrast, the expression of ZO-1 and calcium-adhesive adhesion proteins in BCEC cells cultured on chitosan decreased, indicating that these cells did not develop into terminal phase morphology of corneal endothelial cells.

Example 7 Real-time quantitative polymerase chain reaction (qPCR)

BCEC cell culture for 7 days, the overall cellular RNA was extracted using GeneJET ^TM RNA purification kit (Fermentas). Next, the complementary DNA synthesis kit ^{(RevertAid TM H Minus First Strand cDNA} Synthesis Kit, Fermentas) and the resulting RNA was reverse transcribed to complementary DNA. The amount of signal RNA was detected by a sequence detection system (ABI Prism 7500 HT Sequence Detection System, Applied Biosystem), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control group. Data were collected on instruments equipped with Applied Biosystem SDS 2.1 software and analyzed using threshold cycle value (Ct) correlation quantification. Table 1 shows the sequence of primer pairs used to detect the expression of the target gene.

Using qPCR kits ( SYBR Green qPCR Master Kit) detects the amount of target gene expression. The experimental results are presented as a ratio of the amount of cellular gene expression on the admixture to the amount of cellular gene expression on the chitosan.

Based on the different adhesion and proliferation characteristics of BCEC cells on chitosan/PCL blends, we further explored the RNA of extracellular matrix (ECM) protein (referred to as type IV collagen) on day 7 of culture. which performed. The results were presented as the ratio of the amount of gene expression between the BCEC cells cultured on the admixture and the BCEC cells cultured on pure chitosan. The BCEC cells cultured on the admixture showed higher expression of type IV collagen than the BCEC cells cultured on pure chitosan, and the multiples of performance were PCL 25:6.10±0.48 times, PCL 50: 7.79 ± 0.21 times and PCL 75: 3.91 ± 0.14 times (Figure 8).

Example eight Statistical Analysis

All groups of all experiments were repeated at least 3 times. Statistical analysis was performed using SPSS software (SPSS Inc.). All results of each experiment were normalized based on the data of the chitosan group. The average is shown along with the standard deviation. Cell adhesion, cell proliferation, and comparative analysis of patterns of test results based qPCR analysis using a two-tailed t-test (two-tailed t -test) performed. A p value of less than 0.05 or 0.01 represents a statistically significant difference between the two groups.

A person skilled in the art will readily appreciate that the present invention can be easily accomplished with the results and advantages and those present in the present invention. The blends of the present invention and the processes and methods for their manufacture are representative of the preferred embodiments, which are exemplary and not limited to the field of the invention. Those skilled in the art will be aware of the modifications and other uses therein. These modifications are intended to be within the spirit of the invention and are defined in the scope of the claims.

The present invention has been described in detail with reference to the embodiments of the present invention, and the invention may be Spirit and scope.

All patents and publications mentioned in the specification are subject to the general skill of the art in the field of the invention. All patents and publications are hereby incorporated by reference to the same extent as if each individual publication is specifically and individually indicated.

The invention as exemplified herein may be practiced in the absence of any element, or a plurality of elements, limitations, or limitations. The nouns and expressions used are as a description and not a limitation of the description, and are not intended to be used to exclude any nouns and expressions that are equivalent to the features or parts thereof shown and described, but Various changes are possible within the scope of the patent application of the invention. Therefore, it is to be understood that the present invention has been disclosed and described herein in accordance with the preferred embodiments and the features of the present invention. Within the scope.

<110> National Taiwan University

<120> Chitosan-biodegradable polymer blending composition and use thereof

<130> 1684-NTU-TW

<160> 4

<170> PatentIn version 3.4

<210> 1

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> Type IV collagen forward primer

<220>

<221> misc_feature

<222> (1)..(18)

<400> 1

<210> 2

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Type IV collagen reverse primer

<220>

<221> misc_feature

<222> (1)..(20)

<400> 2

<210> 3

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> GAPDH Forward Initiator

<220>

<221> misc_feature

<222> (1)..(20)

<400> 3

<210> 4

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> GAPDH reverse primer

<220>

<221> misc_feature

<222> (1)..(20)

<400> 4

Figure 1 shows the results of cell culture substrate transparency and light penetration test. The number on the force measurement meter is still clearly visible after passing through chitosan, PCL 25, PCL 50 and PCL75; however, it is worth noting that the cell culture medium prepared by PCL 100 has an opaque condition (AF) and its light wear The permeability is significantly lower than that of other cell culture media (G).

Fig. 2 shows the ratio of the number of cell attachments after 4 hours of culture on each cell culture medium; wherein the number of cell attachments of the pure chitosan group was set to 100% as a control.

Figure 3 shows the results of MTT assays on BCEC cell cultures on days 1, 3, and 7 of chitosan, PCL 25, PCL 50, and PCL 75. Cell proliferation was directly proportional to the resulting absorbance. Data were obtained from four independent experiments and presented as mean ± standard deviation (mean ± SD). * represents p < 0.05 and ** represents p < 0.01.

Figure 4 shows the results of morphological analysis of BCEC cells after 4 hours of culture, presented as a form factor. ** represents p < 0.01 compared to the chitosan group.

Figure 5 (A-H) shows the results of actin fiber and nucleus staining when BCEC cells were cultured on different cell culture media, with a scale representing 100 microns.

Figure 6 shows the morphological changes of BCEC cells after (A) chitosan, (B) PCL 25, (C) PCL 50 and (D) PCL 75 for seven days, with scale bars representing 200 microns.

Figure 7 shows the expression of (AD) ZO-1 and (EH) calcium-binding protein (N-cadherin) after BCEC cells were cultured on chitosan, PCL 25, PCL 50 and PCL 75 for seven days. The scale bar represents 100. Micron.

Figure 8 shows the proportion of type IV collagen message RNA expression after BCEC cells were cultured on chitosan, PCL 25, PCL 50 and PCL 75 for seven days. Data were obtained from three independent experiments and presented as mean ± standard deviation (mean ± SD). * represents p < 0.05 and ** represents p < 0.01.

Figure 9 shows a micrograph of a chitosan/PCL blend showing that it does not have the characteristics of a hole, and the scale represents 1 micron.

Figure 10 shows a micrograph of (A) chitosan/PHB blend and (B) chitosan/PLA blend showing that it does not have pore characteristics, and the scales all represent 1 micron.

[A brief description of the attachment]

Annex I (A-H) shows the results of staining actin fibers (red) and nuclei (blue) when BCEC cells are cultured on different cell culture media. The scale represents 100 microns.

Annex 2 shows the expression of (AD) ZO-1 and (EH) calcium-adhesive adhesion protein (N-cadherin) after BCEC cells cultured on chitosan, PCL 25, PCL 50 and PCL 75 for seven days. The scale represents 100. Micron. In (A-D), green represents ZO-1 protein, blue represents nucleus; in (E-H), green represents a calcium-adhesion protein (N-cadherin), and blue represents a nucleus.

Claims (16)

The invention discloses a non-porous mixed composition comprising a chitosan and a polyester biodegradable polymer, having a transmittance of more than 30% and a thickness of not more than 2000 micrometers. The blending composition of claim 1, wherein the transmittance is greater than 50%. The blending composition of claim 1, wherein the transmittance is between 70% and 99.99%. The blended composition of claim 1, wherein the thickness is no greater than 1000 microns. The blended composition of claim 1, wherein the thickness is between 5 microns and 250 microns. The blending composition of claim 1, wherein the polyester biodegradable polymer accounts for 25% to 100% of the total. The blending composition of claim 1, wherein the polyester biodegradable polymer accounts for 25% to 75% of the total. The blending composition of claim 1, wherein the polyester biodegradable polymer is selected from the group consisting of polycaprolactone, polylactic acid, and polyhydroxybutyrate. A use of a non-porous blended composition as claimed in claim 1 as a corneal substitute, wherein the blended composition is cultured with a plurality of corneal cells. The use of the ninth aspect of the patent application, wherein the blended composition has a high transmittance At 50%. The use of claim 9, wherein the blended composition has a transmittance of between 70% and 99.99%. The use of claim 9, wherein the blended composition has a thickness of no greater than 1000 microns. The use of claim 9, wherein the blended composition has a thickness of between 5 microns and 250 microns. The use of the ninth aspect of the patent application, wherein the polyester biodegradable polymer in the blended composition comprises from 25% to 100% of the total proportion of the blended composition. The use of the ninth aspect of the invention, wherein the polyester biodegradable polymer in the blended composition comprises from 25% to 75% of the total proportion of the blended composition. The use of the ninth aspect of the invention, wherein the polyester biodegradable polymer in the blend composition is selected from the group consisting of polycaprolactone, polylactic acid and polyhydroxybutyrate.