Compositions for preparing organic-inorganic composite hydrogels and kits containing the same for preparing organic-inorganic composite hydrogels

Abstract

This invention relates to compositions for preparing organic-inorganic composite hydrogels and kits comprising the compositions for preparing organic-inorganic composite hydrogels. Specifically, it relates to compositions for preparing organic-inorganic composite hydrogels and kits comprising the compositions for preparing organic-inorganic composite hydrogels, the kits comprising: a biocompatible polymer having photocrosslinkable functional groups; and calcium phosphate-based ceramic powder.

Description

Technical Field

[0001] This invention relates to compositions for preparing organic-inorganic composite hydrogels and kits comprising the compositions for preparing organic-inorganic composite hydrogels. Background Technology

[0002] Tissue engineering is recognized as a method for regenerating or replacing human biological tissues or organs damaged by disease or accident. This method typically uses the patient's own cells and polymer scaffolds to create artificial tissues and organs.

[0003] In short, after collecting a sample of biological tissue from a patient, only the necessary cells are isolated and cultured to ensure a sufficient number of cells are obtained. These cells can be grown into biological tissue within a porous polymer scaffold with a three-dimensional structure and then surgically transplanted back into the patient. Alternatively, the cells can be mixed with a support such as a hydrogel and then injected into the living organism to regenerate the biological tissue. The polymer scaffold used here plays various roles as the extracellular matrix (ECM) of the biological tissue. The polymer scaffold participates in cell attachment, proliferation, and differentiation, and controls the function and structure of the biological tissue to be regenerated. The polymer scaffold also regulates the diffusion of water-soluble factors, nutrients, and metabolites. In particular, the interaction between the polymer scaffold and cells is a very important factor in tissue engineering. Various studies are currently underway, including biological interactions, the physical properties of polymer scaffolds, and controlling the release of soluble factors from the scaffold to modulate the interaction between cells and the scaffold. By appropriately adjusting these factors to control cell growth and differentiation, the desired biological tissue regeneration can be successfully achieved.

[0004] Hydrogels, as such polymer scaffolds, possessing a three-dimensional hydrophilic polymer network structure capable of containing large amounts of water, have recently attracted the most attention due to their high water content and structural properties similar to the extracellular matrix (ECM). However, due to the low mechanical strength of hydrogels, efforts to improve their properties have been ongoing.

[0005] As a related prior art, Patent Document 1 discloses a method for producing a cell-loaded biocompatible polymer-biocompatible natural material hybrid scaffold, comprising the following steps: (a) forming a strut layer by distributing two or more biocompatible polymer struts side by side on a plate; (b) distributing biocompatible polymer struts side by side on the distributed biocompatible polymer strut layer at intervals in a direction intersecting the direction of the distributed biocompatible polymer struts; (c) distributing struts containing at least one natural biocompatible material selected from the group consisting of cell-loaded gelatin, fucoidan, collagen, alginate, chitosan, and hyaluronic acid between the distributed biocompatible polymer struts in step (b), while not contacting the biocompatible polymer struts, while forming crosslinks in the distributed biocompatible natural material: cell-loaded gelatin, fucoidan, collagen, alginate, chitosan, and hyaluronic acid; and (d) forming a hybrid structure by sequentially repeating steps (b) and (c) above.

[0006] In addition, Patent Document 2 proposes a polymer-ceramic hybrid membrane and its preparation method, wherein flexibility and mechanical properties are controlled by adjusting the mixing ratio of polymer and ceramic within a specific range.

[0007] However, the method proposed in Patent Document 1 (Korean Patent No.: 10-1360942) has a drawback: it requires a heat treatment process, making it difficult to manufacture at room temperature and preventing the formation of various shapes. The method proposed in Patent Document 2 (Korean Patent Publication No.: 10-2019-0057268) can improve the mechanical properties of hydrogels to some extent, but it suffers from a problem: when more ceramic is used to increase mechanical strength, the hydrogel is difficult to form.

[0008] To address the aforementioned problems, the inventors developed a composition for preparing organic-inorganic composite hydrogels and a kit containing the composition and a curing solution, thus completing the present invention. As a composition for preparing organic-inorganic composite hydrogels with high water content and exhibiting excellent mechanical properties, the composition comprises a photopolymerizable biocompatible polymer and calcium phosphate ceramic powder that can be cured by hydration reaction at room temperature. Summary of the Invention

[0009] In one aspect, the object of the present invention is to provide an organic-inorganic composite hydrogel composition for forming tissue regeneration scaffolds and a kit comprising the composition.

[0010] To achieve the above objectives, in one aspect of the present invention, a composition for preparing an organic-inorganic composite hydrogel is provided, comprising a biocompatible polymer having photocrosslinkable functional groups and calcium phosphate-based ceramic powder.

[0011] The weight ratio of the biocompatible polymer to the calcium phosphate-based ceramic powder can be greater than 1:20 and less than or equal to 1:1, preferably greater than 1:20 and less than or equal to 1:2, more preferably 1:15 to 1:5, and most preferably 1:12 to 1:7.

[0012] The biocompatible polymer may be selected from at least one of the following: alginate, hydroxypropyl methylcellulose (HPMC), hydroxyethyl cellulose (HEC), methylcellulose, carboxymethyl cellulose (CMC), gelatin, collagen, fibrinogen, chitosan, agar, matrigel, starch, pectin, polyvinyl alcohol, polyurethane, polyethylene glycol, polypropylene glycol, hyaluronic acid, and polyvinylpyrrolidone.

[0013] The calcium phosphate-based ceramic powder may be at least one selected from the following: TCP (tricalcium phosphate), hydroxyapatite, DCPD (dicalcium phosphate dihydrate), MCPM (monohydrate calcium phosphate), DCPA (anhydrous dicalcium phosphate), and BCP (biphasic calcium phosphate), and preferably α-TCP (α-tricalcium phosphate).

[0014] The compositions used to prepare organic-inorganic composite hydrogels may also contain at least one functional component and a cell.

[0015] In another aspect, the present invention provides a kit for preparing organic-inorganic composite hydrogels, comprising a composition for preparing organic-inorganic composite hydrogels and a curing solution, said composition comprising a biocompatible polymer having photocrosslinkable functional groups and calcium phosphate-based ceramic powder.

[0016] The weight ratio of the biocompatible polymer to the calcium phosphate-based ceramic powder can be greater than 1:20 and less than or equal to 1:1, preferably greater than 1:20 and less than or equal to 1:2, more preferably 1:15 to 1:5, and most preferably 1:12 to 1:7.

[0017] The curing solution can be selected from at least one of the following: saline, PBS (phosphate buffered saline), MCPM (calcium phosphate monohydrate), DSP (disodium phosphate dehydrated), MSP (disodium phosphate dehydrated), a-MEM (minimum essential medium), and HBSS (Hank's balanced salt solution).

[0018] The curing solution may also include calcium ions (Ca). 2+ ).

[0019] The kit may also include at least one functional component and a cell.

[0020] In another aspect of the invention, an organic-inorganic composite hydrogel is provided, comprising a biocompatible polymer having photocrosslinkable functional groups and a calcium phosphate-based ceramic bonded to the biocompatible polymer.

[0021] Calcium phosphate-based ceramics are CDHA (Ca-deficient hydroxyapatite).

[0022] In another aspect, the present invention provides a biomaterial comprising an organic-inorganic composite hydrogel.

[0023] Beneficial effects

[0024] According to one aspect, the composition used to prepare organic-inorganic composite hydrogels can be used to produce organic-inorganic composite hydrogels with high water content and excellent mechanical properties at room temperature.

[0025] According to one aspect, the composition for preparing organic-inorganic composite hydrogels can be used to produce organic-inorganic composite hydrogels with excellent mechanical properties by photocrosslinking of biocompatible polymers by UV irradiation and curing of calcium phosphate-based ceramics by curing liquid.

[0026] According to another aspect, the kit for preparing organic-inorganic composite hydrogels comprises a composition for preparing organic-inorganic composite hydrogels and a curing solution. This composition has high fluidity, making it easy to inject into the human body, and exhibits excellent cell adhesion after curing at room temperature. Therefore, after injection into the human body, this kit, through curing using UV irradiation and the curing solution, allows for the delivery of drugs or cells to the desired location within the body with minimal surgical intervention, and the resulting organic-inorganic composite hydrogel can be used as a scaffold for tissue regeneration.

[0027] The effects of the present invention are not limited to those described above, but should be understood to include all effects that can be inferred from the detailed description of the present invention or from the configuration of the present invention described in the claims. Attached Figure Description

[0028] Figures 1 to 5 The graph shows the results of measuring the dimensions of hydrogels according to the examples and comparative examples before and after curing with a curing solution. These hydrogels were prepared by varying the weight ratio of the biocompatible polymer and calcium phosphate ceramic powder.

[0029] Figures 6 to 11 This is a graph comparing the compressive strength values ​​of each hydrogel according to the examples and comparative examples, which were prepared by varying the weight ratio of the biocompatible polymer and calcium phosphate ceramic powder and the type of curing liquid.

[0030] Figure 12 This is a graph showing the results of measuring the crystal structure of hydrogels according to the examples and comparative examples using an X-ray diffraction (XRD) apparatus before curing with a curing solution. These hydrogels were prepared by varying the weight ratio of biocompatible polymer and calcium phosphate ceramic powder.

[0031] Figure 13 This is a graph showing the results of measuring the crystal structure of hydrogels according to the examples and comparative examples using an X-ray diffraction (XRD) device after curing with a curing solution. These hydrogels were prepared by changing the weight ratio of biocompatible polymer and calcium phosphate ceramic powder.

[0032] Figure 14 This is a graph showing the results of water content measurements of hydrogels according to the examples and comparative examples prior to freeze-drying, which were prepared by varying the weight ratio of biocompatible polymer and calcium phosphate ceramic powder.

[0033] Figure 15 This is a graph showing the results of water content measurements of hydrogels according to the examples and comparative examples after freeze-drying, which were prepared by varying the weight ratio of biocompatible polymer and calcium phosphate ceramic powder.

[0034] Figure 16 These are photographs showing the surfaces of hydrogels according to the examples and comparative examples as observed under a scanning electron microscope (SEM). These hydrogels were prepared by varying the weight ratio of the biocompatible polymer and calcium phosphate ceramic powder.

[0035] Figure 17 These are photographs showing the results of observing the cell adhesion ability of octadecyl rhodamine B-stained cells on hydrogels according to the examples and comparative examples under a fluorescence microscope. These hydrogels were prepared by varying the weight ratio of biocompatible polymers and calcium phosphate ceramic powder.

[0036] Figure 18 These are photographs showing the photocrosslinked and ionic crosslinked states of the compositions according to the Examples and Comparative Examples, observed to determine whether a hydrogel has formed.

[0037] Figure 19 This is a photograph showing the state of a composition according to one embodiment, in which a biocompatible polymer and calcium phosphate ceramic powder are mixed at a weight ratio of 1:20, after photocrosslinking, as observed to determine whether a hydrogel has formed.

[0038] Figure 20This is a graph showing the initial drug loading efficiency of hydrogels prepared according to the examples and comparative examples by changing the weight ratio of biocompatible polymer and calcium phosphate ceramic powder.

[0039] Figure 21 This is a graph showing the release behavior of hydrogels prepared according to the examples and comparative examples by changing the weight ratio of biocompatible polymer and calcium phosphate ceramic powder one month after drug loading.

[0040] Figure 22 This is a graph showing the results of measuring the surface cell DNA content after culturing MG-63 cells on the hydrogel surface for 2 weeks. The purpose is to demonstrate the possibility of inducing osteogenic differentiation with hydrogels prepared according to the examples and comparative examples by changing the weight ratio of biocompatible polymer and calcium phosphate ceramic powder.

[0041] Figure 23 This is a graph showing the results of measuring the ALP activity of surface cells after culturing MG-63 cells on the hydrogel surface for 2 weeks. The purpose is to demonstrate the possibility of inducing osteogenic differentiation with hydrogels prepared according to the examples and comparative examples by changing the weight ratio of biocompatible polymer and calcium phosphate ceramic powder.

[0042] Figure 24 These are photographs showing the experimental process of confirming UV curing after injecting the compositions according to the examples and comparative examples into living tissue.

[0043] Figure 25 It is shown Figure 12 A photo of the experimental results.

[0044] Figure 26 This is a graph showing the compressive strength values ​​of the hydrogels according to the embodiments and comparative examples after UV curing when injected into living tissue. Detailed Implementation

[0045] Preferred embodiments of the present invention are described below with reference to the accompanying drawings. However, embodiments of the present invention can be modified and altered in various ways, and the present invention is not limited to the following description. It will be apparent to those skilled in the art that embodiments of the present invention are given for the purpose of more accurately explaining the invention. Therefore, for clarity, the shape and size of elements in the drawings may be enlarged, and elements indicated by the same reference numerals in the drawings are the same elements. In all drawings, factors showing similar functions or activities are also indicated by the same reference numerals. Furthermore, the term "includes" an element throughout the specification does not exclude other elements, but may include other elements unless otherwise specifically stated.

[0046] In one aspect of the invention, the invention provides a composition for preparing an organic-inorganic composite hydrogel, comprising a biocompatible polymer having photocrosslinkable functional groups and calcium phosphate-based ceramic powder.

[0047] According to one aspect, the composition used to prepare organic-inorganic composite hydrogels can be used to prepare organic-inorganic composite hydrogels with high water content and excellent mechanical properties.

[0048] Furthermore, according to one aspect, the composition used to prepare the organic-inorganic composite hydrogel can be used to prepare the organic-inorganic composite hydrogel with excellent cell adhesion ability.

[0049] Therefore, according to one aspect, the composition used to prepare the organic-inorganic composite hydrogel can be a composition used to prepare hydrogels for use as biomaterials such as scaffolds for tissue regeneration, but is not always limited to this.

[0050] Below, a composition for preparing an organic-inorganic composite hydrogel will be described in detail according to one aspect.

[0051] According to one aspect, compositions for preparing organic-inorganic composite hydrogels include biocompatible polymers having photocrosslinkable functional groups.

[0052] The biocompatible polymer may be selected from at least one of the following: alginate, hydroxypropyl methylcellulose (HPMC), hydroxyethyl cellulose (HEC), methylcellulose, carboxymethyl cellulose (CMC), gelatin, collagen, fibrinogen, chitosan, agar, matrix gum, starch, pectin, polyvinyl alcohol, polyurethane, polyethylene glycol, polypropylene glycol, hyaluronic acid, and polyvinylpyrrolidone.

[0053] The molecular weight of the biocompatible polymer can be from 5,000 Daltons to 5,000,000 Daltons, more preferably from 10,000 Daltons to 1,000,000 Daltons.

[0054] Biocompatible polymers have photocrosslinkable functional groups, through which they can be photocrosslinked.

[0055] At this time, the photocrosslinkable functional group can be at least one selected from the following: methacrylate group, acrylate group, vinyl group, epoxy group and tyrosine group.

[0056] For example, a biocompatible polymer could be an alginate with methacrylate groups.

[0057] If the biocompatible polymer does not contain photocrosslinkable functional groups, the preparation of organic-inorganic composite hydrogels using the above composition may lead to some problems, such as the inability to form hydrogels due to poor crosslinking or the low mechanical strength of the prepared hydrogels.

[0058] According to one aspect, the composition used to prepare the organic-inorganic composite hydrogel includes calcium phosphate-based ceramic powder.

[0059] The calcium phosphate-based ceramic powder may be selected from at least one of the following: α-TCP (α-tricalcium phosphate), β-TCP (β-tricalcium phosphate), hydroxyapatite, DCPD (dicalcium phosphate dihydrate), MCPM (monocalcium phosphate monohydrate), DCPA (anhydrous dicalcium phosphate), and BCP (biphasic calcium phosphate). However, it may be more preferable that the calcium phosphate-based ceramic powder is α-TCP, which can be cured by a hydration reaction at room temperature.

[0060] Specifically, α-TCP can be cured at room temperature by hydration reaction with a curing solution such as PBS, simultaneously undergoing a phase transition to CDHA (Ca-deficient hydroxyapatite), which possesses excellent cell adhesion and mechanical strength.

[0061] According to one aspect, the composition used to prepare the organic-inorganic composite hydrogel can form an organic-inorganic composite hydrogel at room temperature by light irradiation and immersion in a curing solution. The formed hydrogel can possess very excellent mechanical properties because it is combined through photocrosslinking of the hydrogel and curing of the ceramic powder.

[0062] According to one aspect, the composition used to prepare the organic-inorganic composite hydrogel contains an amount of calcium phosphate-based ceramic powder equal to or greater than the weight of the biocompatible polymer.

[0063] The composition may include a biocompatible polymer and calcium phosphate-based ceramic powder in a weight ratio greater than 1:20 and less than or equal to 1:1, preferably greater than 1:20 and less than or equal to 1:2, more preferably 1:15 to 1:5, and most preferably 1:12 to 1:7.

[0064] If the weight of the calcium phosphate-based ceramic powder is less than the weight of the biocompatible polymer, the resulting organic-inorganic composite hydrogel may have lower mechanical strength, making it difficult to use for applications requiring higher mechanical strength, and also difficult to use as a biomaterial, such as a scaffold for tissue regeneration, due to its low adhesion.

[0065] Furthermore, if the weight of the calcium phosphate-based ceramic powder is more than 20 times that of the biocompatible polymer, the photocrosslinking of the polymer may not be completed properly, and therefore the organic-inorganic composite hydrogel may not be formed.

[0066] According to one aspect, the composition for preparing organic-inorganic composite hydrogels may also include at least one functional component and a cell.

[0067] Functional components may include drugs, proteins such as growth factors, etc.

[0068] According to one aspect, the composition for preparing the organic-inorganic composite hydrogel may also include at least one functional component and cell for the purpose of delivering cells, growth factors, drugs, etc. in vivo, and may also be used as a biomaterial, such as a scaffold for tissue regeneration in vivo.

[0069] In another aspect, the present invention provides a kit for preparing organic-inorganic composite hydrogels, the kit comprising a composition for preparing organic-inorganic composite hydrogels, the composition comprising a biocompatible polymer having photocrosslinkable functional groups and calcium phosphate-based ceramic powder; and a curing solution.

[0070] Below, a kit for preparing organic-inorganic composite hydrogels according to another aspect will be described in detail.

[0071] A kit for preparing organic-inorganic composite hydrogels may include a composition for preparing organic-inorganic composite hydrogels and a curing solution for curing calcium phosphate-based ceramic powder included in the composition at room temperature.

[0072] The composition for preparing organic-inorganic composite hydrogels may include the above-described configuration of the composition for preparing organic-inorganic composite hydrogels.

[0073] Aqueous solutions can be used as curing solutions, but culture media for cell culture are preferred to increase the biocompatibility of the organic-inorganic hydrogel to be prepared.

[0074] The curing solution can be selected from at least one of the following: saline, PBS (phosphate buffered saline), MCPM (calcium phosphate monohydrate), DSP (disodium phosphate dehydrate), MSP (disodium phosphate dehydrate), α-MEM (minimum essential medium), and HBSS (Henkes balanced salt solution).

[0075] The kit for preparing organic-inorganic composite hydrogels is a kit for preparing hydrogels for use as biomaterials such as scaffolds for tissue regeneration, and may also include at least one functional component and cellular component. Functional components may include drugs, proteins such as growth factors, etc.

[0076] Organic-inorganic composite hydrogels can be prepared using a kit for preparing organic-inorganic composite hydrogels by the following methods.

[0077] Specifically, the preparation process includes the following steps: photocrosslinking a composition containing a biocompatible polymer with photocrosslinkable functional groups and calcium phosphate-based ceramic powder for preparing an organic-inorganic composite hydrogel by irradiation; and immersing the photocrosslinked composition in a curing solution.

[0078] First, the composition used to prepare the organic-inorganic composite hydrogel is photocrosslinked by irradiation with light, such as UV.

[0079] This step involves photocrosslinking the biocompatible polymer contained in the composition to form a hydrogel.

[0080] This composition can be irradiated with light to form a hydrogel with excellent mechanical strength.

[0081] For example, when a solution containing alginate and α-TCP in a weight ratio of 1:10 is irradiated with UV light, cross-linking of alginate can be formed well, but when ionic cross-linking is performed by immersion in a solution containing calcium ions, cross-linking of alginate may not form properly.

[0082] Because the compositions used to prepare organic-inorganic composite hydrogels are fluid, they can be easily delivered to target sites within the body in a minimally invasive manner. Therefore, after delivery of the composition to the target site in the body, a photocrosslinking step can be performed by irradiating the composition with light (such as UV).

[0083] The composition may include a biocompatible polymer and calcium phosphate-based ceramic powder in a weight ratio greater than 1:20 and less than or equal to 1:1, preferably greater than 1:20 and less than or equal to 1:2, more preferably 1:15 to 1:5, and most preferably 1:12 to 1:7.

[0084] The composition for preparing organic-inorganic composite hydrogels, which includes a biocompatible polymer and calcium phosphate-based ceramic powder in the above weight ratio, can be used to prepare hydrogels with high water content and significantly superior mechanical strength.

[0085] Next, the photocrosslinked composition is immersed in the curing solution.

[0086] This step is to solidify the ceramic powder contained in the hydrogel formed by photocrosslinking.

[0087] At this point, an aqueous solution can be used as the curing solution, but it is preferable to use a culture medium for cell culture to increase the biocompatibility of the organic-inorganic hydrogel to be prepared.

[0088] Hydrogels containing ceramic powder formed by UV irradiation can be cured by injecting a culture medium or body fluids after being injected into the human body, without the need for a separate curing liquid.

[0089] The curing solution is preferably PBS (phosphate-buffered saline) to further improve the mechanical strength of the organic-inorganic hydrogel to be prepared, and more preferably contains calcium ions (Ca). 2+ PBS (phosphate-buffered saline).

[0090] In another aspect of the invention, an organic-inorganic composite hydrogel is provided, comprising a biocompatible polymer having photocrosslinkable functional groups, and a calcium phosphate-based ceramic bonded to the biocompatible polymer.

[0091] The organic-inorganic composite hydrogel comprises a biocompatible polymer with photocrosslinkable functional groups and a calcium phosphate-based ceramic bonded to the biocompatible polymer, wherein the calcium phosphate-based ceramic may preferably be CDHA (calcium-deficient hydroxyapatite).

[0092] Organic-inorganic composite hydrogels have high water content and excellent mechanical strength and cell adhesion ability, so they can be used as biomaterials, such as scaffolds for tissue regeneration.

[0093] In another aspect, the present invention provides a biomaterial comprising an organic-inorganic composite hydrogel.

[0094] The present invention will now be described in detail through the following embodiments and experimental examples.

[0095] However, the following embodiments and experimental examples are only for illustrating the present invention, and the content of the present invention is not limited thereto.

[0096] <Preparation Example 1>

[0097] 3 g of sodium alginate (molecular weight: 200,000-300,000; FMC Biopolymers) was dissolved in 300 mL of 0.1 M 2-(N-morpholino)ethanesulfonic acid (MES) buffer (0.3 M NaCl, pH 6.5). 1.628 g of N-hydroxysuccinimide (sulfo-NHS), 2.875 g of dichloroethane (EDC), and 1.234 g of N-(2-aminoethyl)methacrylamide hydrochloride (AEMA) were mixed in the buffer, and the mixture was then synthesized for 20 hours. Following synthesis, the alginate was dialyzed against distilled water for 4 days, treated with activated carbon, filtered through a 0.22 μm filter, and lyophilized to prepare alginate with methacrylate groups.

[0098] <Example 1> Composition for preparing organic-inorganic composite hydrogels

[0099] An alginate solution was prepared by dissolving 0.087 g of the alginate obtained in Preparation Example 1 in distilled water. Then, 0.0435 g of α-TCP (α-tricalcium phosphate) was added to the solution and dispersed using a three-dimensional ultrasonic mixer to prepare a composition for preparing an organic-inorganic composite hydrogel with a weight ratio of alginate to α-TCP of 1:0.5.

[0100] <Example 2> Composition for preparing organic-inorganic composite hydrogels

[0101] A composition for preparing an organic-inorganic composite hydrogel with a weight ratio of alginate and α-TCP of 1:1 was prepared in the same manner as in Example 1, except that the weight of α-TCP in Example 1 was changed to 0.087 g.

[0102] <Example 3> Composition for preparing organic-inorganic composite hydrogels

[0103] A composition for preparing an organic-inorganic composite hydrogel with a weight ratio of alginate and α-TCP of 1:2 was prepared in the same manner as in Example 1, except that the weight of α-TCP in Example 1 was changed to 0.174 g.

[0104] <Example 4> Composition for preparing organic-inorganic composite hydrogels

[0105] A composition for preparing an organic-inorganic composite hydrogel with a weight ratio of alginate and α-TCP of 1:5 was prepared in the same manner as in Example 1, except that the weight of α-TCP in Example 1 was changed to 0.435 g.

[0106] <Example 5> Composition for preparing organic-inorganic composite hydrogels

[0107] A composition for preparing an organic-inorganic composite hydrogel with a weight ratio of alginate and α-TCP of 1:10 was prepared in the same manner as in Example 1, except that the weight of α-TCP in Example 1 was changed to 0.87 g.

[0108] <Example 6> Composition for preparing organic-inorganic composite hydrogels

[0109] A composition for preparing an organic-inorganic composite hydrogel with a weight ratio of alginate and α-TCP of 1:20 was prepared in the same manner as in Example 1, except that the weight of α-TCP in Example 1 was changed to 1.74 g.

[0110] <Example 7> Organic-Inorganic Composite Hydrogel

[0111] The solutions of Examples 1 to 6 were photocrosslinked by UV irradiation, and then immersed in the following curing solutions at 37°C for about 25 hours to prepare organic-inorganic composite hydrogels.

[0112] At this point, α-MEM (minimum essential medium), PBS (phosphate-buffered saline), PBS (phosphate-buffered saline) containing approximately 0.1 g / L calcium chloride (CaCl2), HBSS (Hankes balanced salt solution), and saline were used as curing solutions.

[0113] <Comparative Example 1>

[0114] An alginate solution was prepared by dissolving 0.087 g of the alginate obtained in Preparation Example 1 in 5 mL of distilled water.

[0115] <Comparative Example 2>

[0116] The solution of Comparative Example 1 was photocrosslinked by UV irradiation, and then immersed in the following curing solutions at 37°C for about 25 hours to prepare hydrogels.

[0117] At this point, α-MEM (minimum essential medium), PBS (phosphate-buffered saline), PBS (phosphate-buffered saline) containing approximately 0.1 g / L calcium chloride (CaCl2), HBSS (Hankes balanced salt solution), and saline were used as curing solutions.

[0118] <Comparative Example 3>

[0119] By adding calcium ions (Ca) 2+ The solutions of Comparative Example 1 (α-TCP / alginate = 0), Example 2 (α-TCP / alginate = 1), Example 5 (α-TCP / alginate = 10), and Example 6 (α-TCP / alginate = 20) were ion-crosslinked and then immersed in the following PBS (phosphate buffered saline) curing solutions at 37°C for about 25 hours to prepare organic-inorganic composite hydrogels.

[0120] <Experimental Example 1> Dimensional Change Analysis

[0121] In the preparation of organic-inorganic hydrogels according to one aspect, in Example 7 and Comparative Example 2, the dimensions of the hydrogels before and after curing with the curing solution were measured using the ImageJ program by varying the weight ratio of the biocompatible polymer and the calcium phosphate-based ceramic powder, as well as the type of curing solution. The results are shown in... Figures 1 to 5 middle.

[0122] Figures 1 to 5 The horizontal axis represents the weight ratio of α-TCP to alginate contained in the composition.

[0123] like Figures 1 to 5 As shown, it can be seen that the size of the hydrogel after curing with the curing solution shows a slight decreasing trend compared to after UV light crosslinking and before curing with the curing solution, but there is no significant difference. Therefore, when a composition according to one aspect is injected into the body as a tissue replacement and regeneration material and then cured in vivo, it can be expected that it will maintain its initial volume without volume reduction due to the curing solution.

[0124] <Experimental Example 2> Evaluation of Mechanical Properties

[0125] To confirm the differences in mechanical properties of the organic-inorganic hydrogel of the present invention due to the use of curing liquid, the weight ratio of biocompatible polymer and calcium phosphate-based ceramic powder, and the type of curing liquid, the compressive strength of the organic-inorganic composite hydrogel prepared in Example 7 and the hydrogel prepared in Comparative Example 2 were measured using a rotational rheometer (TA Instruments) in axial compression mode. The low modulus values ​​of the stress-strain curves obtained from the above measurements were compared. Figures 6 to 11 As shown in the image.

[0126] As a result, Figures 6 to 11 As shown, when no curing liquid is used (before setting), even increasing the α-TCP content does not improve the compressive strength, while when curing liquid is used, the compressive strength improves with increasing α-TCP content.

[0127] This reveals that the mechanical properties of the organic-inorganic composite hydrogel containing calcium phosphate-based ceramic powder are superior to those without. This can be attributed to the fact that the organic-inorganic composite hydrogel photocrosslinked by UV irradiation was not cured by the curing solution when α-TCP was not included, while it was cured by the curing solution when α-TCP was included.

[0128] Furthermore, regardless of the type of curing solution, the hydrogels of Examples 1 to 5 (α-TCP / alginate weight ratio = 0.5–10) exhibited higher compressive strength compared to the hydrogel of Comparative Example 1 (α-TCP / alginate weight ratio = 0) without α-TCP. In particular, the compressive strength was very high when the weight ratio of alginate to α-TCP was 5–10, and significantly higher when the weight ratio of alginate to α-TCP was 10.

[0129] This leads to the discovery that when the weight ratio of biocompatible polymer to calcium phosphate-based ceramic powder is 5 to 10, especially when the weight ratio is 10, the mechanical properties can be significantly improved.

[0130] In addition, when using calcium ions (Ca) 2+ In the case of PBS (PBS w / Ca), compared with the use of other curing solutions and the use of PBS without calcium ions (PBS w / o Ca), the mechanical properties are significantly improved when the weight ratio of biocompatible polymer to calcium phosphate-based ceramic powder is 5 to 10, especially when the weight ratio is 10.

[0131] <Experimental Example 3> Analysis of Crystal Structure Changes

[0132] To confirm the crystal structure before and after curing with a curing solution during the preparation of an organic-inorganic hydrogel according to one aspect, X-ray diffraction was used to measure the crystal structure before and after immersion in the curing solution in Example 7 and Comparative Example 2. Results using PBS as the curing solution are shown in... Figure 12 and Figure 13 middle.

[0133] As a result of crystal structure analysis, the main components of the composition, alginate and α-TCP, were present before immersion in the curing solution, such as Figure 12 As shown, after immersion in the curing solution, alginate and CDHA are present, as... Figure 13 As shown.

[0134] This can be attributed to the curing liquid transforming the α-TCP phase contained in the composition into CDHA.

[0135] Mechanical property evaluation and crystal structure analysis revealed that mixing biocompatible polymers and α-TCP, photocrosslinking via UV irradiation, converting α-TCP to CDHA using a curing solution, and curing at low temperature can significantly improve the mechanical properties of the hydrogel.

[0136] <Experimental Example 4> Evaluation of Water Content

[0137] To evaluate the water content of the organic-inorganic hydrogel according to one aspect, the water content of the organic-inorganic composite hydrogel prepared in Example 7 and the hydrogel prepared in Comparative Example 2 before lyophilization was measured. Figure 14 ) and freeze-dried ( Figure 15 The water content of PBS was shown. Results using PBS as the curing solution are illustrated in... Figure 14 and Figure 15 middle.

[0138] like Figure 14 and Figure 15As shown, as a comparison of the results before and after freeze-drying, the water content decreased slightly with the increase of the α-TCP content used, but even when the α-TCP content was 10 times the weight of alginate, the water content was still higher than 80%. This shows that even when the α-TCP content is 10 times the weight of alginate, the hydrogel can maintain excellent water content.

[0139] Furthermore, when the α-TCP content was 10 times the weight of alginate, it was found that it could contain more than 4.2 times the amount of water before freeze-drying compared to after freeze-drying. This suggests that it may exhibit high cell affinity, which would be beneficial for nutrient transport and gas supply within the body.

[0140] <Experimental Example 5> Surface Analysis

[0141] To analyze the surface structure of the organic-inorganic hydrogels according to one aspect, the surfaces of the organic-inorganic composite hydrogel prepared in Example 7 and the hydrogel prepared in Comparative Example 2 were observed using scanning electron microscopy (SEM). Results using PBS as the curing solution are shown in... Figure 16 middle.

[0142] like Figure 16 As shown, with the increase of α-TCP content, the exposure of α-TCP particles on the surface increases, and the α-TCP particles aggregate and connect with each other. It can be seen that the mechanical properties of the organic-inorganic composite hydrogel are improved through this connection. Therefore, it is found that a certain proportion of α-TCP should be included in the organic-inorganic composite hydrogel composition to achieve the effect of improving mechanical properties through ceramic powder.

[0143] <Experimental Example 6> Evaluation of Cell Adhesion Ability

[0144] To confirm the cell adhesion properties of the organic-inorganic hydrogel according to one aspect, cells stained with octadecylrhodamine B were seeded on the surfaces of the organic-inorganic composite hydrogel prepared in Example 7 and the hydrogel prepared in Comparative Example 2. After culturing for 24 hours, the degree of cell adhesion was determined by observation under a fluorescence microscope. The results using PBS as the curing solution are shown in... Figure 17 middle.

[0145] like Figure 17As shown, cell culture was almost impossible on the hydrogel prepared using the composition of Comparative Example 1 (α-TCP 0) without α-TCP, while cell culture proceeded to a minimal extent on the organic-inorganic composite hydrogel prepared using the composition of Example 2 (α-TCP 1) (where the weight ratio of α-TCP to alginate was 1). Large numbers of cells were cultured on the organic-inorganic composite hydrogel prepared using compositions containing more α-TCP, particularly on organic-inorganic composite hydrogels prepared using compositions with α-TCP at a weight ratio of 5 to 10 times that of alginate.

[0146] This can be attributed to the fact that, according to one aspect, CHDA generated by α-TCP crystallization is exposed on the surface of an organic-inorganic composite hydrogel, and cell adhesion ability is significantly improved by CHDA.

[0147] <Experimental Example 7> Differences between photocrosslinking and ionic crosslinking

[0148] To demonstrate the difference between photocrosslinking and ionic crosslinking in the formation of organic-inorganic hydrogels, the compositions of Examples 2, 5, 6, and Comparative Example 1 were photocrosslinked using the method of Example 7 to form hydrogels, and the shapes after ionic crosslinking using the method of Comparative Example 3 were compared and observed. The results are shown in... Figure 18 and Figure 19 middle.

[0149] Figure 18 The results show comparative observations of the shapes of hydrogels formed by photocrosslinking the compositions of Examples 2 and 5 and Comparative Example 1 using the method of Example 7, and by ionic crosslinking using the method of Comparative Example 3. Figure 18 As shown, the composition of Comparative Example 1, which does not contain α-TCP, forms a hydrogel through photocrosslinking and ionic crosslinking, while the compositions of Examples 2 and 5, which contain α-TCP, form a hydrogel through photocrosslinking, but do not form a hydrogel through ionic crosslinking due to the poor crosslinking properties of calcium ions. This indicates that photocrosslinking enables the compositions containing alginate and α-TCP to form a hydrogel structure more effectively than ionic crosslinking.

[0150] Figure 19 The results of observing the shape of the composition of Example 6 after photocrosslinking using the method of Example 7 are shown. Figure 19 As shown, when the weight ratio of alginate to α-TCP was 1:20, it was confirmed that no hydrogel was formed because cross-linking was not properly achieved even by photocross-linking methods.

[0151] The results show that when the weight ratio of α-TCP / alginate is less than 20, the composition containing alginate and α-TCP can form a hydrogel through photocrosslinking.

[0152] <Experimental Example 8> Growth Factor Delivery Characteristics

[0153] To demonstrate the feasibility of delivering growth factors (a protein form used to induce tissue differentiation), BSA was added to the solutions of Comparative Example 1 and Examples 2 through 5, photocrosslinked by UV irradiation, and then immersed in PBS (phosphate-buffered saline) curing solution at 37°C for approximately 24 hours to prepare an organic-inorganic composite hydrogel, and its initial drug loading efficiency and release behavior over one month were confirmed. Results are shown in... Figure 20 and Figure 21 middle.

[0154] Figure 20 The graph shows the initial drug loading efficiency, confirming that the amount of protein remaining 24 hours after initial curing increases with the increase of the proportion of ceramic material in the organic-inorganic composite hydrogel.

[0155] also, Figure 21 The figure shows the release behavior over a month, confirming that protein release can be induced from the organic-inorganic composite hydrogel for more than a month.

[0156] <Experimental Example 9> Osteogenic Differentiation Induction Characteristics

[0157] To confirm the potential of induced osteogenic differentiation as a functional bone substitute, MG-63 cells were cultured for 2 weeks on the surface of an organic-inorganic composite hydrogel prepared by the method of Example 7 using solutions from Examples 2 to 5, and the DNA and ALP activities of the cells on the surface were confirmed. The results showed that... Figure 22 and Figure 23 middle.

[0158] like Figure 22 and Figure 23 As shown, the higher the proportion of ceramic, the higher the activity of osteogenic differentiation factor ALP, especially when the weight of α-TCP relative to alginate is more than 5 times, it confirms that osteogenic differentiation is induced very rapidly.

[0159] <Experimental Example 10> Evaluation of curing characteristics after injection into living tissue

[0160] To confirm the curing characteristics of the material after it is injected into living tissue using UV and curing solution, the following experiments were conducted.

[0161] Figure 24 These are photographs showing the experimental process of confirming the curing properties using UV light after injection into living tissue.

[0162] like Figure 24 As shown, the compositions for preparing organic-inorganic composite hydrogels of Examples 1 to 5 and the composition of Comparative Example 1 were dropped onto a glass substrate, and the skin of an experimental rat was placed under the skin of the rat. The rat's skin was then irradiated with UV light to determine whether the UV light could pass through the rat's skin and cause the composition to form a hydrogel.

[0163] As experimental results confirmed, UV radiation penetrated the skin of rats, and hydrogels were formed from the compositions of Examples 1 to 5 used for preparing organic-inorganic composite hydrogels and the composition of Comparative Example 1. The results of Example 5 and Comparative Example 1 are shown in... Figure 25 middle.

[0164] Furthermore, after immersing the hydrogels formed from the compositions of Examples 1 to 5 for preparing organic-inorganic composite hydrogels and the composition of Comparative Example 1 in physiological saline for approximately 24 hours, a compressive strength measurement experiment was conducted in the same manner as in Experimental Example 2, and the low modulus values ​​of the resulting stress-strain curves were compared. The results showed that... Figure 26 middle.

[0165] like Figure 26 As shown, as a result of the compressive strength test, the hydrogels formed from the compositions of Examples 1 to 5 (α-TCP / alginate weight ratio = 0.5 to 10) exhibited higher compressive strength than the hydrogels formed from the composition of Comparative Example 1 (α-TCP / alginate weight ratio = 0), which did not contain α-TCP. In particular, the compressive strength was higher when the weight of α-TCP relative to the weight of alginate was 5 to 10 times, and significantly higher when it was 10 times.

[0166] The results above confirm that the composition for preparing an organic-inorganic composite hydrogel according to one embodiment can be injected into an animal, then cured into a hydrogel by UV irradiation, and its strength can be improved by a curing solution such as saline.

Claims

1. An organic-inorganic composite hydrogel, prepared by a method comprising the following steps: The steps include photocrosslinking the composition used to prepare the organic-inorganic composite hydrogel by irradiation with light; and immersing the photocrosslinked composition in a curing solution. The composition used to prepare the organic-inorganic composite hydrogel comprises a biocompatible polymer having photocrosslinkable functional groups and α-tricalcium phosphate (α-TCP) powder, wherein the weight ratio of the biocompatible polymer to the α-TCP powder is 1:7 to 1:12; the curing solution is configured to cure the α-TCP powder, wherein calcium ions (Ca) are added to the curing solution. 2+ ).

2. The organic-inorganic composite hydrogel according to claim 1, wherein the curing solution is selected from at least one of the following: saline, phosphate-buffered saline (PBS), monocalcium phosphate monohydrate (MCPM), disodium phosphate dehydrated (DSP), monosodium phosphate dehydrated (MSP), α-minimum essential medium (α-MEM), and Hanks balanced salt solution (HBSS).

3. The organic-inorganic composite hydrogel according to claim 1, wherein the organic-inorganic composite hydrogel further comprises at least one of functional components and cells.

4. The organic-inorganic composite hydrogel according to claim 1, wherein the organic-inorganic composite hydrogel comprises a photocrosslinked biocompatible polymer; and a calcium phosphate-based ceramic bonded to the biocompatible polymer.

5. The organic-inorganic composite hydrogel according to claim 4, wherein the calcium phosphate-based ceramic comprises Ca-deficient hydroxyapatite (CDHA).

6. A biomaterial comprising the organic-inorganic composite hydrogel according to any one of claims 1 to 5.

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