KR20150029988A - Biomaterials for cell delivery and tissue regeneration and method for preparing them - Google Patents

Abstract

The present invention relates to a cellular therapy product carrier for regenerating damaged tissues and to a scaffold for tissue regeneration and, more specifically, to a modified hyaluronic acid derivative for transplanting the cellular therapy product to damaged cartilage. The hyaluronic acid: is in a gel type; can be used by being mixed with the cellular therapy product; has viscoelasticity capable of maintaining cells and 4 weeks or more of decomposition period, thereby effectively treating damaged cartilage areas without being easily decomposed in joints. The gel type hyaluronic acid according to the present invention can be used for cartilage regeneration and as a cell carrier of soft and hard tissues such as skin, bone, and the likes.

Description

TECHNICAL FIELD [0001] The present invention relates to a biomaterial for cell delivery and tissue regeneration,

The present invention relates to a biomaterial for cell delivery and tissue regeneration, and more particularly to a gel prepared by cross-linking, purification, and sterilization of hyaluronic acid for use as a cell carrier and a support for tissue regeneration, And a biomaterial for cell delivery and tissue regeneration having a cell disruption function and tissue regeneration effect, and a manufacturing method thereof.

Cartilage damage is a disease in which cartilage is damaged due to aging, accidents, and the like. Cartilage is a tissue composed of extracellular matrix without neurons and blood vessels, and self-healing is extremely limited, resulting in osteoarthritis. The traditional method for treating damaged cartilage is bone marrow stimulation to drain the bone marrow of the cartilage. Bone marrow stimulation can be divided into bone drilling, microfracture, and abrasion arthroplasty depending on the method. have. However, bone marrow stimulation is problematic in that regenerated cartilage is regenerated as fibrocartilage rather than hyaline cartilage, which is normal cartilage.

To overcome this problem, autologous chondrocytes implantation (ACI) has been developed. Autologous cartilage grafting is a method of collecting cartilage from the uninvolved cartilage and separating and culturing chondrocytes in an in vitro environment. The cartilage is then implanted into the injured area. . However, the periosteum may overproduce the cartilage, causing pain in the affected part and causing secondary cartilage damage. In addition, there is a need for a two-step surgical procedure of collecting and injecting chondrocytes, and it is troublesome to seal the periosteum very tightly.

To overcome the drawbacks of autologous chondrocyte transplantation, there have been commercialized or ongoing therapies using cells that release chondrocytes, umbilical cord blood stem cells, and transforming growth factor beta (TGF-beta) from others. This type of cell transplantation does not have the inconvenience of collecting autologous chondrocyte cells and has no immune response, thus replacing autologous chondrocyte transplantation. In particular, a method of injecting umbilical cord blood-derived stem cells into articular cartilage damage sites has been extensively studied. Korean Patent Registration No. 10-0494265 discloses an injection preparation composed of a cell, a culture medium, and a biocompatible polymer, wherein the stem cell is isolated, proliferated and differentiated from cord blood. Korean Patent Laid-Open Publication No. 2000-0072904 proposes the production of TGF-beta cDNA-inserted cells and cartilage regeneration using the same.

In order to overcome the problems of periosteum, collagen and hyaluronic acid-derived membranes have been developed and sold. Anika's Hyalograft is a product in the form of a hyaluronic acid three-dimensional nonwoven, which is used for cartilage damage. However, sponge and membrane types are difficult to fix on cartilage and arthroscopic procedures are limited. US Publication No. 2007/0202084 discloses a method of applying a crosslinked hyaluronic acid nonwoven fabric to cartilage damage. In order to solve the problems of such a sponge and a membrane type cell carrier, gel type cell carriers using fibrin, hyaluronic acid and collagen have been studied. The gel-type cell transporter is easy to mix with cells and injectable, and has the advantage that there are no restrictions on arthroscopic procedures. Korean Patent Laid-Open Publication No. 10-2012-0046430 discloses a composition for cartilage transplantation using a hydrogel such as hyaluronic acid, collagen, and fibrinogen. However, there are problems that it is difficult to maintain the cells until the tissue is regenerated at the injured area after the gel is decomposed or diluted by the joint synovial fluid in the surrounding environment, especially in the cartilage, after the injection.

Sponge and membrane technologies have excellent strength and disintegration time in the cartilage environment, but they are difficult to perform in three-dimensional form, and the gel type has a limitation that normal cartilage regeneration is difficult to be resolved and absorbed before the cartilage tissue is completely regenerated.

Hyaluronic acid is a kind of polysaccharide, and is a biomolecule material in which repeating units composed of N-acetyl-glucosamine and D-glucuronic acid are linearly connected. In 1934, Meyer and Palmer were introduced for the first time, separated from the vitreous of cattle, and named for the synthesis of hyalos, meaning vitreous, and uronic acid, one of its components. Hyaluronic acid, which is a major component of extracellular matrix, increases in volume during tissue proliferation and regeneration, wound healing, and has the ability to contain water to maintain the extracellular space by maintaining the moisture content of the tissue, It promotes the movement of nutrients. Hyaluronic acid is present in the body in free radical form and in a tissue-bound form. Most of the stabilizers are cross-linked with collagen and other macromolecules, and therefore the concentration of free hyaluronic acid is relatively low but increases rapidly immediately after tissue injury.

These hyaluronic acid-based biomaterials can be easily mixed with cells as a cell carrier and have the advantage of being able to perform operations through the arthroscope. In addition, it maintains the physiological environment such as the three-dimensional support environment in which the cells attach and grow after transplantation and maintains the nutrient supply pH, and prevents the cell from leaking out from the damaged area, thereby maximizing the regeneration of the cartilage have.

In addition, when the present hyaluronic acid biomaterial is used for the existing bone marrow stimulation, it plays a role of preventing the stem cells from the bone marrow and the blood clots formed by the bone marrow from being decomposed and damaged from synovial fluid and pressure. In other words, hyaluronic acid biomaterial can maximize regeneration of cartilage by preventing stem cells from leaking out.

Accordingly, in order to solve the problem that the conventional fibrin, collagen and hyaluronic acid gel type cell carriers are easily degraded or diluted in the joint synovial environment and the degradation and absorption are completed before the cartilage tissue is completely regenerated, .

In other words, the object of the present invention is to provide a gel prepared by cross-linking, purification and sterilization of hyaluronic acid for use as a cell carrier and a tissue regeneration support, and exhibits a viscoelastic property, a degradation period of 4 weeks or more, A cell carrier and a biomaterial for tissue regeneration, and a method for producing the same.

According to the present invention, the crosslinked hyaluronic acid derivative has a half-life in the body of 10 to 60 days, a viscosity of 100,000 to 6,000,000 cp measured at a frequency of 0.02 to 1 Hz, and an elasticity of 10 to 800 pa, (See Fig. 1).

(A) reacting hyaluronic acid and a crosslinking agent in an alkali metal hydroxide solution at -4 to 60 ° C for 1 to 72 hours; (b1) neutralizing and alcohol precipitation of the reactant to obtain a gel-type dried material, or (b2) neutralizing, pulverizing and alcohol-precipitating the reactant to obtain bead type dried material; And (c) dissolving the gel-type dried material of (b1), the bead-type dried material of (b2) or a mixture thereof in a solvent at a weight ratio of 1 to 30% A method for producing a biomaterial for regeneration is provided.

According to the present invention, it is possible to provide a modified hyaluronic acid derivative for transplanting a cell therapy agent into injured cartilage, which can be mixed with a cell therapy agent in a gel type of hyaluronic acid, The above-mentioned disintegration period can be effectively treated by the damaged cartilage area without being easily broken down in the joints. In particular, gel-type hyaluronic acid can be used not only as cartilage regeneration but also as a cell carrier for soft tissues and hard tissues such as skin and bone.

1 and 2 are graphs showing the results of analysis of the mechanical properties of the support obtained according to the present invention.
FIG. 3 is a graph showing the results of the accelerating enzyme experiments obtained according to the present invention.
4 is a graph showing the results of cytotoxicity experiments obtained according to the present invention.
5 is a graph and a photograph showing the results of in vitro cell growth rate confirmation test obtained according to the present invention.
FIG. 6 is a photograph showing the results of in vivo degradation characteristics obtained according to the present invention. FIG.
7 is a photograph showing degradability confirmation (in vivo) using a rabbit model obtained according to the present invention.
8 to 9 are photographs showing validation (in vivo) using the rabbit model obtained according to the present invention,
Figure 10 is a validation (in vivo) related ICRS rating analysis table using a rabbit model obtained according to the present invention.

Hereinafter, the present invention will be described in more detail.

The present invention relates to a crosslinked hyaluronic acid derivative, which has a half-life in the body of 10 to 60 days, a viscosity measured at a frequency of 0.02 to 1 Hz of 100,000 to 6,000,000 cp, a resilience of 10 to 800 pa, Material.

For reference, the term " half-life in the body " refers to a period of time required for the biomaterial of the present invention to dissolve half the content in the body unless otherwise specified.

The term " biomaterial for cell delivery and tissue regeneration " refers to a cell carrier, a support for tissue regeneration, or a biomaterial used by both of them, unless otherwise specified.

The hyaluronic acid is one of the constituents of cartilage and has excellent biocompatibility and is completely decomposed by enzymes in vivo, and may be in the range of 1 to 30 wt% based on the total weight of the biomaterial.

The hyaluronic acid is applicable irrespective of the molecular weight. For example, the hyaluronic acid may have a weight average molecular weight (Mw) in the range of 100,000 to 5,000,000 Dalton.

Examples of the crosslinking agent include 1,4-butanediol diglycidyl ether, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, N-hydroxysuccinimide, ethylene glycol diglycidyl ether, , 6-hexanediol diglycidyl ether, propylene glycol diglycidyl ether, polypropylene glycol diglycidyl ether and diglycerol polyglycidyl ether.

The cross-linking agent may be contained in an amount of 5 to 30 mol% or 10 to 20 mol% based on the amount of the hyaluronic acid derivative. The crosslinking agent content may be in the range of 2 to 14 wt%, or 4 to 10 wt% in terms of wt%.

The biomaterial may be, for example, a bead type, a gel type, or a mixed type thereof.

The bead type biomaterial can be, for example, a viscosity of 1,000,000 to 5,000,000 cp and an elasticity of 100 to 600 Pa.

In particular, the beads may be of the microbead type having a particle diameter of 100 to 1000 mu m.

Specific examples of the gel type biomaterial may include a viscosity of 100,000 to 1,500,000 cp and an elasticity of 10 to 250 Pa.

The mixed type biomaterial may be, for example, 100,000 to 6,000,000 cp and an elastic average of 10 to 800 pa.

The mixed type biomaterial is, for example, 1 to 29% by weight of the bead type biomaterial, and 29 to 1% by weight of the gel type biomaterial. The total weight percentage of the final product may be 1 to 30% by weight.

For reference, the formula for using the bead type alone is as follows:

In addition, the calculation formula when using the gel type alone is as follows:

Further, the formula for using bead and gel-type mixed types is as follows:

The mixed type is such that 1 g of gel is mixed with 1 g of bead type, 1 g of gel is mixed with 29 g of bead, and the like, and the total wt% satisfies 1 to 30 wt%.

The biomaterial can be, for example, a soft tissue selected from cartilage, skin, muscle, vocal cords, or a hard tissue regeneration support such as a bone.

In addition, the biomaterial can be used as a cell transporter or a cell transporter alone, and the cell can be at least one selected from all transplant cells such as chondrocytes, stem cells, umbilical cells, bone marrow cells, and adipose stem cells.

(A) reacting hyaluronic acid and a cross-linking agent in an alkali metal hydroxide solution at -4 to 60 ° C for 1 to 72 hours; (b1) neutralizing and alcohol precipitation of the reactant to obtain a gel-type dried material, or (b2) neutralizing, pulverizing, alcohol precipitation and drying the reactant to obtain bead type dried material; And (c) dissolving the dried gel-type material of (b1), the bead-type dried material of (b2) or a mixture thereof at a weight ratio of 1 to 30%, and then autoclaving.

Specifically, in step (a), the solution having a pH of 9 or more may be a solution of an alkali metal hydroxide solution such as a NaOH solution or a KOH solution at a pH of about 11.

In the step (a), hyaluronic acid is mixed in a solution having a pH of 9 or higher, and then the cross-linking agent is homogeneously mixed in the mixed solution for 40 to 80 minutes, and the mixture is stirred at -4 to 60 ° C for 1 to 72 hours or at 30 ° C to 37 ° C For 18 to 30 hours.

The physical properties can be controlled by adjusting the temperature and time in the reaction conditions.

Specifically, the step (b1) or (b2) may include a step of removing the unreacted crosslinking agent and neutralizing the basic mixture to terminate the crosslinking reaction as a purification step to be carried out after the crosslinking step of (a). The purification step is carried out at least 5 times at least 40 times the volume of the solid reacted with purified water, and the pH is to be neutralized to between 6 and 8 after washing.

In the step (b2), pulverization may be performed by using a molecular sieve to select a particle size of 100 to 1,000 mu m.

Neutralization in (b1) and (b2) was performed by changing the saline solution from saline at least 7 times to 18 times with stirring for 30 minutes twice, then 60 minutes, and the final measured pH was in the range of 6 to 8 have. For example, the neutralization may be an average of seven times of purified water exchange for the bead type, and an average of 14 times of the gel type.

In the step (b1), the precipitation may be carried out with 70 to 100%, or 90 to 100% of alcohol, specifically ethanol.

In (b2), the precipitation may be carried out with an alcohol concentration of 10 to 70%, or 60 to 70%, specifically ethanol.

In step (c), the dried material may be mixed with 1 to 30 wt% of at least one solvent selected from the group consisting of purified water, PBS, and saline solution, sterilized after filling the syringe into the syringe. The sterilization treatment can be carried out by a high pressure steam sterilization method (autoclave, usually at 121 ° C for 15 to 20 minutes), and sterilization and stickiness of the resulting product can be increased.

For example, the syringe may be a pre-field syringe containing a fixed mixing ratio or a double pre-field syringe kit containing a gel tube of tube A and a bead tube of tube B.

[ Example ]

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are for the purpose of illustrating the present invention, and the present invention is not limited thereto.

< Manufacturing example  1> Hyaluronic acid support reaction

To the distilled water, a solution of pH 9 or more was prepared with 0.25N NaOH, and then 10 wt% of hyaluronic acid was mixed. To the mixed solution, 1,4-butanediol diglycidyl ether as a crosslinking agent was added in an amount of 10 mol% (4.5 wt%) relative to hyaluronic acid and mixed homogeneously for 60 minutes.

The reaction was then terminated by placing the reaction mixture in a saline solution at least 40 times its volume at 30 ° C for 24 hours.

< Manufacturing example  2 > Hyaluronic acid support tablets and Bead / Gel Type Support Production

When the hyaluronic acid scaffold obtained in Preparation Example 1 was refined, saline was replaced at least 7 to 18 times for the first two times for 30 minutes and then for 60 minutes. The purified water used was at least 40 times the volume of the obtained hyaluronic acid supporter, and it was confirmed that the pH was 6 to 8 at the final purification.

After the physiological saline was purified, the solids were pulverized with a pulverizer, and the particles were sized at a particle size of 100 to 500 μm using molecular sieves. The selected beads were precipitated with ethanol having a concentration of 90 to 99% A dry, bead-type support was obtained.

After the physiological saline was purified, it was precipitated in ethanol at a concentration of 60 to 70% without a pulverization step, followed by natural drying to obtain a gel type support.

< Manufacturing example  3> Sterilization of hyaluronic acid support

The bead type supporter prepared in Preparation Example 2 and the gel type supporter were mixed at a weight ratio of 1: 4 and mixed in PBS at 5 wt%, filled in a prefilled syringe, and then subjected to high pressure steam sterilization (autoclave, 20 min).

< Manufacturing example  4> Hyaluronic acid support Comparative Example

The biodegradability of the commercially available hyaluronic acid product 10 mg / ml, which exhibits a sustained effect over 6 months to 1 year, and the final dose of the final product prepared in Preparation Example 3 were compared in vivo 7. Also, comparison with the fibrin of the green plast through the efficacy experiment is shown in Fig. 8 to Fig.

<Test items>

1. Analysis of mechanical properties of supports:

A high temperature sterilized bead type single support, a gel type single support, a gel type single support, and a 1: 4 ratio of the preparation of Example 3 were tested to see if they exhibited biocompatibility and mechanical strength of the cartilage not to be degraded by synovial fluid The viscosity and elasticity of the mixed support were measured three times using a rheometer (HAAKE MARS II, Thermo Scientific, inc) at a frequency of 0.02 to 1 Hz, and the average and standard deviation were summarized in Tables 1 and 2 below.

Bead type Gel type Mixed type Viscosity average 1.5 million cp 31 million cp 1.7 million cp Standard Deviation ± 11 million ± 3 million ± 11 million Elasticity average 194 Pa 40 Pa 227 Pa Standard Deviation ± 16 Pa ± 4 Pa ± 18 Pa

As shown in Table 1, it was confirmed that the bead type supports, gel type supports and mixed supports obtained in the present invention each have a viscosity average of 30 to 2 million cp and an elasticity average of 40 to 300 Pa.

2. Enzyme degradation experiment

It is known that bone marrow cells or transplanted cells need at least a month to regenerate into cartilage tissue. Therefore, in order to predict the persistence of the prepared samples in vivo, accelerated protease experiments using Hyaluronidase (Sigma-Aldrich) were performed. 0.6 g of the sample was treated with 6 쨉 l of hyaluronidase (1 units / 쨉 l), and the viscosity and the time-dependent viscosity at 37 째 C were measured.

As shown in the graph of FIG. 3 showing the result of the accelerated degradation enzyme experiment, the decomposition period of about 7 days was confirmed in the bead type sample.

3. Cytotoxicity experiment

Experiments were carried out in accordance with the ISO 10993-5 cytotoxicity test method, and cytotoxicity was evaluated by adding a comparative liquid and an eluent to L929 cell line. As shown in the graph of FIG. 4 showing the cytotoxicity test results, it was confirmed that the absorbance value of each support was not cytotoxic at a level similar to that of the negative control group.

4. Examination of cell growth rate in vitro

Cells were inoculated on each hyaluronic acid support prepared in Preparation Example 3 and the growth rate was analyzed. The rabbit chondrocytes were separated from the tissue culture flask in a 175 cm2 tissue culture flask and cultured in DMEM (Dulbecco's Modified Eagle Medium) containing 10% fetal bovine serum (FBS) and penicillin. Subsequently, 2x10 &lt; ⁵ &gt; of subcultured rabbit chondrocytes was concentrated to 0.1 ml and mixed.

Insert (SPL, Korea) was used to culture the gel on a 6-well plate at 37 ° C and 5% CO ₂ . At this time, the medium was replaced every 3 days with DMEM containing 10% FBS and penicillin. The cell proliferation rate of the supporter was analyzed using MTT at 0 day, 3 day, and 6 day after cell inoculation. MTT solution was added to each well to form formazan crystals originating from living cells. After centrifugation, 200 μl of hyaluronidase (100 units / μl) was added to decompose the gel at 50 ° C for 2 hours to decompose the gel.

After confirming that the gel was decomposed, it was centrifuged at 3000 rpm for 10 minutes to take only the lower layer solution, and 100 μl of DMSO was added to dissolve the formazan cup. The cell proliferation rate of the supporter was measured by measuring absorbance in a 570 nm microplate reader. Chondrocytes tend to be undifferentiated during monolayer culture and cell proliferation does not tend to occur during three-dimensional culture. Therefore, when compared with monolayer cultured cells grown about 7% between 3 days and 6 days, bead type was 13% 8%, and the mixed type showed a cell growth rate of 8% (see FIG. 5).

5. In vivo in vivo ) Decomposition characteristics

In vivo degradation characteristics were tested using rats (SD Rat, 8W, 250-300 g). After anesthesia, they were epilated and treated with povidone, and 0.6 ml of each candidate group was injected between the dorsal skin and muscle. After 1, 2, 4, and 8 weeks, the extent of inflammation and degree of inflammation were visually and histologically analyzed by autopsy.

As shown in FIG. 6, it was confirmed that the supernatant was present visually from the injection site up to the 8th week, and after the samples were taken 1, 2, 4 and 8 weeks for histological analysis, there was.

Observation of the inflammation reaction was not observed and it was confirmed that the size was larger than the first. This suggests that the tissue fluid was absorbed by the decomposition characteristics of the support. The decrease in size was observed from the 8th week, and it was judged that the support was decomposed. H & E staining showed no specific reaction, and we could confirm that the cells penetrated into the supporter at the transplantation sample and the muscle boundary.

Results The in vivo degradation period confirmed that all supports were greater than 8 weeks.

6. Rabbit  Identification of degradation characteristics using model in vivo )

In order to confirm the degradation characteristics of the chondrocyte transporter, the degradation characteristics and the degree of protection of injured joints were evaluated by rabbit arthritis model.

The rabbits (NZW, male, 4 ± 0.3 kg, 16 weeks) were treated with arthritic model of injured ligaments and the cruciate ligaments were resected, and the joints were washed with physiological saline and then sutured. One week after the operation, the other company injected the joints of the D company three times at 1 week intervals, the sample of the candidate type of gel type and the bead type 4: 1 mixture was injected once a week for 6 weeks and 12 weeks, The cartilage tissues were collected and analyzed for visual acuity and histological analysis. (See FIG. 7).

7. Rabbit  Verification using model ( in vivo )

In order to evaluate the effectiveness of chondrocyte transporter, cartilage regeneration was evaluated by rabbit cartilage injury model.

The damaged cartilage of the knee (4 mm in diameter, 2 ㎜ in diameter) in rabbits (NZW, male, 3 ~ 3.5 kg, 14 weeks) was transplanted with the cell transporter. The transplanted cells were obtained by collecting chondrocytes from young rabbits within 1 week after birth. The rabbits were euthanized at 3 weeks, 6 weeks, and 12 weeks, and cartilage tissue was collected. The degree of cartilage regeneration was analyzed by visual and histological analysis (ICRS grade). Histological analysis confirmed the regeneration effect by analyzing the degree of cartilage regeneration by Masson's trichrome, Safranin-O staining and Type II collagen immunostaining. (See Figs. 8 and 9).

Claims (14)

A hyaluronic acid derivative and a cross-linking agent, and has a half-life in the body of 10 to 60 days,
A biomaterial for cell delivery and tissue regeneration having a viscosity average of 100,000 to 6,000,000 cp measured three times at a frequency of 0.02 to 1 Hz and an elasticity average of 10 to 800 pa.
The method according to claim 1,
Wherein the hyaluronic acid is at least one selected from the group consisting of a bead type, a gel type, and a mixed type thereof.
The method according to claim 1,
Wherein the hyaluronic acid is 1 to 30 wt% based on the total weight of the biomaterial.
The method according to claim 1,
The crosslinking agent may be selected from the group consisting of 1,4-butanediol diglycidyl ether, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, N-hydroxysuccinimide, ethylene glycol diglycidyl ether, -Hexanediol diglycidyl ether, propylene glycol diglycidyl ether, polypropylene glycol diglycidyl ether, and diglycerol polyglycidyl ether. 2. A biomaterial for cell delivery and tissue regeneration, comprising:
The method according to claim 1,
Wherein the crosslinking agent is contained in an amount of 5 to 30 mol% based on the amount of the hyaluronic acid derivative.
The method according to claim 1,
Wherein the biomaterial is a soft tissue selected from the group consisting of skin, muscle, vocal cords, and cartilage, or a hard tissue restorative support.
The method according to claim 1,
Wherein the cells for cell delivery are at least one selected from cartilage cells, stem cells, graft cells, and bone marrow cells.
(a) reacting hyaluronic acid and a cross-linking agent in a solution having a pH of 9 or higher at -4 to 60 ° C for 1 to 72 hours; (b1) neutralizing and alcohol precipitation of the reactant to obtain a gel-type dried material, or (b2) neutralizing, pulverizing, alcohol precipitation and drying the reactant to obtain bead type dried material; And (c) dissolving the gel-type dried material of (b1), the bead-type dried material of (b2) or a mixture thereof in a solvent at a weight ratio of 1 to 30% A method for producing a biomaterial for regeneration.
9. The method of claim 8,
Wherein the step (a) comprises mixing hyaluronic acid in a solution having a pH of 9 or higher and then homogeneously mixing the cross-linking agent in the mixed solution for 40 to 80 minutes and reacting at -4 to 60 ° C for 1 to 72 hours. And a method for producing a biomaterial for tissue regeneration.
9. The method of claim 8,
The method for producing a biomaterial for cell delivery and tissue regeneration according to claim 1, wherein, in the step (b2), the particle size of 100 to 1,000 mu m is selected using the molecular sieve.
9. The method of claim 8,
The neutralization of (b1) and (b2) is performed by changing the physiological saline solution 7 to 14 times with stirring for 30 minutes twice and then for 60 minutes, and the final measured pH is in the range of 6 to 8 A method for manufacturing a biomaterial for cell delivery and tissue regeneration.
9. The method of claim 8,
Wherein the alcohol precipitation in step (b1) is performed with an alcohol at a concentration of 70 to 100%.
9. The method of claim 8,
Wherein the alcohol precipitation in step (b2) is performed with an alcohol at a concentration of 10 to 70%.
9. The method of claim 8,
Wherein the step (c) comprises mixing at least one solvent selected from the group consisting of purified water, PBS, and saline solution, filling the syringe, and sterilizing the mixture with high-pressure steam. Way.

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