KR100484337B1 - Modified chitosan and artificial dermis using the same - Google Patents

Abstract

The present invention relates to a method for producing a modified chitosan dermal structure, and more particularly to a method for producing a modified chitosan dermal structure comprising heating a primary dermal structure containing chitosan at 60 to 130 ° C. for 1 to 72 hours. It is about. According to the method of the present invention, a modified chitosan dermal construct having high cell affinity can be provided.

Description

Modified chitosan and artificial dermis using the same

The present invention relates to a method for producing a modified chitosan dermal structure and a modified chitosan dermal structure produced thereby.

Chitosan is a compound that is biodegradable by partially deacetylated chitin. These chitosans, unlike chitin, are readily soluble in dilute acids. In addition, chitosan has antimicrobial activity, but has been reported to have no effect on the protein synthesis capacity of the cell.

The biodegradable, soluble and antimicrobial activity of such chitosan has been used to prepare artificial dermal structures or artificial dermis comprising the same. For example, artificial dermis including a neutralized chitosan sponge prepared by neutralizing a chitosan solution with an alkaline solution and then freeze-drying has been disclosed. In addition, neutralization prepared by neutralizing the chitosan solution with an alkaline solution, neutralizing the collagen solution with an alkaline solution, mixing the neutralized chitosan solution and the neutralized collagen solution obtained above, and lyophilizing the obtained mixed solution. Artificial dermis including chitosan and collagen mixing sponges has been disclosed. Furthermore, in the manufacturing step of the artificial dermis, a neutralized chitosan sponge artificial dermis and a method for producing the same have been disclosed in which the freeze-drying step is performed before the neutralizing step.

However, conventionally used chitosan is excellent in adhesion with human cells, but the suitability for each human cell has been reported to vary depending on the cell. The suitability can be inferred from the ability of human cells to grow on artificial dermis, i.e. the growth of cells. For example, fibroblasts have been shown to exhibit high adhesion to chitosan films, while growth is markedly reduced, and keratinocytes have been reported to exhibit relatively high growth (C. Chatelet et al., Biomaterials , 22, 262, 2001). ).

Therefore, there was a need to develop artificial dermal structures having improved suitability for human cells or artificial dermis comprising the same.

It is an object of the present invention to provide a method for producing an artificial dermal construct with improved suitability for human cells.

It is also an object of the present invention to provide an artificial dermal construct with improved suitability for human cells.

It is also an object of the present invention to provide an artificial dermis with improved suitability for human cells.

The present invention provides a method for producing a modified chitosan dermal structure comprising the step of heating the primary dermal structure comprising chitosan for 1 to 72 hours at 60 ~ 130 ℃.

The heating can be done under vacuum or under reduced pressure. If the reaction time exceeds 72 hours, it is not preferable because the strength of the obtained dermal structure may be reduced and browning may occur. In the case of heating at 60 ° C. or lower, the reaction is not well achieved, and in the case of 130 ° C. or higher, browning occurs, which is not preferable.

 In addition, the primary dermal structure is prepared by processing an acidic chitosan, and any one of the amine groups in the chitosan is in the form of a quaternary ammonium salt of an organic acid. For example, sponge, nonwoven or porous spherical chitosan dermal structures and mixed dermal structures of chitosan and collagen may be included. The acidic chitosan aqueous solution refers to an aqueous chitosan solution prepared by dissolving chitosan in organic acids such as formic acid, acetic acid and propionic acid. Preferably, the said chitosan aqueous solution is obtained by melt | dissolving chitosan in 0.5%-3.5 w / v% in 0.01%-2% of acetic acid aqueous solution. In addition, the chitosan used in the acidic acetic acid aqueous solution has a degree of deacetylation of 45 to 100%, and preferably a molecular weight of 20,000 to 1,000,000 as a weight average molecular weight.

The sponge-type chitosan dermal structure is prepared by, for example, freezing an acidic chitosan solution at -196 ° C to 0 ° C and then freeze-drying. The mixed dermal structure of chitosan and collagen is prepared by, for example, mixing a chitosan solution and a collagen solution and lyophilizing it at -196 ° C to 0 ° C. The collagen may be a type I collagen including telogen collagen and atelocollagen, type II collagen and acidic or basic gelatin in a form modified by heat treatment or chemical treatment. The telogen collagen contains a teromer portion, and the atelocollagen is collagen in which the telomer portion is removed through pepsin treatment.

The collagen solution may be used as a type I collagen (3 mg / ml pH 3.0; Cellmatrix, Gelatin Corp., Osaka, Japan., Acid treatment of pig collagen) as it is. In addition, collagen solution and reconstituted buffer (2.2 g NaHCO ₃ , 4.77 g HEPES (200 mM) / 100 ml 0.05 N NaOH), 10x culture solution with NaHCO ₃ removed (DMEM: F12 = 3: 1, Gibco BRL.DMEM-catalog) No. 12800-058 and F12-Catalog No. 21700-026) can be mixed at a ratio of 8: 1: 1 to adjust the pH to 3.0 to 6.5.

The amount of collagen used can be appropriately adjusted in consideration of the desired hardness and the tissue properties of the wound site. Preferably, the collagen solution and the chitosan solution are mixed and used at a ratio of 1: 0.001 to 1 (v / v).

The method for producing a modified chitosan dermal structure of the present invention may include a neutralization step for removing acid in the obtained modified chitosan dermal structure. In general, when the primary dermal structure is produced by a method such as freeze drying of acidic chitosan, the amine group in the chitosan is present as the quaternary ammonium salt of the organic acid used. The quaternary ammonium salt of the organic acid is presumed to be separated into an organic acid, a free amine group or an acetylated amine group after the preparation of the modified chitosan dermal structure of the present invention. This neutralization step is for removing this organic acid. This step includes an excess of 0.01 to 2.0 M aqueous sodium hydroxide solution, 0.05 to 2.0 M aqueous ammonium hydroxide solution, 30 to 100% alcohol aqueous solution of 0.01 to 2.0 M sodium hydroxide, or 30 to 100% alcohol aqueous solution of 0.05 to 2.0 M sodium hydroxide. It can be carried out by immersing the modified chitosan dermal structure obtained by the method of the present invention in an alkaline solution, followed by washing the excess alkaline solution with distilled water or a phosphate buffer of pH 7.0-7.4.

The present invention also provides modified chitosan dermal structures (hereinafter also referred to as "heat treated" chitosan dermal structures) prepared according to the above method. Modified chitosan dermal structures of the present invention can take many forms depending on the primary dermal structures used in the manufacturing method. For example, sponge, nonwoven or porous spherical modified chitosan dermal structures and mixed modified chitosan dermal structures of chitosan and collagen may be included. In addition, the modified chitosan dermal structure of the present invention may take the form of a collagen-coated form or a film on a substrate. The modified chitosan dermal structure of the present invention has a moderate degree of acetylation between chitosan and chitin, has crosslinking, and has a higher mechanical strength in the hydrated state than the chitosan sponge.

The present invention also provides artificial dermis comprising the modified chitosan dermal structure. The artificial dermis of the present invention may include cells such as, for example, human fibroblasts and vascular endothelial cells. The cells may be seeded at a density range of about ¹ × ^{10 3 to 1} × 10 ⁷ cells / cm ³ .

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these examples are intended to illustrate the present invention by way of example, the present invention is not limited to these examples.

Example

Comparative Example 1

Preparation of chitosan dermal structures

Chitosan (deacetylation degree: 84%, molecular weight; 400,000) was dissolved in a 1v / v% acetic acid aqueous solution to prepare a 1.5w / v% chitosan solution. The solution was depressurized in a sterile hood to remove air bubbles in the solution. 20 ml of the solution was placed in a plastic culture plate having a diameter of 100 mm, and allowed to stand at -70 ° C for 48 hours. The frozen solution was freeze-dried for 72 hours or longer to obtain a chitosan sponge artificial dermal structure. The resulting chitosan sponge artificial dermis structure (circular sponge 10 cm in diameter) was 2.0 M as an alkaline solution for neutralization. It was washed six more times with an aqueous 50% alcohol solution of ammonium hydroxide. The washed chitosan sponge-derived artificial dermal construct was sterilized by gamma ray irradiation (20 kGy / 5 hr, -light source; Co60) and used for cell compatibility experiments on fibroblasts.

Comparative Example 2

Preparation of Chemically Acetylated Chitosan Dermal Constructs

The chitosan dermal structure prepared in Comparative Example 1 was transferred to 100% ethanol, and the free amine of chitosan and acetic anhydride were reacted at room temperature in a molar ratio of 1: 5 for 1 day. It was washed with ethanol and washed with excess 5% ammonia water for 1 hour. Next, after sufficiently washing with excess ethanol and distilled water, lyophilized to prepare an acetylated chitosan dermal structure.

The resulting acetylated chitosan dermal constructs were used for cell compatibility experiments on fibroblasts.

Example 1

Preparation of Modified Chitosan Dermal Structures

Chitosan (deacetylation degree: 84%, molecular weight; 400,000) was dissolved in 1 v / v% acetic acid aqueous solution to prepare a 1.5 w / v% chitosan solution. The solution was depressurized under a clean environment to remove air bubbles in the solution. 20 ml of the solution was put into the plastic culture dish of diameter 100mm, and it left still at -70 degreeC for 48 hours. The frozen solution was freeze-dried for 72 hours or longer to obtain a chitosan sponge-type artificial dermis structure (a circular sponge having a diameter of 10 cm). The obtained chitosan sponge artificial dermal structure was transferred to a vacuum drier, and the temperature was gradually raised to 80 degreeC. Next, after heat treatment at 80 ° C. for 48 hours, the heat source was removed and cooled to room temperature to inject nitrogen. Excess silver acetic acid alkaline solution (2.0M) after heat treatment Wash at least 6 times with 50% aqueous alcohol solution of ammonium hydroxide). This sponge was sterilized and used by gamma ray irradiation (20 kGy / 5 hr,-ray source; Co60).

The resulting modified chitosan dermal structure had soft and flexible properties and was porous.

Physical properties of the prepared modified chitosan dermal structures were measured. Deacetylation degree was calculated by measuring the content of free amine groups according to PVSK (1 / 400N-potassim poly (vinyl sulfate)) method using toluidine blue (toluidine blue) as an indicator (Fig. 1). Physical and chemical changes were investigated by infrared spectroscopy (FT-IR) and X-ray diffraction (XRD) (FIGS. 2 and 3).

In addition, the tensile strength was measured as follows. Tensile strength of each dermal structure was put in desiccator saturated with steam of saturated sodium nitrite solution, and left for 4 hours at room temperature. Next, using a tensile tester, the gage distance was set to 10 mm, fixed with a clamp of 25 to 50 mm width, and measured at a speed of 1 mm for 1 minute. In the wet state, the tensile strength was measured by removing the dermal structure prepared in the desiccant, immersing in PBS for 1 hour, removing excess moisture on the surface, and then drying (Table 1).

As shown in FIG. 1, when the chitosan dermal structure was heat-treated, the degree of deacetylation was reduced to 45%, and less than 30% after 6 days. From this, it can be seen that the content of the free amine group decreases rapidly as the reaction time elapses. On the other hand, since deterioration occurs with the progress of the reaction, a long time reaction is not preferable.

2, the infrared spectroscopic analysis, the present invention is chitosan, chitosan dermal structures in a glass absorption, 1420cm ^-1 and 1720cm ^-1 due to the acetylation of an amine group from 1540cm ^-1 6 of chitosan and alkylation of Absorption due to the carbonyl group introduced into the carbon was observed. This means that not only the acetylation of chitosan but also the crosslinking reaction proceeded through the heat treatment process. In contrast, acetylated chitosan dermal structures showed the same absorption pattern as chitin.

As shown in Fig. 3, as a result of analysis by X-ray diffraction, the chitosan dermal structures of the present invention (Figs. 3b and 3c) are weakened at 2θ = 9.8 depending on the reaction time, and between 2θ = 9.8 and 20. From the wider interval of, it can be seen that a new functional group carbonyl group was introduced to the free amine group of chitosan. On the other hand, in the acetylated base acid dermal structure (FIG. 3D), (010), (110) and (100), which are the crystal planes of chitin, were observed around 2θ = 9.8, 15 and 20, indicating that chitin was regenerated.

The results of the tensile strength measurements are shown in Table 1 below. As shown in Table 1, the chitosan sponge was excellent in the mechanical strength in the dry state, it was found that the mechanical strength is significantly reduced in the hydrated state. On the other hand, the heat treated chitosan sponge was confirmed that this property is significantly improved. In particular, it was found that the elastic modulus of the heat treated chitosan sponge increased by more than 22% in the dry state and about 70% in the hydrated state as compared to the chitosan sponge.

TABLE 1

Tensile Strength Test of Chitosan Dermal Structure (n = 4)

Chitosan sponge Modulus of elasticity (kPa) Elongation (%) dry Chitosan sponge 681.8 ± 4.24 5.66 ± 1.54 Heat Treated Chitosan Sponge 832.9 ± 53.7 7.42 ± 0.94 Sign Language Chitosan sponge 6.25 ± 1.01 46.96 ± 1.44 Heat Treated Chitosan Sponge 10.59 ± 0.16 38.95 ± 9.56

Example 2

Preparation of Modified Chitosan and Collagen Mixed Dermal Structures

Chitosan (deacetylation degree: 95%, molecular weight; 800,000) was dissolved in an aqueous 1.5v / v% acetic acid solution to prepare a solution of 1.25w / v%. This solution was prepared using Type-I collagen (3 mg / ml pH 3.0; Cell matrix, Gelatin Corp., Osaka, Japan, acid-treated pig collagen) solution, reconstituted buffer (2.2 g NaHCO ₃ , 4.77 g HEPES (200 mM) ) / 100ml 0.05N NaOH), NaHCO 10x culture medium ₃ has been removed (DMEM: F12 = 3:. 1, Gibco BRL DMEM- Cat. No. 12800-058, F12- Cat. No. 21700-026) a 8: 1: 1 ratio The mixture was mixed well with a ratio of 0.5: 1 (v / v) and then the solution was transferred into an oven at 37 ° C. and left overnight.

The solution was subjected to a reduced pressure under a clean environment, and freeze-dried to form a sponge-like chitosan and collagen mixed dermis structure as in Example 1, and heat-treated to prepare a sponge-like modified chitosan and collagen mixed dermis structure.

Example 3

Preparation of Collagen-Coated Modified Chitosan Dermal Structure

The modified chitosan sponge obtained in Example 1 was then immersed in 70% ethanol, followed by Type-I collagen (3 mg / ml pH 3.0; acid treated with pig matrix, Cell matrix, Gelatin Corp., Osaka, Japan). Composition buffer (2.2 g NaHCO ₃ .4.77 g HEPES (200 mM) / 100 ml 0.05 N NaOH), 10x culture without NaHCO ₃ (DMEM: F12 = 3: 1, Gibco BRL.DMEM-Catalog No. 12800-058, F12 -Catalog No. 21700-026) was transferred to the collagen solution mixed in a 8: 1: 1 ratio and mixed well with a shaker. This solution was transferred into an oven at 37 ° C. and left overnight.

The solution was subjected to a reduced pressure under a clean environment, and freeze-dried as in Example 1 to form a collagen-coated sponge chitosan dermal structure, and heat-treated to prepare a sponge-type modified chitosan dermal structure coated with collagen.

Example 4

Preparation and Cell Compatibility of Chitosan Films

In this example, modified chitosan films, chitosan films and acetylated chitosan films were prepared, respectively, and examined for human fibroblasts' cellular suitability for these film dermal structures.

(1) Preparation of Modified Chitosan Film

Modified chitosan film dermal structures were constructed as follows. First, a cover slip (diameter 12 mm, Fisher) was immersed in a 0.2% w / v chitosan solution of 1v / v% acetic acid aqueous solution, and then naturally dried at room temperature for 1 week to prepare an acidic chitosan film. 2.0M acid chitosan film Neutralize with 50% aqueous ethanol solution of ammonium hydroxide and wash well with distilled water. Modified chitosan film of the present invention was prepared through the heat treatment process of the acidic chitosan film of Example 1. Meanwhile, the acetylated chitosan film was prepared through the acetylation process of Comparative Example 2. That is, the acidic chitosan film was neutralized with 50% aqueous ethanol solution of 0.2 M ammonium hydroxide and washed sufficiently with distilled water.

Each chitosan film-coated cover slip obtained as above was sterilized for 48 hours with 70% ethanol, washed twice with phosphate buffer solution and once with culture before use.

(2) Compatibility of Human Fibroblasts with Modified Chitosan Films

The suitability of neonatal foreskin-derived fibroblasts (P = 3) with the chitosan film and acetylated chitosan film of the present invention was examined.

First, fibroblasts isolated from the dermal portion of the foreskin were cultured in F-medium (DMEM: F-12 = 3: 1, 10% FBS, 1% penicillin-streptomycin). The cover slip coated with each chitosan film prepared above was transferred to a 48-well culture dish, and cultured human fibroblasts (P = 3) were inoculated at a concentration of 3 × 10 ⁴ cells / ml / well. As a positive control, a cover slip coated with 1% gelatin was used. The cover slips inoculated with these fibroblasts were allowed to stand for 3 to 5 hours at 37 ° C. with 5% CO _2, and then 50 μl of DMEM culture was added. After incubation for 24 hours, 1 ml of medium was added to each culture dish and incubated.

Morphological changes of the cells 1 and 3 days after cell inoculation were observed under a microscope (FIG. 4). In addition, the growth rate of the cells on each cover slip after 3 days of inoculation was measured using the empty method (Fig. 5).

As shown in FIG. 4, fibroblasts inoculated into the chitosan film and the acetylated chitosan film adhered 1 day after inoculation, but did not grow in a stable form. On the other hand, the modified chitosan film of the present invention showed a form very similar to the control. It was also growing well after three days.

As shown in FIG. 5, the result of the empty analysis showed that the number of fibroblasts grown in the modified chitosan film of the present invention was about twice or more the number of fibroblasts grown in the chitosan film and the acetylated chitosan film.

Example 5

Cell Suitability of Chitosan Dermal Constructs and Modified Chitosan Dermal Constructs

One. Cell suitability observation through hematoxylin and eosin staining

Transfer the dermal structures prepared in Examples 1, 2, 3, Comparative Example 1 and Comparative Example 2 to 24 wells, and then inoculate fibroblasts isolated from the foreskin (foreskin) at a concentration of 2x10 ⁵ cells for each dermal structure. It was. 95% humidity in medium supplemented with 10% fetal bovine serum (JRH Biosciences catalog number 12103-78P) (DMEM: F12 = 3: 1, Gibco BRL. The cells were maintained at 37 ° C. in a 5% CO ₂ incubator for 2 hours to allow the cells to attach to the dermal structures in a thermostat. Thereafter, the medium was added only to the extent that the dermal structures did not dry out, and the culture was continued for one day and the medium was added the next day so that the dermal structures were completely submerged. The medium was changed once every two days. Two weeks later, a portion of the dermal structures inoculated with fibroblasts were taken and fixed with 10% formalin / phosphate buffer solution, followed by dehydration and clarification of the fixed tissue, embedded in paraffin, and sections made of 4 μm thick. Matoxylin and eosin staining were performed. As a result, the morphological characteristics such as the density of fibroblasts were read by staining the nucleus (purple) and cytoplasm (pink), and the remaining part of the form of attached cells and the degree of secretion of extracellular matrix by the attached cells. Was confirmed by scanning electron micrograph. In addition, the cell growth and division capacity of fibroblasts were compared by cell density using the empty cell assay.

As shown in Fig. 6, the fibroblasts mostly form a colony on the chitosan sponge, and it was hardly seen that the cells proliferated in good shape. On the other hand, in the modified chitosan sponge dermal structure of the present invention, the fibroblasts proliferated well on the sponge surface and inside. In particular, the surface proliferated to a monolayer, and the cell morphology appeared relatively stable. In addition, in the case of collagen-coated chitosan sponges and mixed sponges of modified chitosan and collagen, the degree of proliferation differed more clearly.

2. Compliance testing through empty staining and DNA analysis

(One) Empty Analysis

Fibroblasts (2 × 10 ⁵ cells / 50 μl) were dispensed into dermal structures with a diameter of 8 mm as in 1 and incubated for 2 weeks. Next, the medium was removed, MTT (3- [4,5-Dimethyl-2-thiazolyl] -2,5-diphenyl-2H-tetrazol bromide) dye (1ml / 24well) was dispensed, and again at 37 ° C. Incubated for hours. After incubation, the dermal structures were soaked in lysis buffer (EtOH: DMSO = 1: 1) to lyse the cells with stirring for 30 minutes. Among them, the supernatant was taken, and the OD (540 nm) value was measured to confirm the proliferation of the cells. The results are shown in FIG.

(2) DNA analysis

Fibroblasts (2 × 10 ⁵ cells / 50 μl) were dispensed into the dermal structures with a diameter of 8 mm as shown in 1 above, and then the number of proliferated cells was calculated by measuring the change in genomic DNA amount. Two weeks after dispensing, the cultured dermal structures were washed three times or more with PBS, and then lyophilized. To this, Proteinase K (0.5mg / ml proteinase K, 0.1mg / ml SDS in buffer solution of 50mM Tris-HCl, 0.1M EDTA, 0.2M NaCl, pH7.4) was added thereto at 55 ° C. per day. Reacted for a while. The support was removed, the supernatant was removed by centrifugation, suspended in PBS, and 25 µl of 20% SDS and 7 µl of Protease K were added and reacted at 55 ° C. for 1 hour. After the reaction, the same amount of phenol / chloroform was added and centrifuged, followed by the same amount of chloroform and centrifuged. 500 µl of isopropyl alcohol and 100 µl of 3M sodium chloride solution were added to the supernatant, and the mixture was reacted at -20 ° C for at least 1 hour, and then, the supernatant was removed by centrifugation, and 500 µl of 70% ethanol was added and centrifuged again. The supernatant was removed, dried in air and then suspended in 20 μl of tertiary distilled water. Using a flowometer (Kontron SFM 250), the autozero was measured with assay solution A (Hoechst 33258 in buffer solution 2ml) under conditions of excitation 360 nm and emission 458 nm. Caught The calibration curve was captured with detection solution A (2 ml) and 4 μl of calf thymus DNA and a standard curve was drawn with calf thymus DNA. The DNA suspension prepared above was measured under the same conditions, and the number of cells was again calculated by calculating the amount of DNA through a standard curve.

The results are shown in Table 2.

TABLE 2

Growth of fibroblasts according to heat treatment and collagen content

Sponge Collagen Coating Fibroblast count (dog) ^a Chitosan - 296 0.01% 1,778 0.1% 8,078 Heat treatment chitosan - 578 0.01% 8,100 0.1% 9,875 Heat Treated Chitosan and Collagen Blending Sponge 13,975

^a : Value calculated from 7.1 pg fibroblast / cell DNA.

As shown in Figure 7 and Table 2, it was found that the growth of the fibroblasts was 95% only by heat treatment of the chitosan sponge. The number of fibroblasts attached and grown to chitosan dermal structures increased more than five times depending on the content of collagen. When collagen was added to the modified chitosan of the present invention, 13 to 23 times increased depending on the concentration. In other words, even when coated with a low collagen concentration of 0.01% on the heat treated chitosan sponge, it was confirmed that the growth of fibroblasts was more improved than that coated with 10 times the collagen concentration on the chitosan sponge.

According to the method for producing a modified chitosan dermal construct of the present invention, a modified chitosan dermal construct having high cell affinity can be provided.

According to the modified chitosan dermal construct of the present invention, it is possible to provide chitosan dermis effective for wound healing due to high cellular suitability.

According to the artificial dermis comprising the modified chitosan dermal structures of the present invention, it can be used to effectively heal wounds.

1 is a view showing the change in the content of the free amine group of chitosan sponge with heat treatment time,

2 is an infrared spectral spectrum of chitosan and modified chitosan dermal structures of the present invention, (a) modified chitosan, (b) acetylated chitosan, (c) chitin and (d) chitosan,

Figure 3 is a view showing the results of X-ray diffraction analysis of chitosan and modified chitosan dermal structures of the present invention, (a) chitosan, (b) modified chitosan heat-treated for 3 days, (c) modified chitosan heat-treated for 6 days And (d) acetylated chitosan,

Figure 4 is an optical micrograph of human fibroblasts in the chitosan film of the present invention,

5 is a view showing the results of analyzing the affinity of human fibroblasts to the modified chitosan film through the empty spectrum analysis,

Figure 6 is a hematoxylin and eosin stained photograph of human fibroblasts cultured in chitosan sponge and modified chitosan sponge (100-fold photo), A: neutralized chitosan sponge, B: modified chitosan sponge, C: coated with 0.01% collagen Neutralized chitosan sponge, D: modified chitosan sponge coated with 0.1% collagen, E: modified chitosan and collagen mixed sponge,

7 is a diagram showing the results of analyzing the affinity of human fibroblasts to the modified chitosan sponge through an empty spectrum analysis.

Claims (7)

A method for producing a chitosan dermal structure comprising heating a primary dermal structure comprising chitosan under vacuum at 60 to 130 ° C. for 1 to 72 hours. The primary dermis structure may be a sponge-type chitosan dermis structure, a nonwoven chitosan dermis structure, a porous spherical chitosan dermis structure, a sponge-like, non-woven or porous spherical chitosan and collagen-derived dermis structure or a collagen-coated sponge, nonwoven form or Method for producing a porous spherical chitosan dermal structure. delete The method of claim 1, wherein the amine group in the chitosan of the primary dermal structure comprises a quaternary ammonium salt of acetic acid. delete delete delete delete