KR101657235B1 - Polymer-Ceramic Fusion Hybrid Gel And Method For Preparing The Same - Google Patents

Abstract

(2-carboxyethyl) phosphine is added to all or a part of the repeating units of the polymer-acrylate compound and the biocompatible polymer formed by introducing acrylic acid into all or a part of the repeating unit of the biocompatible polymer. TCEP] or a polymer-TCEP compound formed by introducing a lipoic acid or a lipoamide or a polymer-lipoic acid compound or a polymer-lipoamide compound formed by cross-linking by a Michael type addition reaction and a buffer solution containing calcium phosphate A polymer-ceramic hybrid gel in which a calcium phosphate ceramic gel formed by injecting particles is uniformly mixed without phase separation is disclosed. Since the polymer-ceramic hybrid gel of the present invention has a structure in which the polymer hydrogel and the ceramic gel are uniformly mixed without phase separation, the bioactive substance is slowly released when the bioactive substance is incorporated into the polymer hydrogel by incorporation into the living body The drug delivery function can be effectively achieved, and the requirements such as mechanical properties required as a tissue engineering support can be sufficiently achieved, and therefore, they can be usefully used.

Description

TECHNICAL FIELD [0001] The present invention relates to a polymer-ceramic fusion hybrid gel,

TECHNICAL FIELD The present invention relates to a polymer-ceramic fusion hybrid gel and a method for producing the same, and more particularly, to a method for harmoniously fusing the characteristics of a polymer hydrogel and a ceramic gel, Ceramic fusion hybrid gel capable of providing a hybrid biomaterial having various physical properties by controlling the proportions of components, and a method for producing the same.

Polymer and ceramic tissue engineering supports are artificially manufactured in the form of bone or cartilage fillers, surface coatings, scanning gels, or stationary supports and are used in vivo to regenerate damaged or missing tissue. As a method for improving the efficiency of tissue regeneration using such a tissue-engineered support, a method of including a biologically active substance such as a protein, a growth factor, a drug, and a cell on the inside or outside of the support is used in a tissue- The biologically active substance must be gradually released from the support according to time and purpose. Polymer hydrogels or ceramic gels have been developed for this purpose. However, it is applied as a polymeric hydrogel and a support for tissue engineering that requires mechanical properties such as 3-D bio-printing, which is applied to a damaged area requiring a constant load for a certain period of time in applying the polymer hydrogel and a support, But it is somewhat inadequate to meet the requirements to be satisfied.

On the other hand, the ceramic gel or support satisfies the requirements as a tissue engineering support in terms of mechanical properties, but it is difficult to incorporate the biologically active substance into the biologically active substance while maintaining its activity, and biodegradation is not performed or it proceeds very late.

BACKGROUND ART [0002] Supports for regenerating tissues having a function of transferring a bioactive substance having mechanical properties and maintaining activity at specific sites are required in the fields of bioprinting, tissue engineering, and biomedical engineering. The most ideal method for developing such a tissue regeneration support is to develop a polymer-ceramic fusion hybrid gel / support by fusing a polymer hydrogel having bio-active substance loading ability and a ceramic gel having biodegradability and mechanical properties . However, conventionally, in producing a polymer-ceramic fusion hybrid gel or a support in which a ceramic and a polymer are fused, phase-separation occurs in the two types of materials, so that a polymer-ceramic fusion hybrid gel having substantially desired physical properties has been obtained There was no. Particularly, polymer-ceramic fusion hybrid gels did not have an efficient method to convert into a scanning type having the characteristics of controlling the expansion rate, controlling the mechanical properties and containing the bioactive substance.
On the other hand, Patent Registration No. 10-1100803 (registered on December 23, 2011) discloses the formation of a polymer hydrogel by a Michael type addition reaction between a polymer into which a lipoamide functional group is introduced and a polymer into which an acrylate functional group is introduced , Registered Patent No. 10-1161640 (registered on June 26, 2012), discloses a method for producing a biocompatible polymer comprising reacting a first biocompatible polymer compound produced by binding of a biocompatible polymer compound having a carboxy functional group of lipoic acid with an amine functional group and an unsaturated hydrocarbon such as acrylate A polymer hydrogel is formed by a Michael type addition reaction between a second biocompatible polymer having a functional group.
Patent Registration No. 10-1277658 (Registered on Mar. 17, 2013) discloses a method for manufacturing a biomedical ceramic material having multiple drug delivery systems. The disclosed method is to encapsulate a physiologically active substance in a biodegradable polymer to prepare a physiologically active substance-containing fine particle, and to fix the prepared fine particle on the surface of the ceramic carrier.

Accordingly, the present invention has been made to overcome the above-described problems of conventional hybrid gel / support production by phase separation and to produce a polymer gel-ceramic fusion type hybrid gel and a support capable of containing a bioactive substance. Accordingly, it is an object of the present invention to prepare a hybrid gel comprising a polymer and a ceramic, and a hybrid gel in which the properties of the polymer hydrogel and the properties of the ceramic gel are harmoniously fused. The present invention also provides a polymer-ceramic fusion hybrid gel capable of providing a hybrid biomaterial having various physical properties such as controlling the properties of a polymer gel and a ceramic gel by controlling a ratio of a polymer component and a ceramic component, and a method for producing the same. Hybrid biomaterials can be manufactured in various forms such as a porous solid support in which a polymer gel-ceramic gel is fused and a scanning gel. At this time, it is possible to manufacture a hybrid hydrogel by incorporating various bioactive substances such as cells, proteins, growth factors, and drugs into the polymer hydrogel to induce release at a controlled rate of contained bioactive material have. In addition, the hybrid gel can be converted into a scanning type, so that it has been developed for clinical use. That is, the two kinds of solutions were mixed and the polymer solution and the ceramic solution were injected into the syringe, and then injected into the required part.

In order to achieve the above object, a polymer-ceramic fusion hybrid gel according to the present invention comprises a biocompatible polymer compound having a polymer-acrylate structure formed by introducing acrylate into all or a part of a repeating unit of a biocompatible polymer (hereinafter, (2-carboxyethyl) phosphine (hereinafter referred to as " tris (2-carboxyethyl) phosphine] is added to all or part of the repeating units of the biocompatible polymer. (Hereinafter referred to as 'polymer-TCEP compound', 'polymer-lipoic acid compound' or 'polymer-lipoic acid compound') having a polymer-TCEP structure, Referred to as " polymer-lipoamide compound ") solution by simple addition of a solution of calcium carbonate and calcium phosphate into a buffer solution and a buffer solution, and a calcium phosphate ceramic gel formed by adding calcium phosphate particles to the buffer solution Mixed.

The total weight of the biocompatible polymeric hydrogels constituting the biocompatible polymer hydrogel is preferably in the range of 1: 1 to 1: 1, Preferably 1 to 99% by weight.

The biocompatible polymer may be a biocompatible polymer such as a polysaccharide composed of hyaluronic acid, chondroitin sulfate (CS), carboxycellulose, alginate and chitosan, collagen, gelatin, polyethylene oxide, polyvinyl alcohol, polypyrrolidone, poloxamer and fibrin gel It is preferable to select it from a suitable polymer.

Preferably, the calcium phosphate is calcium phosphate, and the calcium phosphate is tricalcium phosphate (TCP) or calcium phosphate.

The polymer-acrylate compound is a polymer-linker-acrylate type structure in which acrylate is introduced into the linker after the linker is introduced into all or a part of the side chain of the repeating unit of the biocompatible polymer, and the polymer- Compound or a polymer-lipoic acid compound or a polymer-lipoamide compound is obtained by introducing a linker to all or a part of the repeating unit of the biocompatible polymer, and then introducing TCEP, lipoic acid or lipoamide into the linker to form a polymer-linker-TCEP form or a polymer- Linker-lipoic acid type or a polymer-linker-lipoamide type.

Wherein the linker is adipic acid dihydrazide (ADH), and the linker reacts with a carboxy functional group in the repeating unit of the biocompatible polymer to form the polymer-linker first, It is preferred that the carboxy functional group or the carboxy functional group of the TCEP or the biocompatible polymer-linker-functional group is reacted with the carboxy functional group of the lipoic acid to combine with acrylate, TCEP or lipoic acid. At this time, it is preferable that the functional group forms acrylate, TCEP and thiol at the terminal.

The present invention also provides a polymer-ceramic hybrid biomaterial made of the above polymer-ceramic hybrid gel.

The biomaterial preferably encapsulates a bioactive substance in the polymer hydrogel and slowly releases the bioactive substance or induces tissue regeneration when the biomaterial is injected into a living body.

The bioactive material may be selected from the group consisting of proteins, growth factors, drugs, and cells.

The hybrid biomaterial preferably controls the release rate and the tissue regeneration rate of the biomolecule in addition to the composition, biodegradability, and physical properties of the biomaterial by controlling the ratio of the polymer component and the ceramic component.

The hybrid biomaterial may have a form selected from the group consisting of a paste, a particle, a porous support, a compressed support, and a film that are eluted by injection.

The present invention also provides a method for producing a polymer-ceramic fusion hybrid gel. The method for preparing a polymer-ceramic fusion hybrid gel according to the present invention comprises the steps of: introducing acrylate into all or a part of the repeating units of the biocompatible polymer to produce a polymer-acrylate compound; (2-carboxyethyl) phosphine is added to all or a part of the repeating unit of the biocompatible polymer. TCEP], lipoic acid or lipoamide to produce a polymer-TCEP compound or a polymer-lipoic acid compound or a polymer-lipoamide compound; And an aqueous solution of the polymer-acrylate compound, an aqueous solution of the polymer-TCEP compound or a polymer-lipoic acid compound or a polymer-lipoamide compound, and a buffer solution to which calcium phosphate particles have been added to prepare a polymer-acrylate compound and the polymer- Formation of a biocompatible polymer hydrogel by crosslinking the compound or the polymer-lipoic acid compound or the polymer-lipoamide compound by the Michael type addition reaction and the formation of the calcium phosphate ceramic gel by the calcium triphosphate particles added to the buffer solution And forming a polymer-ceramic hybrid gel in which the polymer hydrogel and the ceramic gel are uniformly mixed.

The total weight of the biocompatible polymer hydrogels constituting the biocompatible polymer hydrogel and the total weight of the ceramic composition constituting the ceramic gel and the total weight of the biocompatible polymer hydrogels, Is preferably 1 to 99% by weight.

Preferably, the biocompatible polymer is selected from a biocompatible polymer comprising polysaccharides consisting of hyaluronic acid, chondroitin sulfate, carboxycellulose and chitosan, gelatin, collagen, polyethylene oxide, polyvinyl alcohol and fibrin gel.

It is preferable that the calcium phosphate is made of tricalcium phosphate (TCP).

The polymer-acrylate compound is a polymer-linker-acrylate type structure in which acrylate is introduced into the linker after all or a part of the repeating unit of the biocompatible polymer is introduced, and the polymer-TCEP compound or The polymer-lipoic acid compound or the polymer-lipoamide compound may be prepared by introducing a linker to all or a part of the repeating unit of the biocompatible polymer, and then introducing TCEP or lipoic acid or lipoamide into the linker to form a polymer-linker-TCEP form or a polymer- It is preferable to have a structure of a lipoic acid type or a polymer-linker-lipoamide type.

Wherein the linker is adipic acid dihydrazide (ADH), the linker is capable of reacting with the carboxy functional group in the repeating unit of the biocompatible polymer to bind to the polymer and also to form a carboxy functional group of acrylic acid or a functional group of TCEP It is preferred to react with a carboxy functional group or a carboxy functional group of the lipoic acid to bind to the acrylate or TCEP or lipoic acid.

The aqueous solution of the polymer-acrylate compound and the aqueous solution of the polymer-TCEP compound or the polymer-lipoic acid compound or the polymer-lipoamide compound is a PBS (Phosphate buffered solution) or a citrate buffer solution, and the polymer- The compound and the polymer-TCEP compound or the polymer-lipoic acid compound or the polymer-lipoamide compound are preferably present in the aqueous solutions at a concentration of 1 to 20 wt%, respectively.

Preferably, the calcium phosphate buffer solution is a citrate buffer solution, and the buffer is present in the buffer solution at a concentration of 1 to 10 wt%. In particular, in the calcium phosphate buffer solution, Is preferably present.

In utilizing the fusion hybrid sample as a support for tissue regeneration, it is important to prepare a support capable of adhering and adhering cell adhesion and proliferation early cells. A cell adhesion polymer such as collagen, gelatin, fibronectin, a vitronectin fibrin gel, a peptide having a cell adhesion domain, or the like can be coated or included to enhance cell adhesion and proliferation. The coating is preferably used by coating and drying using the polymer solution. The polymer hydrogel such as collagen, gelatin, and fibrin gel, which replaces the hydrogel, is preferably used in the hybrid. In addition, it is preferable to apply the particles as hybrid particles to fill the space filled with a lesion having a large defect and to induce regeneration of tissues by the sustained release of the bioactive substance.

The polymer-ceramic fusion hybrid gel of the present invention has a structure in which a polymer hydrogel and a ceramic gel are mixed, and has the inventive effect that a bioactive material can be enclosed in a polymer solution preparation step using a buffer solution. In addition, when a biologically active substance is incorporated into a polymer hydrogel and the biologically active substance is injected into a living body, the bioactive substance can be released gradually from the gel, thereby effectively achieving the function of delivering a bioactive substance. Hydrogel, 3-D bioprint And the mechanical properties required as a hydrogel support for material and tissue engineering can be sufficiently achieved and can be usefully used. In addition, the polymer-ceramic fusion hybrid gel of the present invention can regulate the release rate of the bioactive substance by appropriately controlling the ratio of the polymer component and the ceramic component, can promote regeneration of the polymer into a polymer scaffold having a high degradability, Biodegradability and mechanical / chemical / biological properties of the hybrid gel / support can be controlled and utilized in various ways.

1 is an electron microscopic observation of cross-sections of polymer hydrogels, ceramic hydrogels and polymer-ceramic fusion hybrid gels prepared by mixing in various ratios according to the present invention. (A), film-type chondroitin-gelatin (10% (w / v)] hydrogel (B), α-TCP ceramic gel (C) HA-CS polymer gel-α-TCP ceramic gel (15: 1; v / v) fusion hybrid gel (D).
FIG. 2 shows the result of the distribution of polymer gel (fluorescent) in the HA-CS polymer gel-ceramic gel fusion hybrid sample using the fluorescence color for the polymer-ceramic hybrid gel according to the present invention. α-TCP ceramic gel (A) and 5% HA-CS polymer gel (w / v) -α-TCP ceramic gel (5%; w / w) fusion hybrid gel (B).
Figure 3 is a graph showing the results of the HA-CS polymer gel-α-TCP ceramic gel fusion hybrid gel [1.25%] with platelet-rich plasma (PRP) added at various concentrations [1.6, 4.8, 8.0% (v / (PDGF-BB (A), bFGF (B)] released from platelet-derived growth factor (w /
4 is HA-CS polymer gel according to the invention - in the cells in the culture flask surface by a ceramic gel fused hybrid growth factor contained in PRP released from the compression support Evaluation of osteoblast proliferation in vitro [Control (A); (B), 4.8% (C), 8.0% (D) (v / v).
Figure 5 - 8, 17 and 25% (v / v)] the HA-CS polymer gel was added to PRP according to the invention a ceramic gel fused hybrid gel (5%; w / w) in accordance with the collagen coating on the surface vitro Evaluation of bone cell adhesion and proliferation.

Hereinafter, the present invention will be described in detail with reference to the drawings.

The present invention relates to a polymer-ceramic fusion hybrid gel in which a biocompatible polymer hydrogel and a calcium triphosphate ceramic gel are uniformly mixed.

To this end, acrylate is first introduced into all or a part of the repeating units of the biocompatible polymer to produce a polymer-acrylate compound. Herein, the biocompatible polymer may include a polysaccharide consisting of hyaluronic acid, chondroitin sulfate, carboxycellulose, alginate and chitosan, collagen, fibronectin, polyethylene oxide, polyvinyl alcohol and fibrin gel.

The above schemes show the process of introducing acrylate and TCEP into chondroitin sulfate and hyaluronic acid.

In the present invention, the polymer-acrylate compound can be formed so as to have a polymer-linker-acrylate type structure by introducing a linker to all or a part of the repeating units of the biocompatible polymer and then introducing acrylate into the linker. Specifically, with reference to biocompatible polymers, e. G., The above schemes, chondroitin sulfate or hyaluronic acid reacts with dihydrazide compounds, particularly adipic acid dihydrazide (ADH). Then, the carboxy functional group in the repeat unit of chondroitin sulfate or hyaluronic acid reacts with a hydrazide amine functional group of ADH to introduce ADH into chondroitin sulfate or hyaluronic acid. It then reacts with acrylic acid. Then, the other hydrazide functional group of ADH reacts with the carboxy functional group of acrylic acid, and the acrylate is introduced into the polymer to form a polymer-ADH-acrylate type structure.

Similarly, TCEP is introduced into all or a part of the repeating units of the biocompatible polymer to produce a polymer-TCEP compound. Alternatively, lipoic acid or lipoamide may be introduced to form a polymer-lipoamide compound (or, in the case of a biocompatible polymer having an amine end group, a polymer-lipoic acid compound). Herein, the biocompatible polymer may include polysaccharides consisting of hyaluronic acid, chondroitin sulfate, carboxycellulose, alginate and chitosan, collagen, polyethylene oxide, polyvinyl alcohol and fibrin gel.

In the present invention, the polymer-TCEP compound can be formed so as to have a polymer-linker-TCEP type structure by introducing a linker to all or a part of the repeating units of the biocompatible polymer and then introducing TCEP into the linker. Specifically, with reference to biocompatible polymers, e. G., The above schemes, chondroitin sulfate or hyaluronic acid reacts with hydrazide compounds, particularly adipic acid dihydrazide (ADH). Then, the carboxy functional group in the chondroitin sulfate or hyaluronic acid repeating unit reacts with a hydrazide amine functional group of ADH to introduce ADH into chondroitin sulfate or hyaluronic acid, and then reacts with TCEP. Then, the other hydrazide functional group of ADH reacts with the carboxy functional group of TCEP, and TCEP is introduced into the polymer to form a polymer-ADH-TCEP type structure.

Similarly, a polymer-lipoic acid compound in the form of a polymer-linker-lipoic acid can be formed. At this time, when ADH is used as the linker, the carboxy functional group of the lipoic acid reacts with a hydrazide functional group of the ADH to form a bond.

On the other hand, the polymer-lipoamide compound can be formed with a linker other than ADH, for example, a polymer-linker-lipoamide compound in the form of a polymer-linker-lipoamide by using a linker having a hydrazide functional group and a carboxy functional group, Amide functional group of amide may be formed of a polymer-lipoamide compound in the form of a polymer-lipoamide by reacting directly with the carboxy functional group in the repeating unit of the polymer to form a bond.

The polymer-acrylate compound, the polymer-TCEP compound, the polymer-lipoic acid compound or the polymer-lipoamide compound prepared in the above manner are each prepared in an aqueous solution state. At this time, the aqueous solution is preferably in the form of a buffer solution, particularly preferably a PBS (phosphate buffered solution) or a citrate buffer solution. The polymer-acrylate compound and the polymer-TCEP compound or the polymer-lipoic acid compound or the polymer-lipoamide compound are preferably present in the aqueous solutions in a concentration of 1 to 20% by weight, respectively, and a phosphate buffered solution (PBS) The buffer in the buffer solution is preferably in a concentration of 1 to 20% by weight. If the concentration of the polymer is too low, the gel formation is not good. If the polymer concentration is too high, the gel formation proceeds too quickly, so concentration control is preferable for controlling the formation of the fusion hybrid gel.

Next, the calcium phosphate particles are put into a buffer solution to prepare a calcium phosphate ceramic solution. At this time, calcium phosphate which is a calcium phosphate ceramic gel is preferably tricalcium phosphate (TCP). The calcium phosphate buffer solution is preferably a citrate buffer solution, and the buffer is preferably present in the buffer solution at a concentration of 1 to 10 wt%. In particular, in the calcium phosphate buffer solution, the buffer is present at a concentration of 4-8 wt% desirable.

Next, as described above, an aqueous solution of a prepared polymer-acrylate compound, an aqueous solution of a polymer-TCEP compound or a polymer-lipoic acid compound or a polymer-lipoamide compound, a calcium phosphate ceramic particle and a citrate buffer solution are mixed to form a polymer- A hybrid gel is obtained. A hybrid gel is also prepared by adding a citrate buffer solution to a mixture composed of a polymer-acrylate compound and a polymer-TCEP compound or a polymer-lipoic acid compound or a polymer-lipoamide compound and calcium phosphate ceramic particles.

In the present invention, the total weight of the biocompatible polymer hydrogels constituting the biocompatible polymer hydrogel, that is, the total weight of the polymer-acrylate compound and the polymer-TCEP compound or the polymer-lipoic acid compound or the polymer-lipoamide compound, It is preferably 1 to 99% by weight based on the total weight of the combined weight of the polymers and the weight of the ceramics constituting the ceramic gel. The present invention can control the physical / biological / chemical properties of the hybrid gel by appropriately controlling the ratio of the polymer component to the ceramic component.

In the present invention, the polymer components and the ceramic components are first dissolved in the buffer solution, respectively. Then, the polymer-acrylate compound and the polymer-TCEP compound or the polymer-lipoic acid compound or the polymer-lipoamide compound are crosslinked by the Michael type addition reaction to form the polymer hydrogel. The ceramic component is dissolved in a buffer solution, and then a calcium phosphate ceramic gel is formed. Polymer hydrogel formation and ceramic gel formation can be accomplished by simultaneous mixing of an aqueous solution of a polymer-acrylate compound, an aqueous solution of a polymer-TCEP compound or a polymer-lipoic acid compound or a polymer-lipoamide compound, a ceramic particle and a buffer solution, Polymer hydrogels and ceramic gels are formed of uniformly mixed polymer-ceramic fusion hybrid gels, particles, and supports. Also, the polymer and ceramic may be mixed in powder and particle state, and the buffer solution may be mixed to prepare a polymer-ceramic fusion hydrogel and a support. In particular, the present invention relates to a concentrate type hybrid gel which is self-formed by simple mixing of a spray type polymer solution and a spray type ceramic solution.

In the present invention, if the concentration of the buffer in the calcium phosphate buffer solution is too high, the solidification of the polymer hydrogel and the solidification of the ceramic gel proceed too quickly, making them difficult to mix and making it difficult to induce the formulation by injection. On the other hand, if the concentration of the buffer in the calcium phosphate buffer solution is too low, the curing time of the polymer-ceramic hybrid gel becomes too long. Therefore, the concentration of the buffer in the calcium phosphate buffer solution is preferably determined appropriately, and is preferably about 4-8% by weight. However, when a polymer gel is coated on a ceramic particle, the concentration of the polymer gel is increased.

On the other hand, the polymer-ceramic hybrid gel prepared as described above can be used as a biomaterial, and a biomaterial can be incorporated into the biomaterial. The bioactive material is contained in the biological material while being enclosed in the polymer hydrogel, and the bioactive material is gradually released after the biological material is injected into the living body. The bioactive material may include an antibiotic, a protein, a growth factor, a drug, and a cell. Since a polymer solution is prepared using a buffer solution in the preparation of a fusion hybrid gel, when the hydrophilic bioactive material to be applied is included in the hybrid gel It has an advantage that denaturation of the bioactive substance is prevented. The release rate, the release amount and the release concentration of the bioactive substance in vivo depend on the amount and concentration of the bioactive substance incorporated, and the bioactive substance contained in the hybrid gel is controlled by controlling the ratio of the polymer component and the ceramic component. And can be used as a condition for controlling the degradability of the fusion hybrid gel and the tissue regeneration characteristics when the amount of the substance is adjusted and applied to a support for tissue engineering. Since the hybrid gel to be produced is characterized in that it maintains its shape even in the course of being injected into water, it can be used as a bioprinting material which is kept in a three-dimensional form containing cells and bioactive materials at room temperature, a filler of a damaged region of the spine, And the like.

Hereinafter, specific embodiments according to the present invention will be described. It should be understood, however, that the embodiments shown are only illustrative of the present invention, and thus the scope of the present invention is not limited thereto.

Example

A solution of a polymer derivative such as chondroitin sulfate (CS) having an acrylate functional group and hyaluronic acid having a TCEP functional group and a-TCP solution at a predetermined ratio to prepare a fusion hybrid gel sample, or to prepare a fusion hybrid gel To form a compressed support, and a method of analysis.

Example  1: polymer-ceramic [2.5% (w / w), 1: 1 (v / v)] fusion hybrid  Gel manufacturing

Step 1: Add 0.302 g adipic acid dihydrazide (ADH), 0.234 g hydroxybenzotrazole hydrate (HOBT), 5 mL dimethylsulfoxide (DMSO), 0.316 mL N- (3 -dimethylaminopropyl) -N'-ethylcarbodiimide (EDC) and reacted with 0.241 mL of acrylic acid. The prepared sample was put into a dialysis membrane, dialyzed with distilled water, precipitated and lyophilized to synthesize a chondroitin sulfate-acrylate compound.

Step 2: Add 0.09 g of ADH and 0.09 mL of EDC to 300 mL of 0.13% hyaluronic acid solution, add 0.43 g of TCEP, and add 0.27 mL EDC. A synthetic sample was placed on the dialysis membrane and dialyzed and precipitated in distilled water to synthesize a hyaluronic acid-TCEP compound.

Step 3: A solution of 0.003 g of an aqueous solution of hyaluronic acid-TECP and 0.003 g of chondroitin sulfate-acrylate compound aqueous solution was added to a microtube, and then 100 μl of citrate (6 wt% sodium citrate buffer solution) containing 0.200 g α- And sufficiently mixed to prepare a fusion hybrid gel. The prepared hybrid hybrid gel was put into a Teflon mold (diameter 1 cm, thickness 1 mm) to prepare a hybrid support.

2.5% (w / w) described in the title of Example 1 represents the ratio of the combined weight of the chondroitin sulfate-acrylate compound and the hyaluronic acid-TCEP compound to the total solid weight of the polymer-ceramic hybrid gel, 1 (v / v) is the ratio of the volume of the solution mixed with the chondroitin sulfate solution and the hyaluronic acid solution to the volume of the ceramic solution. Hereinafter, the same applies.

Example  2: polymer-ceramic [1.25% (w / w)] fusion Hybrid Gel  Produce

Ceramic [1.25% (w / w)] hybrid gel was prepared in the same manner as in Example 1, except that the polymer solution was prepared by mixing the solution of the polymer component and the solution of α-TCP in a ratio of 1.25% (w / w) .

Example  3: polymer-ceramic [5% (w / w), 2: 1 (v / v) Hybrid Gel  Produce

The polymer-ceramic [5%] was prepared in the same manner as in Example 1, except that the polymer solution was prepared by mixing the solution of the polymer component and the solution of α-TCP at 5% (w / w) and 2: 1 (v / (w / w), 2: 1 (v / v)] fusion hybrid gel.

Example  4: Preparation of polymer-ceramic [2.5% (w / w), 1: 1 (v / v)

A polymer-ceramic 2.5% (w / w), 1: 1 (v / v) fusion hybrid gel prepared in the same manner as in Example 1 was transferred to a Teflon mold (1 cm in diameter and 1 mm in thickness) Ceramic (2.5% (w / w), 1: 1 (v / v)] fusion hybrid support was prepared by pressing the slide glass and pressing both sides with a forceps under pressure.

Example  5: Polymer-ceramic [1.25% (w / w), 1: 2 (v / v)] Fusion Compression Support Production

Ceramic 1.25% (w / w) fusion hybrid gel prepared in the same manner as in Example 2 was transferred to a Teflon mold and polymer-ceramic [1.25% (w / w) 1: 2 (v / v)] fusion hybrid support.

Example  6: Polymer-ceramic [5% (w / w), 2: 1 (v / v)] Fusion Compression Supporting Agent Manufacture

Ceramic 5% (w / w) fusion hybrid gel prepared in the same manner as in Example 3 was transferred to a Teflon mold and polymer-ceramic [5% (w / w) 2: 1 (v / v)] hybrid compression support.

Example  7: Manufacture of collagen-coated polymer-ceramic [5% (w / w), 2: 1 (v / v)] fusion compression support

After transferring the polymer-ceramic 5% (w / w) fusion hybrid compressed support prepared in the same manner as in Example 6 to a 24-well plate, 0.3 mg of solid collagen was dissolved in 1 mL of 1% acetic acid, Respectively. The sterilized collagen solution was dispensed in 1 mL each on a polymer-ceramic 5% (w / w) fusion hybrid support, and was then dried for 6 hours in a clean bench to obtain a polymer-ceramic [5% w / w), 2: 1 (v / v)] fusion hybrid support was prepared (Fig.

Example  8: Preparation of chondroitin-gelatin (10% (w / v)) gel (Preparation of Fig. 1-B)

Step 1: Add 0.329 g of adipic acid dihydrazide (ADH), 0.331 mL EDC, and 0.530 g of TCEP to 50 mL of 1.6% gelatin solution and add 331 mL EDC to the reaction. The prepared sample was put in a dialysis membrane, dialyzed with distilled water, precipitated and lyophilized to synthesize a gelatin-TCEP compound.

Step 2: 0.302 g of ADH, 0.234 g of HOBT, 5 mL of DMSO and 0.316 mL of EDC were added to 100 mL of 0.4% chondroitin sulfate solution, and the resulting sample was reacted with 0.241 mL of acrylic acid. The resulting sample was dialyzed against distilled water, And dried to synthesize a chondroitin sulfate-acrylate compound.

Step 3: 0.01 g of a gelatin-TECP powder and 0.01 g of chondroitin sulfate-acrylate powder were placed in a microtube, and then 200 μl of a PBS (Phosphate buffered solution) was added thereto and sufficiently mixed to prepare a 10% (w / v) chondroitin gelatin hydro Gel.

Example  9: HA - CS  Polymer gel-α-TCP Ceramic gel  fusion hybrid  Gel preparation (15: 1; v / v) (Fig. 1-E)

0.023 g of hyaluronic acid-TECP and 0.023 g of chondroitin sulfate-acrylate prepared in steps 1 and 2 of Example 1 were dissolved in 468.75 μl citrate buffer solution, and then 0.12 g of α-TCP buffer solution containing 62 μl was added And mixed thoroughly with a pipette to dispense the mixed solution into a mold to prepare a polymer-ceramic [15: 1 (v / v)] fusion hybrid gel.

.

Analysis 1. Morphological observation by electron microscope

The gel-type dried polymer-ceramic fusion hybrid sample prepared in Examples 3, 8 and 9 and the α-TCP ceramic gel sample were subjected to gold vacuum coating, and then the shape and cross-sectional shape of the sample were measured using a scanning electron microscope As a result of observation (2000, 3000 magnification), it was observed that a porous polymer gel was coated with a ceramic gel (Fig. 1).

Analysis 2. Fluorescent material hybrid  In the sample Hyaluronic acid  Assessment of potential gel and drug content

In Example 3, a hydrogel was prepared by adding zinc nitrate [Zn (NO ₃ ) ₂ ] to a hyaluronic acid solution in the preparation of a hydrogel, and then a 3 mM ligand solution [2- (bis -dimethyl-1H-pyrazol-1-yl) ethyl) amino) -N- (quinolin-8-yl) acetamide; PMQA], and fluorescence by fusion of the nitrate and the PMQA substance was expressed. Thus, it was evaluated whether a polymer gel was present in the fusion hybrid and the drug could be contained in the hybrid gel. The presence of hyaluronic acid hydrogel in the polymer-ceramic fusion hybrid gel sample was observed by fluorescence microscopy to induce fluorescence expression of the fluorescent substance. Fluorescence was observed in the hyaluronic acid hydrogel, but fluorescence was hardly observed in the ceramic gel, so that the polymer hydrogel was present inside the ceramic gel, and the drug inside the polymer gel ≪ / RTI > In addition, among the hybrid samples, it was confirmed that the brightness of the fluorescent light was reduced to 5%, 2.5%, and 1.25% (w / w) in the order of smaller samples in the sample with the largest amount of hyaluronic acid hydrogel added, And the amount of the added hyaluronic acid gel was different (Fig. 2).

2-yl) acetylamino] -N- (quinolin-8-yl) acetamide]

Analysis 3. hybrid  Measure pore size and distribution of sample

Sample characteristics such as pore size, pore density, porosity and the like of the ceramic gel and the fused hybrid gel prepared in Examples 1, 2, 3, 4, 5 and 6 and the compressed support sample were analyzed by the mercury measurement method. As a result, It was confirmed that the porosity of the fused hybrid gel sample can be controlled within the range of 56-72% by adding the gel.

Analysis 4. hybrid  Of the sample Bioactive material  Loading and Release behavior  analysis

A fused hybrid gel sample prepared by adding various concentrations of platelet-rich plasma (PRP) to the polymer solution in Example 2 was prepared. The PRP-containing hybrid sample was precipitated in a buffer solution, and the solution carrying the sample at each time point of 12 hours, 1 day, 3 days, and 7 days was extracted with a micropipette (1 mL). (PDGF-BB, bFGF) released through the sample was measured quantitatively by measuring the amounts of PDGF and bGFG growth factors present in the extracted solution using a growth factor kit and an Elisa absorptiometry instrument As a result, it was observed that the release amount of two growth factors increased over time, and it was observed that the bioactive substance could be incorporated into the fusion hybrid support and the release rate could be controlled (FIG. 3).

Analysis 5. in vitro Bone cell by growth factor release Attachment  And conformance analysis

The osteoblast cells were cultured on a 12-well plate, and a milicell-PCF container containing the PRP-containing hybrid gel prepared in Example 2 was placed on a cell culture plate, and proliferation of osteoblasts following the release of the platelet- Lt; / RTI > Cell proliferation Cell proliferation on the gel and supporter surface at 1, 3, and 7 days was measured by cell count kit-8 assay, and cell proliferation was differentiated by the control of growth factor release (Fig. 4).

Analysis 6. in vitro Analysis of bone cell proliferation rate

Ceramic Fusion Hybrid Compression Supports (d = 1 cm) prepared by adding 15% PRP in Examples 6 and 7 were used to inoculate rat osteoblasts in 24 well plates for up to 14 days in vitro cultured. Cell culture The cellular proliferation on the surface of the supporter at 1, 3, 7 and 14 days was measured by the cell count kit-8 assay, and it was confirmed that the growth rate of the cells could be controlled by the inclusion and release time of the bioactive substance (Fig. 5).

Analysis 7. hybrid  Gel Injection Functionality  Evaluation results

Ceramic Fusion Hybrids were selected by selecting the ratio (volume ratio) of the polymer gel to the ceramic gel to 7.5% (w / w) 10% (w / w), 15% (w / w) and 22.5% (w / As a result of gelation, it was possible to prepare polymer - ceramic fusion hybrid gel and the injection function of gel was maintained with time. Specifically, it was confirmed that injection can be performed in water using a syringe 30G (inner diameter 0.16 mm) and 25G (inner diameter 0.26 mm) within 1-5 minutes after the hybrid gel concentration of 22.5% (w / w) .

In addition, the injection functionality of the fusion hybrid gel was confirmed by observing that injection was possible through a 18G (inner diameter 0.84 mm) syringe after 72 hours from the preparation of the 22.5% (w / w) hybrid gel.

Claims (22)

(Hereinafter referred to as "polymer-acrylate compound") having a polymer-acrylate structure formed by introducing acrylic acid into all or a part of the repeating units of the biocompatible polymer and the whole or a part of the repeating units of the biocompatible polymer Tris (2-carboxyethyl) phosphine; (Hereinafter referred to as 'polymer-TCEP compound', 'polymer-lipoic acid compound' or 'polymer-lipoic acid compound') having a polymer-TCEP structure, A biocompatible polymer hydrogel formed by cross-linking by a Michael type addition reaction and a calcium-phosphate ceramic gel formed by charging calcium phosphate particles into a buffer solution are uniformly mixed with a polymer- Ceramic Hybrid Gel. The method according to claim 1,
The total weight of the biocompatible polymer hydrogels constituting the biocompatible polymer hydrogels is preferably 1 to 100 parts by weight based on the combined weight of the total weight of the biocompatible polymers and the weight of the ceramics forming the ceramic gel, 99% by weight of the polymer-ceramic hybrid gel. The method according to claim 1,
Wherein the biocompatible polymer is selected from biocompatible polymers comprising polysaccharides consisting of hyaluronic acid, chondroitin sulfate, carboxycellulose, alginate and chitosan, collagen, polyethylene oxide, polyvinyl alcohol and fibrin gel. The method according to claim 1,
The polymer-ceramic hybrid gel is characterized in that the calcium phosphate is calcium tricalcium phosphate (TCP). The method according to claim 1,
The polymer-acrylate compound is a polymer-linker-acrylate type structure in which acrylic acid is introduced into the linker after the linker is introduced into all or a part of the repeating unit of the biocompatible polymer, and the polymer-TCEP compound or polymer -Lipoic acid compound or polymer-lipoamide compound may be prepared by introducing a linker to all or a part of the repeating unit of the biocompatible polymer and then introducing TCEP or lipoic acid or lipoamide into the linker to form a polymer-linker-TCEP form or a polymer-linker- Polymer-linker-lipoamide-type polymer or a polymer-linker-lipoamide-type polymer. 6. The method of claim 5,
Wherein the linker is adipic acid dihydrazide, the linker reacts with the carboxy functional group in the repeating unit of the biocompatible polymer and binds to the polymer, and further comprises a carboxy functional group of acrylic acid, lipoic acid, or TCEP Wherein the polymer-ceramic hybrid gel is reacted with a carboxy functional group to bind to acrylate or TCEP or lipoic acid. 7. The method according to any one of claims 1 to 6,
Wherein the polymer-ceramic hybrid gel is coated with a cell adhesion polymer. 8. The method of claim 7,
Wherein the cell adhesive polymer is collagen, fibronectin, bitronectin, fibrin, a peptide having a cell adhesion domain, or gelatin. A polymer-ceramic hybrid biomaterial prepared from a polymer-ceramic hybrid gel according to any one of claims 1 to 6.
10. The method of claim 9,
Wherein the bio-active material is gradually released when the bio-active material is enclosed in the polymer hydrogel and the bio-material is injected into the living body. 11. The method of claim 10,
Wherein the bioactive material is selected from the group consisting of proteins, growth factors, drugs, and cells. 11. The method of claim 10,
Wherein the hybrid biomaterial regulates the rate of release of the bioactive material by controlling the ratio of the polymer component to the ceramic component. 11. The method of claim 10,
Wherein the hybrid biomaterial has a shape selected from the group consisting of a scan function, a porous support, a compression support, and a film. Introducing acrylic acid into all or a part of the repeating units of the biocompatible polymer to produce a polymer-acrylate compound;
Introducing tris (2-carboxyethyl) phosphine (TCEP) or lipoic acid or lipoamide into all or a part of the repeating units of the biocompatible polymer to produce a polymer-TCEP compound or a polymer-lipoic acid compound or a polymer-lipoamide compound; And
An aqueous solution of the polymer-acrylate compound, an aqueous solution of the polymer-TCEP compound or a polymer-lipoic acid compound or a polymer-lipoamide compound, and a buffer solution into which the calcium phosphate particles have been added to obtain a polymer-acrylate compound and the polymer- Or a polymer-lipoic acid compound or a polymer-lipoamide compound is crosslinked by the Michael type addition reaction, the formation of the biocompatible polymer hydrogel and the formation of the calcium phosphate ceramic gel by the calcium phosphate particles injected into the buffer solution are carried out, A method for preparing a polymer-ceramic hybrid gel, comprising: forming a polymer-hydrogel and a polymer-ceramic hybrid gel in which the ceramic gel is uniformly mixed without phase separation. 15. The method of claim 14,
The total weight of the biocompatible polymer hydrogels constituting the biocompatible polymer hydrogels is preferably 1 to 100 parts by weight based on the combined weight of the total weight of the biocompatible polymers and the weight of the ceramics forming the ceramic gel, By weight based on the total weight of the polymer-ceramic hybrid gel. 15. The method of claim 14,
Wherein the biocompatible polymer is selected from biocompatible polymers consisting of polysaccharides consisting of hyaluronic acid, chondroitin sulfate, carboxycellulose, alginate and chitosan, collagen, polyethylene oxide, polyvinyl alcohol and fibrin gel. Gt; 15. The method of claim 14,
Wherein the calcium phosphate ceramic gel is calcium phosphate (Tricalcium phosphate) (TCP). 15. The method of claim 14,
The polymer-acrylate compound is a polymer-linker-acrylate type structure in which acrylic acid is introduced into the linker after the linker is introduced into all or a part of the repeating unit of the biocompatible polymer, and the polymer-TCEP compound or polymer -Lipoic acid compound or polymer-lipoamide compound may be prepared by introducing a linker to all or a part of the repeating unit of the biocompatible polymer and then introducing TCEP or lipoic acid or lipoamide into the linker to form a polymer-linker-TCEP form or a polymer-linker- And a polymer-linker-lipoamide-type structure. The method for producing a polymer-ceramic hybrid gel according to claim 1, 19. The method of claim 18,
Wherein the linker is an aminodihydrazide, the linker is capable of reacting with the carboxy functional group in the repeating unit of the biocompatible polymer to bind to the polymer, and further comprising a carboxy functional group of acrylic acid or a carboxy functional group of the TCEP, Wherein the polymer is reacted with a functional group to bind to acrylate or TCEP or lipoic acid. 15. The method of claim 14,
The aqueous solution of the polymer-acrylate compound and the aqueous solution of the polymer-TCEP compound or the polymer-lipoic acid compound or the polymer-lipoamide compound is a buffer solution using PBS (Phosphate buffered solution) or citrate as a buffer, And the polymer-TCEP compound or the polymer-lipoic acid compound or the polymer-lipoamide compound are present in the aqueous solutions at a concentration of 1 to 20 wt%, respectively. 15. The method of claim 14,
Wherein the calcium phosphate buffer solution is a buffer solution using citrate as a buffer, and the buffer solution is present in a concentration of 1 to 10 wt% in the buffer solution. 22. The method of claim 21,
Wherein the buffer is present in a concentration of 4 to 8% by weight in the calcium phosphate buffer solution.

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