KR20160032638A - Multi-layered hybrid scaffold with porous, biodegradable and controlled-release for simultaneous regeneration of soft and hard tissues - Google Patents

Abstract

Disclosed is a multilayered hybrid scaffold with a structure including polymer hydrogel formed in pores of a porous and biodegradable polymer scaffold and on one surface or both surfaces of the polymer scaffold. The polymer hydrogel is formed by a micro-type addition reaction mechanism which does not additionally use a cross-linking agent. The multilayered hybrid scaffold can regenerate soft and hard tissue at the same time.

Description

Technical Field [0001] The present invention relates to a multi-layered hybrid scaffold having porous, biodegradable and sustained-release properties for simultaneous regeneration of soft tissue and hard tissue,

The present invention relates to a multi-layer hybrid support having porous, biodegradable and sustained-release properties for simultaneous regeneration of soft tissue and hard tissue, and more particularly, to a multi-layer hybrid support having a polymer hydrogel on the inside of a porous and biodegradable polymer scaffold, Layered hybrid support capable of simultaneously regenerating a soft tissue and a hard tissue by allowing the bioactive material to be released at different rates.

Hard tissue, such as musculoskeletal system, is related to soft tissues and maintains human tissue. In recent years, there has been an increasing demand for medical care for elderly patients whose healing ability has been deteriorated due to rapid aging of the world's population and chronic diseases such as diabetes and osteoporosis. Especially for these patients, the soft tissues (epithelial tissues) The restoration to a more complete structure is possible.

In order to regenerate such soft tissues / hard tissues, a method of simultaneously regenerating two tissues is required. However, a multifunctional biomaterial capable of regenerating a hybrid tissue simultaneously has not been developed so far. In particular, bone and soft tissues require different techniques for inducing regeneration of soft tissue / bone tissue, considering mechanical / biological properties and tissue regeneration rates when damaged.

Dental tissue is a representative tissue that must be regenerated at the same time as soft tissues and hard tissues. Especially, dental shielding membranes, which are biomaterials used for regeneration of alveolar bone tissue, are mainly intended to inhibit downward penetration of soft tissues in hard tissue defect sites.

The type of shielding is a non-absorptive shielding membrane which has excellent space maintenance ability but needs reoperation due to the exposure frequency of the shielding membrane and an absorbing shielding membrane which does not need reoperation but has a low space maintenance ability and quicker than necessary absorption. Has been used for alveolar bone regeneration. However, since the non-absorbable and absorbable shielding film produced until now can only be used for bone regeneration, there is a disadvantage that complete periodontal tissue regeneration is impossible. To overcome this, development of a tissue regeneration support capable of simultaneously regenerating hard tissue and soft tissues has not been reported, despite the need for a manufacturing method having an ideal shielding membrane function that promotes epithelial regeneration and promotes bone regeneration.

(PLGA) polymer is commonly used as a polymer to be used as a shielding film material, PLGA polymer is a kind of an absorbent shielding film, and has a stability and absorbability in the body and is not cytotoxic (Burg KJL, Porter S, Kellam JF, Biomaterials development, 2000), although it is known to have the advantage of being able to regulate the point and absorption period and shape the shielding membrane. In order to compensate for the disadvantages of such PLGA shielding membranes, attempts have been made to chemically / physically modify the shielding membranes, one of which is hyaluronic acid (HA) A method of increasing cell affinity and increasing biocompatibility has been reported. (Toole BP, Hyaluronan in morphogenesis, Seminar Cell Dev Biol 2001; 12: 79-87.)

The present invention was conceived to overcome the limitations of the prior art described above. That is, it is an object of the present invention to provide a multi-layered hybrid support capable of simultaneously regenerating a soft tissue and a hard tissue by forming a polymer hydrogel on the inside and on the surface of the pores of the porous and biodegradable polymer scaffold.

It is also an object of the present invention to provide a multilayer hybrid support capable of inhibiting the occurrence of inflammation due to local acidification induced in a biodegradation process of a biodegradable polymer scaffold.

In order to accomplish the above object, the present invention provides a multi-layer hybrid support having a structure in which a polymer hydrogel is formed in pores of a porous and biodegradable polymer scaffold and on one surface or both surfaces of the polymer scaffold, Michael between the polymer having alpha-beta unsaturated hydrocarbon functional group such as acrylic functional group and the polymer having nucleophilic functional group such as tris (2-carboxyethyl) phosphine (TCEP) functional group, amine functional group, peptide having cystine or thiol of lipoamide Is formed by an addition reaction.

The porous and biodegradable polymeric scaffold is preferably selected from the group consisting of polylactide, polyglycolactide, poly (lactide-glacolide) copolymer, polycaprolactone, gelatin and collagen.

It is preferable that the polymer having the acrylic functional group and the polymer having the TCEP or the lipoamide functional group which are subjected to the Michael addition reaction are selected from the group consisting of polyethylene glycol, hyaluronic acid, cellulose, chitosan, fibrin and chondroitin sulfate.

The polymer hydrogel preferably contains a bioactive substance.

The bioactive material may be selected from the group consisting of growth factors for promoting tissue regeneration of hard tissues and soft tissues, inflammation inhibitors, DNA, and drugs.

The growth factor may be selected from the group consisting of an osteogenic growth factor (BMP), a fibroblast growth factor (FGF), and a platelet derived growth factor.

The drug may be selected from the group consisting of tetracycline and dimethyloxalyl glycine (DMOG).

The multi-layer hybrid support is preferably composed of three layers.

The multi-layer hybrid support according to the present invention is characterized in that a natural polymer such as hyaluronic acid capable of neutralization is added to the biodegradable polymer layer in order to inhibit inflammation due to local acidification induced in the biodegradation process of the biodegradable polymer layer induced by porosity (Lactide-glicolide) shielding membrane support caused by acidification by providing a neutralization-type hydrogel on the inside and both surfaces of the induced pores.

In addition, the multilayer hybrid support of the present invention is intended to regenerate a tissue by using a single polymer layer in a conventional shielding film product, while mainly aiming at regeneration of only one tissue, Effect.

In addition, the multi-layer hybrid support of the present invention can be used as a therapeutic drug delivery system, a tissue engineering support, or the like by selectively applying a bioactive substance suitable for hard or soft tissues to a functionally separated surface layer, And can be selectively provided at the same time.

In addition, the method for producing a multilayer hybrid support using two kinds of solutions capable of inducing Michael type addition reaction is a method for producing a multi-layer hybrid support by inducing crosslinking using radical polymerization or enzymes, thereby forming a hydrogel on both surfaces of the support Compared with the hybrid support manufacturing methods, the method using the Michael type addition reaction is a method in which the activity of the bioactive substance is maintained and byproducts / harmful substances generated when inducing crosslinking are not generated, and the spontaneous crosslinking It has the advantage of using a coupling reaction.

In addition, since each of the three-layered polymer scaffolds has a different biodegradation rate, it can provide a function of controlling the release rate of the bioactive substance. Such a three-layer support has the effect of promoting the regeneration of different tissues into different tissues for simultaneous regeneration. For example, dimethyloxalylglycine (DMOG) promotes angiogenesis, tetracycline acts as an anti-inflammatory agent, fibroblast growth factor promotes soft tissue regeneration, and bone morphogenic protein-2 (BMP-2) To provide a three-layered hybrid support for promoting inflammation prevention, angiogenesis, regeneration of soft tissue and hard tissue.

FIG. 1 shows a method of inducing penetration of a hyaluronic acid derivative solution by centrifugation into pores of porous PLGA prepared in Example 1 of Example 2 in accordance with the present invention.
FIG. 2 is a schematic diagram (A) for preparing a three-layer porous / biodegradable hybrid support for simultaneous regeneration of soft tissues and hard tissues of Example 3 prepared according to the present invention and a prepared three-layer hybrid support (B).
FIG. 3 is an electron microscopic observation of the cross section of a three-layer porous / biodegradable support with hyaluronic acid (5%) added to both sides of the PLGA disk and porous pore prepared according to the present invention. (B) a 3-layer porous / biodegradable hybrid support (B) coated with 5% hyaluronic acid, and a hybrid PLGA support (C) having a 5% hyaluronic acid solution penetrated into the pores.
FIG. 4 is a graph showing the change in the pH of a solution by adding a certain amount of hyaluronic acid to an acidic solution (pH 5.0) similar to an acidic condition when a single-layered PLGA is decomposed.
FIG. 5 is a graph showing the adhesion and proliferation properties of in vitro fibroblasts on the surface of a 3-layer porous / biodegradable hybrid support for simultaneous regeneration of PLGA and soft tissue and hard tissue prepared according to the present invention.
6 is an in vitro cell viability evaluation (14 days) of fibroblasts on a 3-layer porous / biodegradable hybrid support surface for simultaneous regeneration of PLGA and soft tissue and hard tissue prepared according to the present invention. Cell viability on a porous PLGA (control) surface Fluorescence image (A) and a 3-layer porous / biodegradable hybrid support (B) coated with 10% hyaluronic acid.
Figure 7 shows the results of drug release behavior measurements from microparticles containing tetracycline prepared according to the present invention.
Figure 8 shows the results of H & E staining of a 3-layer porous / biodegradable scaffold for simultaneous regeneration of soft tissue and hard tissue according to the present invention after transplantation into a rabbit skull 8 weeks later. A three-layered PLGA scaffold (C) with 10% HA gel, and a 3-layer PLGA scaffold (D) with 10% HA gel. (Arrowhead: defect margin, NB: new bone, CM: collagen membrane, AT: adipose tissue, F: fibrous tissue, HS: hydrogel scaffold).

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

The multilayer hybrid support of the present invention is capable of simultaneously reproducing soft tissues and hard tissues.

To this end, the multi-layer hybrid support of the present invention first comprises a biodegradable polymer scaffold. The biodegradable polymer scaffold used in the present invention is a porous polymer scaffold in which pores are formed. A porogen such as a blowing agent or a printing technique is used to form pores. The porous biodegradable polymer scaffold for use in the present invention may be formed of, for example, polylactide, polyglycolactide, poly (lactide-glacolide) copolymer, polycaprolactone, gelatin, have.

The multi-layer hybrid support of the present invention also includes a polymeric hydrogel. Such a polymer hydrogel is coated on the pores formed in the interior of the porous biodegradable polymer scaffold and is coated on one surface or preferably both surfaces of the porous biodegradable polymer scaffold.

The polymer hydrogels formed on the pores and both surfaces of the porous biodegradable polymer scaffold are formed by a Michael addition reaction between a polymer having an acrylic functional group and a polymer having tris (2-carboxyethyl) phosphine (TCEP) or a lipoamide functional group . The cross-linking of the polymers is accomplished by the Michael addition reaction, thereby completing the hydrogel formation. The polymer having an acrylic functional group and the polymer having a TCEP or lipoamide functional group to be subjected to the Michael addition reaction may include polyethylene glycol, hyaluronic acid, cellulose, chitosan, chondroitin sulfate, and the like. Of these, it is preferable to use natural polysaccharides such as hyaluronic acid and chondroitin sulfate. These materials are generally hydrophilic.

In the present specification, a polymer having an acryl functional group is represented by a polymer-acrylate, for example, hyaluronic acid-acrylate. Hyaluronic acid-acrylate can be simply expressed as HA-Ac. Also, polymers with TCEP or lipoamide functional groups can be represented by polymer-TCEP or polymer-lipoamides, such as hyaluronic acid-TCEP or hyaluronic acid-lipoamide. Hyaluronic acid-TCEP can be simply expressed as HA-TCEP.

In the above, a polymer having an acrylic functional group is presented as one of reactants for the Michael type addition reaction, wherein the acrylic functional group represents a representative example of an alpha-beta unsaturated hydrocarbon functional group as a carbonyl functional group, and a methacrylate functional group May be used as a reaction material for the Michael type addition reaction in the present invention. In addition, in the Michael type addition reaction, a polymer having a TCEP or a lipoamide functional group is exemplified as a reactive substance for an acrylic functional group. In addition, a polymer having a nucleophilic functional group such as a peptide having a cystine or a polymer having an amine functional group can be used . Wherein the lipoamide functional group and the cysteine have a thiol functional group therein and this thiol functional group acts as a nucleophile in the Michael addition reaction.

Such a polymer hydrogel may contain a bioactive substance. The bioactive material that can be used in the present invention is not particularly limited as long as it is a substance generally used for tissue regeneration for the human body and includes, for example, growth factors for promoting tissue regeneration, inflammation inhibitors, DNA, . Examples of the growth factor include osteogenic growth factor (BMP), fibroblast growth factor (FGF), and platelet derived growth factor. Examples of the drug include tetracycline and dimethyloxalylglycine (DMOG) . The polymer hydrogel may contain cells as a bioactive substance.

The present invention relates to a porous biodegradable polymer scaffold which is formed by forming polymer hydrogels having different properties on the pores and both surfaces of the porous biodegradable polymer scaffold, so that each layer of one polymer scaffold product can have different biodegradation rates, biological, mechanical and chemical properties, And the hard tissue. The present invention also allows for the simultaneous regeneration of soft and hard tissues by containing different bioactive substances in the polymer hydrogels of each layer.

That is, the multi-layered hybrid support of the present invention can provide a function of providing a support suitable for hard tissue regeneration in one layer and inducing soft tissue regeneration in the other layer. Also, it is easy to control the tissue regeneration rate, the amount of regenerated tissue and the mechanical properties by forming a polymer hydrogel layer containing biologically active substances suitable for soft tissue and hard tissue regeneration on both surfaces of the porous biodegradable polymer scaffold by applying a scanning technique .

In addition, the multilayer hybrid support of the present invention minimizes side effects because the polymer hydrogel component neutralizes the inflammatory reaction caused by the local acidification in the biodegradation and absorption process of the porous biodegradable polymer scaffold.

In addition, when a bioactive substance is to be delivered to an existing shielding membrane for tissue regeneration, for example, a shielding membrane for bone regeneration, a method of adsorbing the bioactive substance on the surface is used. However, There is a problem that the activity of the substance is lost. On the contrary, the multi-layered hybrid support of the present invention enables the biologically active substance to be gradually released by containing the bioactive substance in the polymer hydrogel, and the biologically active substance released so slowly is continuously activated, I can do it. In particular, the present invention prevents the loss of the activity of temperature sensitive biologically active materials by simply mixing the scanning type polymer solution at room temperature to form the hydrogel in the pores and on the surface of the porous biodegradable polymer scaffold.

Hereinafter, the present invention will be more specifically shown by way of examples. However, the following examples are merely examples of the present invention and should not be construed as limiting the scope of the present invention.

Example

Type gel reaction between a chondroitin sulfate having an acrylate or phosphate functional group and a sugar compound derivative such as hyaluronic acid through a chemical bond by a Michael type addition reaction in a pore in a porous PLGA support and inducing gelation by a Michael type reaction at a low temperature, And drying and drying the support to form a hybrid disk type support.

Example  One: disk  Porosity of form PLGA  Support manufacturing

Step 1: 0.18 g of PLGA was dissolved in 1 ml dioxane at 18% (g / ml) at room temperature for one day to completely dissolve the PLGA.

Step 2: The PLGA solution prepared in Step 1 was poured into a polyethylene (PE) mold having a diameter of 10 mm, and 2.4 g of ammonium bicarbonate (AB, particle size of 355 to 500 μm) was added thereto. .

Step 3: The mixture prepared in Step 2 was frozen using liquid nitrogen, the PE mold was removed with a razor blade, and the frozen cylinder sample was dried for 3 days in a freezing vacuum dryer at -80 占 폚. dioxane was removed.

Step 4: The cylindrical PLGA sample dried in step 3 was cut into 1 mm or 2 mm thick discs using a razor blade to prepare a supporter. The supernatant was loaded on 4 L distilled water at 30 ° C, Gas foaming was carried out while replacing it, and AB was removed to prepare a porous disc.

Step 5: In step 4, the PLGA sample from which the AB was removed was stored in a freeze dryer to remove moisture and recover the porous PLGA disk support.

Example  2: Porosity PLGA  Induction of penetration of hyaluronic acid solution into the pores in the support

Step 1: Hyaluronic acid-acrylate (HA-Ac) polymer derivative solution and hyaluronic acid-tricarboxyethyl phosphate (HA-TCEP) functional groups were dissolved in phosphate buffered saline (PBS) % Concentration solution.

Step 2: A disc-shaped porous PLGA support having a diameter of 10 mm and a thickness of 2 mm prepared in Example 1 was placed in a cylindrical PE mold, and the two types of 5% HA-Ac and The HA-TCEP hyaluronic acid derivative precursor solution was mixed at a ratio of 1: 1. The mixture was dispensed onto the surface of the porous PLGA support and centrifuged (3500 rpm, 5 minutes) to induce the hyaluronic acid mixed solution to penetrate into the pores of the PLGA support (Fig. 1).

Step 3: The PLGA supporter impregnated with the hyaluronic acid solution prepared in Step 2 was refrigerated for 4 to 1 days, and further stored at -70 for 1 day, so that the hyaluronic acid solution in the supernatant was added to the Michael type addition reaction Induced gelation.

Step 4: The sample which had been gelated in Step 3 was lyophilized at -40 ° C for one day to induce it to become a porous support, and the sample was recovered.

Example  3: 5% or 10% HA - AC , HA - TCEP  Manufacture of three-layer disc-shaped support using hyaluronic acid solution

Step 1: Hyaluronic acid derivative polymer solid samples of HA-Ac and HA-TCEP were each dissolved in PBS buffer solution to prepare a 5% concentration hyaluronic acid derivative precursor solution.

    Step 2: The porous PLGA support having a diameter of 10 mm and a thickness of 1 mm prepared in Example 1 was centrifuged so that the solution of the hyaluronic acid derivative was infiltrated into the PLGA in the same manner as in Step 2 of Example 2 Respectively.

Step 3: Mixing of 5% HA-Ac solution and HA-TCEP solution at a ratio of 1: 1 on the surface of a porous PLGA support impregnated with a hyaluronic acid mixed solution proceeded in step 2 using a Teflon mold The solution was dispensed to prepare a two-layered sample by coating a layer of hyaluronic acid in the form of a thin disc (0.5 mm thickness) on top of the PLGA support.

  Step 4: A new 5% or 10% hyaluronic acid derivative solution was prepared in the same manner as in the case where the lower layer of the two-layered support sample prepared in step 3 was placed on the upper surface and then coated with the hyaluronic acid derivative solution in the step 3 A 3-layer hyaluronic acid-PLGA-hyaluronic acid polymer sample coated with hyaluronic acid polymer (FIG. 2) was prepared by preparing a thin disc-shaped layer having a thickness of 0.5 mm by dispensing.

Step 5: The 3-layer hyaluronic acid-PLGA-hyaluronic acid sample prepared in step 4 was stored for 4 days to 1 day and then stored at -70 ° C for 1 day to induce hyaluronic acid hydrogelation. Respectively.

Step 6: A 3-layered hyaluronic acid-PLGA-hyaluronic acid sample in which the gelation was completed in step 5 was lyophilized at -40 ° C for one day to obtain a solution containing 5% or 10% HA-AC and 3 with HA-TCEP hyaluronic acid derivative solution Layer-hyaluronic acid-PLGA-hyaluronic acid hybrid support.

Example  4: 10% HA - AC  Solution and HA - Lipoamide  Preparation of Porous / Biodegradable Supports of Three-Layer Structure by Solution

A 10% hyaluronic acid solution was infiltrated into PLGA disk pores using a 10% solution (HA-AC, HA-lipoamide) instead of the 5% hyaluronic acid derivative solution of Example 3 (HA-AC and HA- Then, three layers of porous hybrid supports were prepared by coating hydrogel with 10% hyaluronic acid solution on both surfaces of PLGA disk. The same procedure as in Example 3 was applied to the remaining embodiments.

Example  5. Platelet richness Platelet-rich-plasma (PRP)  Added three-layer porous / biodegradable hybrid  Support manufacturing

The 10% HA-Lipoamide derivative solution of Example 4 was replaced with a 10% HA-Lipoamide derivative solution to which PRP (150 μL / gel mL) was added. The hyaluronic acid gel containing PRP penetrated into the PLGA support pores, A three layer porous / biodegradable hybrid support coated with 10% hyaluronic acid gel with PRP was prepared on the PLGA outer surface. The same procedure as in Example 3 was applied to the remaining embodiments.

Example  6. Antibiotic Drugs ( tetracycline ) Containing microparticles

Step 1: PLGA polymer was added to butadiene diepoxide (BDDE) at a concentration of 10% (v / v) and mixed for 24 hours to prepare a PLGA solution containing a BDDE crosslinking agent (PLGA-BDDE solution).

Step 2: Hyaluronic acid at a concentration of 16% (v / v) was dissolved in a 4% NaOH solution and tetracycline (50 mg / ml) was added to prepare a hyaluronic acid solution containing antibiotics.

Step 3: Spin 80 (Sigma-Aldrich, USA) was dispensed into Hexane at a concentration of 0.5% (v / v) and then dispersed for 5 minutes using an ultrasonic disperser (VCX 750, Sonic & Span80 dispersant is dispersed in Hexane (Hexane-Span80 solution) at an 80% yield of the solution.

Step 4: Mix the solution prepared by mixing PLGA-BDDE solution of step 1 and hyaluronic acid solution containing 2-step antibiotic at a ratio of 1: 1 (v / v) to the Hexane-Span80 solution prepared in step 3 The mixture was dispensed using a syringe, and HA-PLGA microparticles containing antibiotics were prepared by dispersing the mixed solution for 30 minutes at an output of 80% using an ultrasonic dispersing machine.

Step 5: PLGA-HA particles containing tetracycline drug were prepared by centrifugation (3500 rpm, 1 min) of the solution containing the antibiotic-containing (HA-PLGA) microparticles.

Example  7. tetracycline  3-layer porous / biodegradable with microparticles added hybrid  Support manufacturing

The 10% hyaluronic acid derivative solution (HA-AC, HA-Lipoamide) of Example 4

Hybrid support preparation was carried out by replacing 10% hyaluronic acid derivative solution with drug-containing (HA-PLGA) microparticles prepared in Example 6 added thereto. A porous / biodegradable hyaluronic acid hybrid support was prepared by infiltrating a 10% hyaluronic acid solution containing tetracycline drug microparticles into PLGA disk pores. The same procedure as in Example 5 was applied to the hyaluronic acid gel coating method in which the tetracycline drug was contained on both surfaces of the hybrid support.

Example  8: 3-layer porous / biodegradable hybrid  Animal implantation of support

A 16-week-old New Zealand white rabbits were implanted with a 8 mm diameter, 2 mm high bone defect. In each defect, a collagen support (Bio-Gide) (A), a control PLGA support (B), a 5% HA gel-induced 3-layer PLGA support (C) After covering the bone defect with a hybrid shielding membrane made of PLGA support (D), the periosteum was closed with an absorbable suture to fix the shielding membrane and then bred for 8 weeks (References, Biomaterials Research , Yoo Hoon et al., Accepted 2014-08-08). Animals were sacrificed and the samples were extracted and analyzed.

Analysis 1. Morphological observation by electron microscope

Each of the disk-shaped supports prepared in Examples 1, 2 and 3 was subjected to gold vacuum coating on the surface and the front surface thereof in a dried state, and the shape of the sample was observed by an electron microscope. As a result of observing the morphology of the surface and cross section of the sample at a magnification of 200 using a scanning electron microscope, it was confirmed that pores having a diameter of about 400 μm were uniformly formed in the PLGA porous support, and the inside of the 3-layer shielding film was filled with HA gel And HA gel coating on the surface were observed. In addition, as the concentration of HA gel increased from 5% to 10%, it was confirmed that hyaluronic acid gel was filled in the PLGA disk pores (FIG. 3).

Analysis 2. By hyaluronic acid addition pH  Change measurement

10 mL of distilled water was adjusted to pH 5.0 with 0.1 M hydrogen chloride, and then the pH change of the sample solution was measured using a pH meter while adding hyaluronic acid (0.012 g) Respectively. As a certain amount of hyaluronic acid was added, it was confirmed that the pH rose from 5.9 to 6.1 (FIG. 4).

Analysis 3. in vitro Fibroblast Attachment  And proliferative

Fibroblast cells (100,000) were inoculated on the surface of the three-layer porous biodegradable membrane for simultaneous regeneration of soft tissue and hard tissue prepared in Example 3, and were inoculated for 14 days in The cell proliferation on the surface of the supporter at each time point on days 1, 3, and 14 after the in vitro culture was measured by a cell count kit-8 assay (FIG. 5).

Analysis 4. in vitro  Fibroblast Survival  Confirm

On the surface of the 3-layer porous / biodegradable hybrid support prepared in Example 3, fibroblasts were inoculated for 14 days in vitro cultured was then confirmed that the results, survival to constitute the large cell clumps of fibroblasts cultured in three-layer porous / biodegradable PLGA hybrid support surface than the surface of the sample was analyzed by live & dead assay (Fig. 6).

Analysis 5: 3-layer porous / biodegradable hybrid  The support and microparticle drug ( Tetracycline ) Loading and Release behavior  analysis

A 3-layer porous / biodegradable hybrid support containing the drug (tetracycline) prepared in Example 5 and the microparticles were precipitated in PBS (10 ml) buffer solution, and samples were collected at 12 hours, 1 day, The supported solution was extracted with a micropipette (5 mL). UV-vis absorbance at 365 nm was measured to determine if tetracycline was present in the eluted solution from the sample (Figure 7).

Analysis 6: 3-layer porous / biodegradable hybrid  Support Bioactive material  Loading and Release behavior  analysis

  The three-layer porous / biodegradable hybrid support to which the platelet-rich plasma (PRP) prepared in Example 6 was added was precipitated in a buffer solution (0.1% BSA), and after 12 hours, 1 day, 3 days, (1 mL) was extracted with a micropipette. The PDGF and bFGF growth factors contained in the platelets in the solution extracted using the growth factor kit (R & D system DY220 (PDGF-BB kit), R & D system DY233 (FGF basic kit) and ELISA microplate reader Was measured at a wavelength of 450 nm. The amount of platelet-derived growth factor (PDGF-BB, bFGF) released through the sample was quantitatively measured. As a result, the release amount of the two growth factors was measured to increase with time. Thus, it has been observed that a bioactive material such as a growth factor can be included in the coating layer present on both surfaces of the porous / biodegradable hybrid support of three layers, and the release rate of the growth factor can be controlled.

Analysis 7: drug microparticles three layers porous / biodegradable hybrid  On both sides of the support Loaded  Analysis of forms

The cross-section of the three-layer porous / biodegradable hybrid support coated with the drug microparticle-added hyaluronic acid derivative solution prepared in Example 8 was cut with a razor and observed under an optical microscope and an electron microscope. As a result, The particles were loaded.

Analysis 8. in vivo  Eight weeks after transplantation of rabbit skull defects, Hematoxylin Eosin  Histological analysis through staining

    A three-layer hybrid support (C) coated with 5% HA gel, a three-layer hybrid support (D) coated with 10% HA gel, a porous / biodegradable support prepared in Examples 1, 2 and 3 ) And collagen-shielded (Bio-Gide) [(A) control samples were harvested eight weeks after implantation in a 8 mm diameter rabbit skull defect. The extracted sample was immobilized with 10% neutral formalin solution, and then impregnated with 5% nitric acid solution. Samples were grafted onto paraffin and cut to a thickness of 7 m and stained with hematoxylin and eosin (H & E) to observe new bone formation, absorption of hybrid supports, and infiltration of inflammatory cells. It was confirmed by an optical microscope that the biodegradable / porous hybrid support sample of the three-layer structure was absorbed more slowly than the collagen shielding membrane and the single layer PLGA shielding membrane sample, and the sample was retained for a longer time in the animal (Fig. 8 ). (40 on the left, 100 on the right)

(arrowhead: defect margin, NB: new bone, CM: collagen membrane, AT: adipose tissue, F: fibrous tissue, HS: hydrogel scaffold)

Claims (9)

Wherein the polymer hydrogel has a structure in which a polymer hydrogel is formed in the pores of the porous and biodegradable polymer scaffold and on one surface or both surfaces of the polymer scaffold and the polymer hydrogel does not contain a crosslinking agent and is used as an alpha-beta unsaturated hydrocarbon Wherein the polymer is formed by a Michael type addition reaction mechanism between a polymer having a functional group and a polymer having a nucleophilic functional group of a Michael type addition reaction. The method according to claim 1,
The Michael type addition reaction mechanism may include a Michael addition reaction between a polymer having an acrylic functional group and a polymer having a tris (2-carboxyethyl) phosphine (TCEP) functional group or an amine functional group, or a polymer having a thiol functional group in a lipoamide or cystine form ≪ RTI ID = 0.0 > 1, < / RTI > The method according to claim 1,
Wherein the porous and biodegradable polymeric scaffold is selected from the group consisting of polylactide, polyglycolactide, poly (lactide-glicolide) copolymer, polycaprolactone, gelatin and collagen. Support. The method according to claim 1,
Wherein the polymer having the acrylic functional group and the polymer having the TCEP or the lipoamide functional group for the Michael addition reaction are selected from the group consisting of polyethylene glycol, hyaluronic acid, cellulose, chitosan and chondroitin sulfate. The method according to claim 1,
Wherein the polymer hydrogel comprises a bioactive substance. 6. The method of claim 5,
Wherein the bioactive substance is selected from the group consisting of growth factors, inflammation inhibitors, DNA, and drugs for promoting tissue regeneration. The method according to claim 6,
Wherein the growth factor is selected from the group consisting of an osteogenic growth factor (BMP), a fibroblast growth factor (FGF), and a platelet derived growth factor. The method according to claim 6,
Wherein the drug is selected from the group consisting of tetracycline and dimethyloxalyl glycine (DMOG). The method according to claim 1,
Wherein the multi-layer hybrid support comprises three layers.

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