KR20160125640A

KR20160125640A - Photocrosslinked collagen hydrogel and method for manufacturing the same

Info

Publication number: KR20160125640A
Application number: KR1020150056309A
Authority: KR
Inventors: 황석연; 허지승; 하철원
Original assignee: 서울대학교산학협력단; 사회복지법인 삼성생명공익재단
Priority date: 2015-04-22
Filing date: 2015-04-22
Publication date: 2016-11-01

Abstract

Provided are a photocrosslinked collagen hydrogel, and a preparation method thereof. The photocrosslinked collagen hydrogel includes a photocrosslinked bond induced by a light-sensitive agent. The preparation method of the photocrosslinked collagen hydrogel comprises the following steps: preparing a collagen solution; adding a light-sensitive agent to the collagen solution; forming a collagen hydrogel by culturing the collagen solution; and emitting ultraviolet rays to the collagen hydrogel.

Description

[0001] PHOTOCROSSLINKED COLLAGEN HYDROGEL AND METHOD FOR MANUFACTURING THE SAME [0002]

The present invention relates to a photo-crosslinked collagen hydrogel and a process for preparing the same, and more particularly to a photo-crosslinked collagen hydrogel including a photo-crosslinking induced by a photosensitizer and a process for producing the same.

Meniscus is a fibrocartilaginous tissue present between the knee joints, which contributes significantly to knee health by playing an important role in shock absorption, load distribution, and joint stabilization. The cartilage tissue consists of water (72%), collagen fibers (22%), and proteoglycan (0.8%). Their biochemical composition varies depending on the anatomical region of meniscus with different characteristics. The external meniscus consists mostly of type I collagen and provides tensile strength. The internal meniscus consists of type II collagen and proteoglycan, which plays an important role in shock absorption.

Manicus rupture is known to occur easily due to aging and severe exercise. When a meniscus rupture occurs, the regeneration capacity of the inner meniscus region is limited because it is an avascular tissue with limited natural healing ability. In the past, meniscus was considered to be an unimportant tissue residue that arises from the joint formation process. However, it has recently been shown that the elimination of meniscus amplifies the development of degenerative arthritis, thus emphasizing the importance of meniscus in homeostasis. A variety of studies have been conducted to develop treatments for meniscus rupture. Allograft transplantation, meniscectomy, and meniscus substitution have been proposed, but the regeneration has not yet been clinically solved. Recently, collagen-based scaffolds have been applied to meniscus tissue engineering. Despite its well-known technology, the use of natural collagen scaffolds in the clinical field is very limited due to the significantly low strength and rapid deterioration of physical properties.

In order to solve the above problems, the present invention provides a photo-crosslinked collagen hydrogel having excellent physical properties.

The present invention provides a photo-crosslinked collagen hydrogel which can be used for biotissue regeneration.

The present invention provides a method for producing the photo-crosslinked collagen hydrogel.

Other objects of the present invention will become apparent from the following detailed description and the accompanying drawings.

The photocrosslinked collagen hydrogel according to embodiments of the present invention includes photocrosslinking induced by a photosensitizer.

A method of preparing a photocrosslinked collagen hydrogel according to embodiments of the present invention includes the steps of preparing a collagen solution, adding a photosensitizer to the collagen solution, culturing the collagen solution to form a collagen hydrogel, And irradiating the collagen hydrogel with ultraviolet light.

The collagen hydrogel according to the embodiments of the present invention is excellent in physical properties such as mechanical properties since it includes photo-crosslinking. In addition, cytotoxicity is very low because the photocrosslinking is induced by a biocompatible material such as riboflavin. The collagen hydrogel can retain its physical properties and shape for a long time even if it is inserted into a human body. Therefore, the collagen hydrogel can be used as a scaffold for regeneration of living tissues, and can be applied to meniscus regeneration in particular.

Figure 1 schematically shows the photocrosslinking of collagen with riboflavin in steps.
FIG. 2 is a flow chart schematically showing a method of producing a photo-crosslinked collagen hydrogel according to an embodiment of the present invention.
Figure 3 shows the mechanical properties of riboflavin induced light cross-linking of collagen hydrogel.
4 is a graph showing the results of measurement of CD for each type of collagen hydrogel.
Figure 5 shows the expansion ratios of the collagen hydrogel according to the embodiments of the present invention and the collagen hydrogel according to the comparative example.
Figure 6 shows the elastic modulus of six types of collagen hydrogels with different treatment times.
Figure 7 shows the viscous modulus of six types of collagen hydrogels with different treatment times.
Figure 8 shows the effect of ultraviolet exposure time on cell viability.
FIG. 9 shows the viability calculated by the ratio of the number of living cells to the total number of cells.
10 is a graph comparing shrinkage ratios of collagen hydrogel (COL) and riboflavin-induced light-crosslinked collagen hydrogel (COL-RF).
Figure 11 shows the effect of riboflavin-induced photocrosslinked collagen hydrogel on enzyme degradation.
Figure 12 schematically shows the synthesis of cross-linked hyaluronic acid and its application in collagen hydrogel.
13 shows the result of carbazole analysis for confirming the release of hyaluronic acid from the collagen hydrogel.
Figure 14 shows FT-IR spectra of cross-linked hyaluronic acid, photo-crosslinked collagen hydrogel containing crosslinked hyaluronic acid, and photo-crosslinked collagen hydrogel not containing hyaluronic acid.
FIG. 15 shows the results of PCR analysis of fibroblast chondrocytes cultured in COL hydrogel, COL-RF hydrogel, COL-RF-HA hydrogel.
Figure 16 shows H & E staining and saprinin-o staining of rabbit chondrocytes in collagen hydrogel after 3 weeks of culture.
Figure 17 shows the entire image of fibrocartilage cells encapsulated by COL, COL-RF, and COL-RF-HA hydrogels after 4 weeks of injection.
Fig. 18 shows the results of various coloring analysis of in vivo tissues.

Hereinafter, the present invention will be described in detail with reference to examples. The objects, features and advantages of the present invention will be easily understood by the following embodiments. The present invention is not limited to the embodiments described herein, but may be embodied in other forms. The embodiments disclosed herein are provided so that the disclosure may be thorough and complete, and that those skilled in the art will be able to convey the spirit of the invention to those skilled in the art. Therefore, the present invention should not be limited by the following examples.

The sizes of the elements in the figures, or the relative sizes between the elements, may be exaggerated somewhat for a clearer understanding of the present invention. In addition, the shape of the elements shown in the drawings may be somewhat modified by variations in the manufacturing process or the like. Accordingly, the embodiments disclosed herein should not be construed as limited to the shapes shown in the drawings unless specifically stated, and should be understood to include some modifications.

In the drawings, the number on the left of the slash indicates the incubation time, and the number on the right side of the slash indicates the ultraviolet exposure time. For example, 10/1 shown in the figure represents a collagen hydrogel prepared by setting the incubation time to 10 minutes and the ultraviolet exposure time to 1 minute.

The photosensitizer may comprise riboflavin. The riboflavin is a biocompatible vitamin B2 and has very little cytotoxicity as compared with the photo-initiator used in the prior art. The collagen photo-crosslinking with riboflavin improves the mechanical properties and delays the deterioration of the properties of the collagen scaffold. The riboflavin may induce intrahelical crosslinking of amino acids such as arginine, histidine, and lysine.

The photo-crosslinked collagen hydrogel may further comprise crosslinked hyaluronic acid powder. The hyaluronic acid may be introduced into the collagen hydrogel to supplement the beneficial physiological activity factor for meniscus regeneration. Further, in order to continuously retain hyaluronic acid in the riboflavin-induced light-crosslinked collagen hydrogel, crosslinked hyaluronic acid may be used.

The photocrosslinked collagen hydrogel can be used as a scaffold for regenerating tissues in the human body. The tissue may be a meniscus.

FIG. 1 schematically shows photo-crosslinking of collagen having riboflavin in a stepwise manner, and FIG. 2 is a flow chart schematically showing a method of producing a photo-crosslinked collagen hydrogel according to an embodiment of the present invention.

Referring to FIGS. 1 and 2, the method for preparing a photo-crosslinked collagen hydrogel comprises the steps of preparing a collagen solution (S10), adding a photosensitizer to the collagen solution (S20), culturing the collagen solution Forming a collagen hydrogel (S30), and irradiating the collagen hydrogel with ultraviolet light (S40).

The photosensitizer may comprise riboflavin. The riboflavin may induce internal helical bridging of the amino acid in the collagen by the ultraviolet irradiation.

The method of preparing the photo-crosslinked hydrogel may further include adding crosslinked hyaluronic acid powder to the collagen solution. The crosslinked hyaluronic acid powder may be formed by mixing hyaluronic acid and hexamethylenediamine, followed by culture, lyophilization, and grinding.

[ Example ]

Fiber chondrocyte Isolation and Culture ( Fibrochondrocytes isolation and culture)

Fibrous chondrocytes were isolated from New Zealand white rabbits. The meniscus tissue was removed from the knee joints of the rabbits, and a disinfected razor blade was used to dissect the inner margin of the meniscus. Separated cartilage pieces were stained with 0.2% collagenase in DMEM / F12 (Dulbecco's modified Eagle's medium, nutrient mixutre F-12, Gibco) supplemented with 10% fetal bovine serum (Gibco) Strept (Pen strep) for 16 hours. The cells were filtered through a 70 [mu] m cell filter. The isolated cells were maintained in DMEM / F12, 10% FBS, 100 U / ml Fenstreb, 50 μg / ml vitamin C, and 100 μM NEAA and incubated in 5% CO 2 at 37 ° C.

Collagenous Light bridging Cell encapsulation (Photo- crosslinking of collagen and cell encapsulation)

Collagen hydrogel (0.27% w / v) was prepared by mixing 10x PBS (10x phosphate-buffered saline, Gibco) and collagen solution (0.3% w / v in 0.01N acetic acid) Riboflavin solutions (five different final concentrations of 0.001, 0.006, 0.01, 0.02, and 0.1%) were added and neutralized with 1 M NaOH. The hydrogel was incubated at 37 ° C for 10 minutes or 20 minutes and exposed to ultraviolet light (3.5 mW / cm ² ) for 1, 3, or 5 minutes. Collagen hydrogel without riboflavin was used as a control (control). For fiber chondrocyte encapsulation, the cells are pipetted with a neutralized collagen precursor solution (150 μl) at a concentration of 1 × 10 ⁶ cells / construct. The cell-containing collagen solution with riboflavin is then incubated for 20 minutes and photo-crosslinked for 3 minutes. For the control (control), the cell-containing collagen solution was incubated for 1 hour. The constructs were incubated in a fibroblast chondrocyte incubator at 37 < 0 > C for 3 weeks with CO ₂ incubation.

Bridged Synthesis of Hyaluronic Acid crosslinked hyaluronic acid)

4% w / v hyaluronic acid in distilled water was mixed with HMDA (hexamethylenediamine, Sigma). The molar ratio of HMDA to the carboxyl group of hyaluronic acid was 1: 1. EDC (1-ehtyl-3- [3- (dimethylamino) propyl] carbodiimide, Thermo Scientific) and HOBt (1-hydroxybenzotriazole monohydrate, GL Biochem Ltd.) were added to a mixed solution of hyaluronic acid and HMDA, Lt; / RTI > for 2 hours. After the cross-linking reaction was completed, the hydrogel was dialyzed against 1x PBS (1x phosphate-buffered saline, Gibco) for 3 days to remove unreacted material and lyophilized for 2 days. The dried hydrogel was pulverized into powder and stored at -20 < 0 > C. To confirm the crosslinking of hyaluronic acid, the hydrogel was degraded by hyaluronidase (100 U / ml) and 1 H NMR analysis was performed. Cross-linked hyaluronic acid was added to the collagen scaffold.

[Property Analysis]

Circular Dichroism Measurement dichroism (CD) measurement)

The circularly dichromatic (CD) spectrum was measured to analyze the time-dependent triple helix structure of the photocrosslinked collagen scaffold. A CD detector (Applied Photophysics Chirascan Plus) equipped with a 150-W xenon lamp was used for the measurement and the spectrum range was 210 to 250 nm.

Rheology Rheological analysis

The viscoelastic properties of the hydrogel were measured using a strain-controlled rotational rheometer (TA Instrument, ARES). The hydrogel was prepared with a diameter of 8 mm and a height of 3 mm. For frequency sweep measurements, the strain was maintained at 0.2% and the frequency was varied from 0.1 to 100 rad / s. The temperature was maintained at 30.0 [deg.] C during the measurement.

Swelling ratio

The hydrogel was swollen overnight in PBS and wet weight was measured after removal of moisture from the surface with weighing paper. The dry weight of the lyophilized hydrogel was measured. The expansion ratio was calculated by the following equation.

Expansion ratio (Q) = wet weight of equilibrium hydrogel in PBS / weight of dry hydrogel

Live / dead assay and contraction assay

Biopsy / cell survival / cytotoxicity kits were used according to the manufacturer's protocol. Live cells were stained with green fluorescent calcein AM and dead cells were stained with red fluorescence ethidium homodimer-1 (Ethd-1). Cell images were collected by a Zeiss LSM 720 confocal microscope. In order to determine the degree of shrinkage, the cell-containing hydrogel (7x10 ⁵ cells / construct) were measured for the Image J software, at various times the size.

Scaffold Scaffold degradation

The hydrogel was treated with 10 U / ml Type I collagenase. Time course data was collected by weighing the remaining hydrogel (n = 3) while changing the fresh collagenase daily. Collagen hydrogel was used as a control (control).

Fourier-transform infrared spectroscopy (FT-IR)

The FT-IR spectrum of each hydrogel was obtained to determine the retention of hyaluronic acid in the collagen hydrogel. Spectra were recorded using ATR-FTIR (attenuated total reflection infrared spectrometer, Bruker Tensor 27) from 4000 to 650 cm ^-1 . For comparison, lyophilized cross-linked hyaluronic acid and collagen hydrogels were also analyzed.

Carbazole ( Carbazole assay)

In order to determine the retention of crosslinked hyaluronic acid in the hydrogel, the release amount of crosslinked hyaluronic acid by carbazole analysis was measured. Non-crosslinked (non-crosslinked) hyaluronic acid was used as a control (control). Uncrosslinked or crosslinked hyaluronic acid in a final concentration of 1% w / v in collagen hydrogel was cultured in distilled water. Water was collected after 6 days and carbazole analysis was performed using a known concentration of D-glucuronic acid to draw a standard curve.

real time PCR Analysis (Real-time PCR analysis)

Gene expression of type I collagen, type II collagen, and aggrecan was analyzed after incubation for 3 weeks in a hydrogel structure (n = 3). Total RNA was extracted from each hydrogel as Trizol and reverse transcribed into cDNA using the M-MLV cDNA synthesis kit. Using the PCR Master Mix (SYBR Green PCT Mastermix) and the real time PCR system (ABI StepOnePlus Real time PCT system), the cDNA was amplified by rabbit specific primers for type I collagen, type II collagen, and aggrecan. GAPDH was used as a control (control) and gene expression levels were calculated. Rabbit specific primers are shown in Table 1 below.

gene The primer 5'-3 ' GAPDH F: TCA CCA TCT TCC AGG AGC GA
R: CAC AAT GCC GAA GTG GTC GT Type I collagen F: CTG ACT GGA AGA GCG GAG AGT AC
R: CCA TGT CGC AGA AGA CCT TGA Type II collagen F: TTC ATG AAG ATG ACC GAC GA
R: GAC ACG GAG TAG CAC CAT CG Aghrecane F: CCT TGG AGG TCG TGG TGA AAG G
R: AGG TGA ACT TCT CTG GCG ACG T

Histological analysis Histological analysis)

After culturing the chondrocyte cells in the hydrogel for 3 weeks, the constructs were fixed in 4% paraformaldehyde, snap-cooled in OCT embedding media cooled with liquid nitrogen (snap -frozen), cryosectioned into 10μm thick sections. The sections were stained with hematoxylin for 3 minutes and then stained with Eosin Y for 1 minute. For the Safranin-O staining, the sections were stained with 0.1% sapranin-O solution after hematoxylin staining for 3 minutes. Sections were also stained with Masson's trichrome for collagen detection.

Statistical analysis

All data are presented as means ± SD. The statistical significance between the groups was determined by the students' t test using Microsoft Excel.

[Analysis]

Photocrosslinked Collagen Hydrogel Preparation and characterization of photo-crosslinked collagen hydrogels

Acid dissolved collagen solution can form physical cross-linking in neutralization and 37 ° C culture. A two step gelation method was used for riboflavin induced chemical crosslinking. First, the neutralized collagen solution was incubated at 37 [deg.] C to form self-aligned triple intrahelical crosslinks. The incubation time was changed from 10 minutes to 20 minutes to achieve complete physical crosslinking from partial physical crosslinking of the collagen. Collagen hydrogel was formed by physical crosslinking of collagen through neutralization and 37 ℃ incubation. The collagen hydrogel was exposed to ultraviolet light (3.5 mW / cm ² ) along with riboflavin for cross-linking amino acids between collagen fibrils. Riboflavin-mediated photo-crosslinked collagen hydrogels exhibit stronger material properties compared to physically cross-linked collagen hydrogels. In the presence of oxygen and ultraviolet light, riboflavin can induce internal spiral bridging of amino acids such as arginine, histidine, and lysine.

To determine the appropriate riboflavin concentration to produce a photocrosslinked collagen hydrogel (COL-RF) with improved mechanical properties as compared to a physically crosslinked collagen hydrogel (COL), the riboflavin concentration was adjusted to 0.001, 0.005, 0.01 , 0.02, and 0.1%, respectively. After performing photocrosslinking of collagen by ultraviolet exposure for 3 minutes, frequency sweep measurement was performed. In Figure 3, the results show that photo-crosslinking with riboflavin can significantly increase the elastic modulus of collagen. In physically cross-linked collagen hydrogel (COL), the elastic modulus increased steadily depending on the frequency, but the photo-crosslinked collagen hydrogel (COL-RF) was more elastic than the physically crosslinked collagen hydrogel (COL) The elastic modulus value was constant in the frequency range of 0.1 to 100 Hz. The optimum riboflavin concentration to increase the modulus was 0.01% and was 5.5 times stronger than the physically cross-linked collagen hydrogel (COL) at a frequency of 0.25 rad / s.

The effect of incubation time was investigated prior to UV exposure. The neutralized collagen solution was incubated at 37 占 폚 for 10 minutes or 20 minutes prior to exposure to ultraviolet light (1, 3, or 5 minutes). As the incubation time increased from 10 minutes to 20 minutes, the hydrogel became turbid, indicating increased triple helix formation. The ultraviolet exposure time did not affect the hydrogel appearance.

Figure 4 shows the results of a circular dichroism measurement performed to determine triple helix formation at 10 min and 20 min culture. 4, a collagen solution (acidic condition), COL (physically crosslinked collagen), COL-RF (riboflavin induced light crosslinked collagen) were incubated at 37 DEG C for 10 or 20 minutes prior to UV exposure, . The ultraviolet exposure time for COL-RF was fixed at 3 minutes. COL showed a negative peak at 190 nm and a positive peak at 230 nm. Similarly, the CD spectrum of COL-RF exhibited a maximum peak at 230 nm and a minimum peak at 190 nm. COL-RF had a triple helix structure despite pre-incubation time.

Hydrogels were characterized for water retention. COL had a higher expansion ratio of 104 than the COL-RF group (see FIG. 5). In addition, the preincubation period did not change the expansion ratio prior to the photo-crosslinking. Riboflavin and ultraviolet light treated collagen gel had a much higher initial viscosity. COL-RF exhibited a small but significant increase in elastic modulus (see FIGS. 6 and 7). As the incubation time and exposure time increased, the elastic modulus tended to increase. In particular, the elastic modulus increased significantly when incubated for 20 minutes and exposed to ultraviolet light for 3 and 5 minutes. For all samples, the loss modulus, which is indicative of viscous loss of energy, was nearly one order lower than the storage modulus. This result indicates that the collagen gel behaves almost like a solid structure.

Next, the biocompatibility of the scaffolds depended on ultraviolet exposure time was investigated by encapsulating rabbit chondrocytes in COL-RF (see Figures 8 and 9). Free radicals generated during photopolymerization can cause cell death. COL-RF with fibrocartilage cells were exposed to ultraviolet light for 1, 3, and 5 minutes, respectively, and viability analysis was performed within 24 hours. The viability of the cells at 1 and 3 minutes was almost 93 to 94%, and no significant difference was found between the two conditions. However, when cells were exposed to ultraviolet light for a longer time (5 minutes), cell viability decreased rapidly to 70%. These results indicate that riboflavin affects cell viability when light irradiation time exceeds 5 minutes. Experiments performed with 20 min incubation and 3 min UV exposure showed improved mechanical properties and higher viability.

Contraction assay and degradation rate analysis

To assess cell mediated hydrogel shrinkage, 7x10 ⁵ fibrocartilage cells were encapsulated in COL and COL-RF hydrogels. In the COL hydrogels, cell mediated hydrogel constriction decreased to less than 60% of the original size within 4 hours. In contrast, the COL-RF hydrogels resisted chondrocyte mediated contraction and the COL-RF hydrogel retained 90% of its original size even after 66 hours (see FIG. 10).

COL and COL-RF hydrogels (see Fig. 11). COL and COL-RF hydrogels were placed in 0.1 mg / ml Type I collagenase and incubated at 37 ° C. The remaining hydrogel mass was measured at each hour. The weight of the COL hydrogel was significantly reduced compared to the weight of the COL-RF hydrogel. COL-RF hydrogels were more resistant and showed increased stability compared to COL even in an enzyme-enriched environment.

COL -RF Bridged Incorporation of hyaluronic acid crosslinked HA into COL -RF)

Hyaluronic acid was introduced into the collagen hydrogel to supplement the beneficial physiologically active elements for meniscus regeneration. Also, in order to keep hyaluronic acid constantly in COL-RF, crosslinked hyaluronic acid was used (see Fig. 12). With support from EDC and HOBt, HMDA was reacted with a carboxylic acid group cross-linking agent in hyaluronic acid repeat units. At the end of the reaction, the product was dialyzed, lyophilized, and ground in powder form. The crosslinked hyaluronic acid in powder form was mixed with the collagen precursor solution, incubated for 20 minutes, and irradiated with ultraviolet light for 3 minutes. The hyaluronic acid contained in COL-RF was formed into a visible bead shape.

To determine hyaluronic acid retention in the construct, carbazole analysis and FT-IR were performed. First, carbazole analysis was performed to determine the amount of crosslinked hyaluronic acid released from COL-RF (see FIG. 13). Uncrosslinked hyaluronic acid contained in COL-RF was used as a control (control). The initial amount of hyaluronic acid was 1.5 mg / construct, and each sample was immersed in distilled water for 6 days. After 6 days, distilled water was collected and the release of hyaluronic acid was analyzed by carbazole. As a result, the crosslinked hyaluronic acid and non-crosslinked hyaluronic acid exhibited mass percentages of 4.49 and 31.41% of released hyaluronic acid, respectively.

FT-IR measurement was performed for the structural analysis of COL-RF-HA, and the amount of hyaluronic acid present in the hydrogel was confirmed after 7 days (see FIG. 14). Cross-linked hyaluronic acid and COL-RF were analyzed for comparison. Peaks at 1252 and 1408 cm < ^-1 > in the COL-RF-HA spectrum may be due to symmetrical CO stretching modes of the planar carboxyl groups in glucuronic acid and hyaluronic acid.

COL And COL -RF Within Fibrocartilage Gene expression analysis and histological staining of fibrochondrocytes in COL and COL-RF)

Isolated rabbit chondrocytes (P2) were encapsulated in COL hydrogel, COL-RF hydrogel, and COL-RF-HA hydrogel for 3 weeks. Real-time PCR was performed on the chondrocyte cells cultured for three weeks in three groups of collagen hydrogels (COL, COL-RF, and COL-RF-HA) (see FIG. Expression of type I collagen, type II collagen, and aggrecan was assayed to assess whether the fibrocartilage cells in each hydrogel could promote ECM production. The COL-RF-HA group showed an increase in total gene expression levels compared to the other two groups. There was a significant increase in COL-RF-HA, except for slightly reduced type I gene expression in COL-RF. In addition, the expression of aggrecan gene was significantly increased in COL-RF-HA as compared to COL and COL-RF. To evaluate ECM matrix production, histological sections of each hydrogel with fibrocartilage cells cultured for 3 weeks were stained with H & E and Saf-O (see FIG. 16). Compared to COL-RF hydrogels, large cell populations were found in COL-RF-HA. After 3 weeks of culture, sGAG, one of the major cartilage ECM components, was more expressed in COL-RF-HA than COL-RF and COL. These results indicate that hyaluronic acid enhances cartilage ECM production in riboflavin induced photocrosslinked hydrogel systems.

Body Menicus Play vivo meniscus regeneration)

The possibility of COL hydrogel, COL-RF hydrogel, and COL-RF-HA hydrogel to support body tissue formation was investigated. Rabbit fiber chondrocytes were encapsulated in COL, COL-RF, and COL-RF-HA and implanted for 4 weeks subcutaneously in mice without hair. The COL-RF-HA hydrogel remained in its original form for more than 4 weeks (see FIG. 17). The fibrocartilaginous ECM deposition was evaluated by histological analysis (see FIG. 18). Histological staining showed high cell density in COL-RF-HA hydrogel, while cell density was low in COL and COL-RF. The entire image of the host cell filtration was observed at the periphery of the COL-RF-HA hydrogel. As observed in vitro, COL-RF-HA exhibited significantly enhanced cytoplasmic and sapranin-ovarian staining compared to other hydrogels, thus confirming hyaluronic acid-induced proteoglycan secretion.

Hereinafter, specific embodiments of the present invention have been described. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

Claims

Wherein said photocrosslinked collagen hydrogel comprises a photo-crosslinking induced by a photosensitizer.

The method according to claim 1,
Wherein said photosensitizer comprises riboflavin. &Lt; RTI ID = 0.0 > 11. < / RTI >

The method according to claim 1,
7. A photocrosslinked collagen hydrogel further comprising a crosslinked hyaluronic acid powder.

The method according to claim 1,
Wherein said collagen hydrogel is used as a scaffold for regenerating tissue in a human body.

5. The method of claim 4,
RTI ID = 0.0 > 1, < / RTI > wherein the tissue is meniscus.

Preparing a collagen solution;
Adding a photosensitizer to the collagen solution;
Culturing the collagen solution to form a collagen hydrogel; And
And irradiating the collagen hydrogel with ultraviolet light. The method for producing a photocrosslinked collagen hydrogel according to claim 1,

The method according to claim 6,
Wherein the photosensitizer comprises riboflavin. &Lt; RTI ID = 0.0 > 11. < / RTI >

The method according to claim 6,
Wherein the riboflavin induces internal helical cross-linking of the amino acid in the collagen by ultraviolet irradiation.

The method according to claim 6,
Further comprising the step of adding a cross-linked hyaluronic acid powder to the collagen solution.

10. The method of claim 9,
The crosslinked hyaluronic acid powder may contain,
Characterized in that it is formed by mixing hyaluronic acid and hexamethylenediamine, followed by cultivation, freeze-drying, and pulverization.