KR20130091824A - The fabrication method of porous hyaluronic acid-gelatin hydrogel scaffolds for bone tissue engineering and the hydrogel scaffolds fabricated thereby - Google Patents

Abstract

The present invention uses lyophilization at a ratio of 15:85, 50:50, 85:15 to obtain a porous biocompatible support for applying hyaluronic acid (HyA) -gelatin (Gel) support to bone tissue engineering. It is manufactured by. The composite structure showed the highest porosity when the HyA-Gel ratio was 15:85. The pores of the support were interconnected, the porosity was 28.59 to 53.46% and the pore size was 50 to 100 μm. The compressive strength of the support is 4.39 ± 0.2 MPa when the ratio of HyA-Gel is 15:85. The proliferation of MG-63 osteoblasts and the behavior and biocompatibility of HyA-Gel compounds were measured by MTT analysis, optical microscopy and confocal microscopy. Extraction of Collagen type 1 from the cultured cells of the support was performed using immunoblotting. The support shows good biocompatibility, mechanical properties and high porosity, and is suitable for use as a porous support for bone tissue engineering.

Description

Method for preparing porous hyaluronic acid-gelatin hydrogel scaffold for tissue engineering and its support manufactured by

The present invention relates to a method for producing a porous hydrogel scaffold using a freezing method and a lyophilization method, and to the application of bone tissue engineering in a form that mimics the extracellular matrix of spongy bone.

The initial structure of the support prepared in bone tissue engineering can play an ideal substrate for cell growth, metabolism and matrix formation in the physiological environment of bone depending on the composition and development of bone tissue. Therefore, designing the structure of the scaffold is an important process for the purpose of treating biomaterials, and various kinds of polymers such as synthetic polymers and natural polymers have recently been applied to bone tissue engineering. Natural polymers such as collagen, gelatin, oxidized alginate, chondroitin chitosan, fibrin, hyaluronic acid become components of extracellular matrix or contain important domains of cells. Has biodegradable properties.

The porosity and pore size of biocompatible scaffolds also play an important role in bone formation in cell and animal experiments. These porosities and pore sizes are macroscopic and fine due to the morphological properties of the scaffolds for bone regeneration. All levels are shown, and many natural polymers have the advantages of good biocompatibility and biodegradability.

Hyaluronic acid is found in the joint capsules of soft tissues of higher organisms and the vitreous of the eye. Hyaluronic acid and gelatin complexes play an important role in many biological processes such as tissue hydration, proteoglycan organs, cell differentiation and angiogenesis, and protective actions around cell membranes. In addition, hyaluronic acid has a significant effect on cell motility and cell-to-cell interactions, and gelatin is obtained from partial hydrolysis of collagen, in which the triple helix structure of collagen is split into single-stranded molecular weights during hydrolysis. Gelatin has been applied in the field of tissue engineering because it has the characteristics of enhancing cell adhesion, differentiation, and proliferation rate.

It is an object of the present invention to provide a method for producing a porous support having high porosity and excellent biocompatibility using freezing and lyophilization techniques.

The present invention mixes the ratio of hyaluronic acid (Hyaluronic acid, HyA) -gelatin to 15:85, 50:50, and 85:15, respectively, and uses a freeze method and a freeze-drying method to form a hydrogel in the form of extracellular matrix of spongy bone. It is manufactured and applied to bone tissue engineering. Hyaluronic acid-gelatin can provide an optimal environment for enhancing cell growth and bone tissue formation, and porous hyaluronic acid-gelatin supports are expected to show excellent effects in the treatment for bone defect patients based on the present invention.

The present invention relates to the preparation of a support for a new construct comprising a solution of gelatin (10 wt%, molecular weight 300) dissolved in distilled water at 30 ° C. for application to bone tissue engineering. Add 0.5% by weight of the gelatine solution to the hyaluronic acid solution until it is in the form of a slurry and mix in a stirrer at 30 ° C. for 1 hour so that each ratio (wt%) is 15:85, 50:50, 85:15 Give it. A well mixed 1.0 ml slurry solution was put into a polyethylene mold as a resin mold having a diameter of 3 cm, then frozen at a temperature of -80 ° C for 2 hours and freeze-dried overnight at a temperature of -70 ° C. The freeze-dried porous support was crosslinked overnight at 4 ° C. with 0.5wt% 1-ethyl-3 (3-dimethylaminopropyl) carbodiimide (EDC) solution and sonicated in distilled water to remove the remaining EDC. After 5 times of 3 minutes, it is frozen for 2 hours at a temperature of -20 ℃ and lyophilized at a temperature of -70 ℃. The crosslinking agent is treated twice in order to prevent degradation of the support when performing a cell experiment to evaluate the biocompatibility of the prepared support.

MTT (3- [4, 5-dimethylthiazol-2) to evaluate the viability or toxicity of cells cultured on a hyaluronic acid-gelatin (HyA-Gel) scaffold prepared at the ratio of 15:85, 50:50 and 85:15 -yl] -2,5-diphenyltetrazolium bromide) assay was performed. This assay is a method based on ISO 10993, in which cytotoxicity evaluation is performed using a solution eluted from a manufactured hyaluronic acid-gelatin support, compared to a standard control (control). The eluate (100%) prepared by putting the hyaluronic acid-gelatin support in DMEM medium (animal cell culture medium) and shaking at 100 rpm for 72 hours in an incubator at 37 ° C. was mixed with DMEM medium 0%, 12.5 Make up in percentage of 25% and 50%. Simultaneously prepare the eluate and incubate 1 × 10 4 cells / well of MG-63 osteoblasts in 96 wells for 1 day. Pure DMEM medium containing no eluate is the control (control), and 200 μl of each ratio (0%, 12.5%, 25%, 50%, 100%) is added to 96well of osteoblasts cultured for one day. Incubate for 3 days in an incubator. After 3 days, 100 μl of MTT solution was added to each well and incubated for 4 hours, and then absorbance was measured at 595 nm using an ELISA reader (EL, 312, Biokinetics reader, Bio-Tek instruments).

The MTT assay is a measure of cell viability and is based on the formation of purple color by the reaction of MTT solution with dehydrogenase of surviving cells. This metabolism and surviving cells produce mitochondrial dehydrogenase during culture in the medium, which is quantified by measuring the degree of purple turn using a spectrophotometer. The absorbance measured in this way is related to the number of cells that survived.

Eluate and MG-63 osteoblasts (1x103 cells / ml) were incubated in 24 wells for 1 day, 3 days, and 7 days. New eluates were replaced during each culture period. Osteoblasts cultured in the eluate were 70-80% of the wells. When the proliferation was about the number of cells by desorption with 0.25% Trypsin-EDTA. Subsequently, MG-63 osteoblasts, which were incubated for 1 day, 3 days, and 7 days in the eluate of the hyaluronic acid-gelatin support with an inverted optical microscope (Olympus, 1 X 71), were observed. As the duration increases, the number of cells increases.

Confocal microscopy was used to observe the growth ability of MG-63 osteoblasts cultured on the surface of the hyaluronic acid-gelatin support. To observe confocal microscopy, the cells were cultured with hyaluronic acid-gelatin scaffold twice with phosphate buffer (PBS), treated with 4% paraformaldehyde (Sigma-aldrich) for 15 minutes at room temperature, and then 0.25% for 10 minutes. Treat with Triton-X (Sigma-aldrich) and block for 30 minutes with 2.5% BSA solution (bovine serum albumin solution). Cells were immunostained with the combination of two compounds, Fluorescein Isothiocyanate (FITC) and Paloidine (25 μg / ml-Sigma) for 2 hours at room temperature. The nucleus of the cell was DAPI (4′-6′-). contrast stained with diamidine-2-phenyl indole dihydrochloride). In the last step, the support was placed on a glass slide and sealed, and observed at 10 × and 60 × magnification using a confocal microscope (FV10i-W).

After washing the osteoblasts cultured on the hyaluronic acid-gelatin support, lysis buffer (Tris 50mM, pH7.4, NaCl 40mM, EDTA 1mM, Triton X-100 0.5%, Na3VO4 1.5mM, NaF 50mM, sodium pyrophosphate 10mM, glycerolphosphate 10mM) , PMSF 1mM, Protease inhibitor cocktail 10mM) cells were washed and collected by centrifugation for 10 minutes at 4 ℃ at a speed of 13,000rpm. Immunohistochemical staining was performed using Collagen Type I (Santa Cruz) diluted 1: 200 with 30 μg of protein quantified from cells by Western blotting. Anti-β actin (1: 1000) antibody was used as a control, and the polyvinylidene difluoride membrane (Millipore) was loaded with diluted protein on 8% SDS-PAGE gel and loaded with anti-β actin on 12% SDS-PAGE gel. ). The membrane was blocked in a Tris buffered saline containing fat-free milk powder and 0.1% Tween 20 (Polyoxyethylene Sorbitan monolaurate), and the diluted primary antibody was incubated overnight at 4 ° C for chemiluminescence solution (ECL). , Amersham).

Other characteristics and aspects will be revealed through detailed descriptions, figures, and claims.

Hydrogel manufacturing process of the present invention for application to bone tissue engineering has a bio-friendly characteristics by producing a porous support, showing the interconnected pore structure, excellent mechanical strength and excellent growth rate of MG-63 osteoblasts.

Next, the method of synthesizing the porous hydrogel support is described with the accompanying drawings.
Figure 1 shows the HyA-Gel at 15:85 ratio (a) and magnification (d), 50:50 ratio (b) and magnification (e), 85:15 ratio (c) and magnification (f) As observed.
2 shows a graph of porosity of HyA-Gel in a ratio of 15:85 (a), a ratio of 50:50 (b), and a ratio of 85:15 (c).
Figure 3 shows the porosity and compressive strength of each HyA-Gel.
4 shows PBS uptake of the porous HyA-Gel support at 37 ° C.
Figure 5 shows the cell viability of each of the HyA-Gel by MTT analysis.
Figure 6 shows the cell proliferation after culturing the HyA-Gel support for 1 day (a), 3 days (b), 7 days (c) by MTT analysis.
FIG. 7 shows HyA: Gel at 15:85 ratio of 1 day (a1), 3 days (a2), 7 days (a3), 50:50 ratio of 1 day (b1), 3 days (b2), 7 days (b3), the proliferation of osteoblasts was observed under an optical microscope when cultured for 1 day (c1), 3 days (c2), and 7 days (c3) at a ratio of 85:15.
FIG. 8 shows DAPI (red) when incubated for 3 days (a, b, c) and 7 days (d, e, f) on a support having a ratio of HyA: Gel of 15:85, 50:50, and 85:15. Confocal microscopy of the nucleus) and Phalloidin (membrane into the green portion) are shown.
FIG. 9 shows that collagen was expressed in cells by Western blot analysis when HyA-Gel was incubated for 3 days and 7 days, respectively.

Specific details for carrying out the present invention will be described in detail with reference to the drawings.

Example 1

Pore is necessary for the formation of bone tissue because it allows the movement and proliferation of osteoblasts. For example, the porous surface aids in the mechanical bonding between the implant and the surrounding natural bone, making it more stable. HyA-Gel scaffolds are made by freezing and lyophilization. The raw compound support shows high porosity. 1 is an SEM image of the support, showing that its shape depends on the ratio of HyA and Gel. When the ratio of 15:85 is used, the pores are uniformly distributed (Fig. 1A). In the ratio 50:50, the shape and the size of the pores are not uniform, and the orientation of the fibers is also uneven (Fig. 1B). When HyA: Gel is used at 85:15, the shape of the support is significantly different from the other two. As shown in Fig. 1 (c), the material is vertically aligned and has a different shape from the sponges.

The distribution of pores in the HyA-Gel support is shown in FIG. The pores have a diameter of 50 μm in a 50:50 ratio of HyA-Gel and a diameter of 100 μm at a ratio of 15:85 and 85:15. It can be seen from FIG. 2 that the pore sizes of all HyA: Gel supports are uniform. The support is well connected between the pores, and it is well known that the pores promote bone ingrowth. The pores play an important role in implanting artificial bones, making them effective in protein absorption with bone formation. And the rough surface promotes cell adhesion, proliferation, and differentiation. For successful transplantation of hard tissues, the material must be bioactive and can be converted into natural hard tissues. The former materials have high porosity and pore size, which allow for the transfer and storage of osteoblasts throughout the skeleton. The high connectivity of each of the pores allows for better cell proliferation and influx of physiological fluids. The skeleton exhibits high porosity in the range of porosity 28.59-53.46% (FIG. 3).

Mechanical properties are very important in tissue engineering because one of the important roles of the scaffold is to penetrate cells and tissues to give them mechanical effects and to grow skeletal structures.

Figure 3 shows the compressive strength and porosity of each of the HyA-Gel. At 15:85 ratio, the compressive strength and porosity are the highest. Compressive strength is 4.39 ± 0.2, 1.47 ± 0.2, 0.95 ± 0.1 MPa when HyA: Gel is 15:85, 50:50 and 85:15, respectively. As a result, it can be seen that the mechanical strength is the greatest when HyA and Gel are in the ratio of 15:85. The results also showed that mechanical strength is important for regenerative applications and can be controlled by the composition of the mixture. Porosity values decrease with gel concentrations from 85 to 50 and increase to 15. This is due to the unique form of HyA: Gel = 85:15. Longitudinal shapes at 85:15 ratio increase porosity and decrease compressive strength.

Nutrients must be able to pass through the support. As such, the ability of a support to absorb moisture is an important factor for its support for biological activity. In this study, PBS was used as cell culture media to evaluate the uptake of fluid at 37 ° C. into a support. 4 shows that the PBS uptake of all supports is initially greater than 13. However, after 6 days of incubation, it decreased to 10, and after 10 days, to 9. The rate of absorption depends on the three-dimensional structure and hydrophilicity of the support. These results indicate that absorption capacity is necessary to maintain biological function for nutrient delivery.

Viability of MG-63 cells in the porous support is shown in FIG. 5. The results show good cell viability of all porous supports (more than 80% at 100% extraction). The porous HyA-Gel scaffold is harmless to MG-63 cells and has been used for tissue engineering scaffolds.

Osteoblast proliferation in different ratios of HyA-Gel scaffolds was cultured for 1, 3, 7 days, and then used for MTT analysis. Cell proliferation (7 days culture) of HyA: Gel = 15: 85 and 85:15 is higher than the ratio of 50:50 (FIG. 6). After one day, the number of cells is greater than the initial number of cells (103 cells / well). As shown in Figure 6, the number of increased osteoblasts is much greater than the number of cells after the first three days incubation. While incubating for 1-7 days, MG-63 cells continued to proliferate on the support.

MTT assay was performed after determining the ratio of viable cells to extract solution after the incubation period. The change in the number and shape of the cells was measured using an optical microscope. The optical microscope image of the cell shown in FIG. 7 used the solution incubated for 1, 3, 7 days. On day 1, a small number of cells were observed. However, in all samples, the number of cells gradually increased over the three and seven days. On day 7, the cells nearly merged. The sample showed high cell proliferation with a HyA: Gel ratio of 15:85. This result is consistent with the results observed in cell survival and proliferation assays. The cells were used to produce the shape and size (10-20 μm in diameter) of osteoblasts.

Confocal microscopy shows that osteoblasts adhere to and proliferate on the surface of the support. The increase in the number of cells in FIG. 8 shows that the culture for 7 days (FIG. 8D-F) adhered well to the porous surface compared to the culture for 3 days (FIGS. 8A-C). This image shows that cells are used to build structures. As a result, the porosity and surface morphology affect the proliferation and migration of cells. High porosity and pore structure have advantages in terms of cell growth and survival, respectively. High porosity increases the surface area of the support, allowing it to adhere well to the material, retaining ECM proteins on the surface, and helping to propagate osteoblasts.

Along with porosity, pore size plays an important role in the diffusion of osteoblasts into the material. Large and small porous structures help osteoblasts migrate and cling. This result has always been consistent with previous studies. Cells need oxygen and nutrients, and their proliferation and diffusion increase with the porosity of the support.

More than 90% of bone protein, Collagen Type I, acts as a bone matrix and controls nucleation. Collagen expression was analyzed by Western blot using Collagen Type 1 antibody. This Western blot analysis showed that MG-63 made Collagen Type 1 in all materials (FIG. 9). After 3 days, collagen extracted from cells incubated at the ratio of HyA: Gel = 15: 85 was higher than HyA: Gel = 50: 50 and 85:15. After 7 days, Collagen type 1 showed no difference from other materials.

Claims (5)

In the method of manufacturing a porous support for tissue engineering,
A first step of preparing a gelatin solution and a hyaluronic acid solution as biodegradable polymers for bone tissue engineering;
A second step of preparing a slurry solution by adding and mixing the gelatin solution until the hyaluronic acid solution is in the form of a slurry;
Method for producing a porous hyaluronic acid-gelatin hydrogel support for tissue engineering, characterized in that it comprises a third step after the slurry solution to the synthetic resin mold, followed by a freezing and freeze drying process sequentially.
The method of claim 1, wherein the gelatin solution of the first step is gelatin 10% by weight, and the hyaluronic acid solution is 0.5% by weight of hyaluronic acid for the preparation of a porous hyaluronic acid-gelatin hydrogel support for tissue engineering.
The porous hyaluronic acid-gel for tissue engineering according to claim 1, wherein the mixing ratio of the hyaluronic acid solution and the gelatin solution in the second step is any one of 15:85, 50:50, and 85:15 ratios (wt%). Method for preparing a latin hydrogel support.
The method of claim 1, further comprising: crosslinking the porous support obtained after the third step with a 0.5 wt% 1-ethyl-3 (3-dimethylaminopropyl) carbodiimide (EDC) solution at 4 ° C. for one day;
A fifth step of sonicating five times in distilled water three times for the decomposition of the remaining EDC after the fourth step;
And a sixth step of freezing at a temperature of −20 ° C. for 2 hours and lyophilizing at a temperature of −70 ° C., wherein the porous hyaluronic acid-gelatin hydrogel support for tissue engineering is further included.
A porous hyaluronic acid-gelatin hydrogel support for tissue engineering prepared by the method of any one of claims 1 to 4.

Images