KR102105549B1 - A manufacturing method for calcium phosphate hydrogel composition containing a constant content of calcium phosphate - Google Patents

Abstract

The present invention relates to a method for preparing a hydrogel composition containing a specific amount of calcium phosphate. Since the hydrogel composition prepared by the method has a fixed content of calcium phosphate, it is possible to quantify phosphate contained in the hydrogel composition.

Description

A manufacturing method for calcium phosphate hydrogel composition containing a constant content of calcium phosphate}

It relates to a method for producing a hydrogel composition containing a certain amount of calcium phosphate.

Osteoporosis is a skeletal disease in which fractures increase due to weakening of bone strength, and is a senile disease, and has emerged as an important health problem worldwide due to the aging of the population. Korea is also one of the countries that is aging rapidly, and osteoporosis patients are also expected to increase rapidly. According to the 4th National Health and Nutrition Survey of Korea, about 35% of women over 50 are osteoporosis patients, and the number is expected to increase in the future. Therefore, with the increase in osteoporosis patients, many osteoporosis treatment drugs have already been developed and are currently being developed.

 On the other hand, the animal model refers to the use of animals in the study of diseases or injuries similar to humans. Researchers can apply treatment methods and procedures that are difficult to implement to humans through animal models, and show the therapeutic effects. Can be confirmed. In the field of medicine, as molecular biological, genetic, and biotechnological factors play an important part in the study of specific diseases, the role of disease model animals became important and clinical trials involving subjects were conducted to conduct research on specific diseases. Preclinical testing has become an essential step before doing so. However, as ethical issues emerge with respect to animal models, it is necessary to develop experimental models that can replace animal models. In addition, currently, it takes at least six months to produce an animal model of osteoporosis, and expensive equipment such as CT is used to confirm the effect of an osteoporosis treatment using an animal model. In this case, it takes a lot of time and expertise to use and analyze equipment. Therefore, it is necessary to develop an evaluation method for confirming osteoporosis and efficacy of a therapeutic agent in a simpler and faster time.

One aspect is a step of preparing a first composition by dissolving chitosan in a gelatin solution; Dispersing calcium phosphate in a calcium chloride solution to prepare a second composition; And it is to provide a method for preparing a calcium phosphate hydrogel composition by mixing the first composition and the second composition.

Another aspect is to provide a hydrogel composition prepared by the above method.

Another aspect is dissolving the hydrogel composition in an acidic solution; And measuring the content of phosphate freed from the composition.

One aspect is a step of preparing a first composition by dissolving chitosan in a gelatin solution; Preparing a second composition by dissolving calcium phosphate in a calcium chloride solution; And a method for preparing a calcium phosphate hydrogel composition by mixing the first composition and the second composition.

The term "hydrogel (hydrogel)" in the present specification may mean a three-dimensional network structure made by crosslinking a hydrophilic polymer by a covalent or non-covalent bond. Due to the hydrophilicity of the constituent materials, it absorbs and swells a large amount of water in an aqueous solution and under an aqueous environment, but has a property of not dissolving by a crosslinked structure. Therefore, depending on the composition and manufacturing method, hydrogels having various shapes and properties can be made, and generally contain a large amount of moisture, so that they may have liquid and solid intermediate properties.

The term "chitosan" in the present specification may mean a linear polysaccharide composed of D-glucosamine and N-acetylglucosamine. The chitosan may be represented by the following Structural Formula 1 and may be obtained by treating a shellfish, shellfish and crustacean shells with a sodium hydroxide base, but is not limited thereto. The chitosan may include chitosan derivatives in addition to pure chitosan. For example, the chitosan derivative may include at least one of phthalated chitosan, esterified chitosan, amidated chitosan, or formylated chitosan:

[Structural Formula 1]

In the present specification, the term "calcium phosphate (Calcium Phosphate)" may refer to various phosphates of calcium and may be represented by the following structural formula 2. The calcium phosphate may be selected from the group consisting of amorphous calcium phosphate, tricalcium phosphate (TCP), and calcium phosphate (Tetracalcium phosphate, TTCP):

[Structural Formula 2]

The method according to one embodiment comprises dissolving chitosan in a gelatin solution to prepare a first composition. The chitosan is 0.1 to 10% by weight, 0.1 to 8% by weight, 0.1 to 5% by weight, 0.1 to 0.25% by weight, 0.25 to 0.3% by weight, 0.15 to 0.2% by weight, 1 to 8% based on 100% by weight of the first composition It may be included by weight%, or 1 to 5% by weight. At this time, when the content of the chitosan is less than the above range, the viscosity of the first composition is low, so it is difficult to prepare a hydrogel composition having a constant shape, and there is a problem that calcium phosphate can easily leak from the formed hydrogel composition. If it exceeds, the viscosity of the first composition is high, and it is difficult to manufacture the hydrogel in an even form because it is difficult to uniformly distribute calcium phosphate and bubbles are formed.

The method according to one embodiment comprises dispersing calcium phosphate in a calcium chloride solution to prepare a second composition. The step may further include sonicating the calcium chloride solution in which calcium phosphate is dissolved. The calcium phosphate is 0.1 to 10% by weight, 0.1 to 8% by weight, 0.1 to 5% by weight, 0.5 to 6% by weight, 0.5 to 3.5% by weight, 4.5 to 7.5% by weight, or 1.5 relative to 100% by weight of the second composition It may be included at 2.5% by weight. At this time, when the content of calcium phosphate is less than the above range, there is a problem that it is difficult to apply calcium phosphate hydrogel to an osteoporosis model, etc., and if it exceeds the above range, it may cause toxicity when applied to cells and animals, as well as other biological stimuli There is a problem that can cause.

The method according to one embodiment includes the step of preparing a hydrogel bead composition by dropping the hydrogel composition into a basic solution. The basic solution may be pH 8 to 11. For example, it may be pH 8 to 11, pH 8 to 10, pH 8 to 9, pH 9 to 11, pH 9 to 10, or pH 10 to 11. At this time, when the pH of the solution is less than the above range, there is a problem in that the instantaneous crosslinking reaction does not occur to maintain the shape of the beads and is dissolved in a solvent, that is, a basic solution, and when it exceeds the above range, crosslinking occurs too quickly. Not only is it difficult to implement the form of a hydrogel with reproducibility, there is a problem that when cultured with cells, it can cause cytotoxicity due to pH. In addition, the basic solution may be, for example, sodium hydroxide solution, calcium hydroxide solution or ammonia solution.

Another aspect provides a calcium phosphate hydrogel composition prepared by the above method. The hydrogel composition may be a hydrogel bead composition. The calcium phosphate may be included in 0.1 to 10% by weight based on 100% by weight of the hydrogel composition. For example, the phosphate comprises 0.1 to 10% by weight, 0.1 to 8% by weight, 0.1 to 5% by weight, 0.5 to 8% by weight, 0.5 to 5% by weight, 2.5 to 5% by weight, or 1 to 5% by weight It can be. At this time, when the content of phosphate is less than the above range, there is a problem in that it is difficult to confirm the osteoclast effect of the cells by reducing the contact area between osteoclast and calcium phosphate, and when it exceeds the above range, the amount of calcium phosphate that can be included in the hydrogel As this is supersaturated, there is a problem that calcium phosphate leaks from the formed hydrogel composition. That is, since the composition has a constant content of calcium phosphate, the amount of decrease in calcium phosphate can be quantitatively confirmed by measuring the content of phosphate contained in the hydrogel composition.

In addition, the hydrogel composition may be a composition for bio ink. The chitosan contained in the hydrogel composition has relatively good solubility in an acidic environment of pH 6 or lower, but when the content of chitosan is high, the hydrated chitosan polymer body has a high viscosity, and thus can be used as an ink for 3D printing.

Another aspect is dissolving the hydrogel composition in an acidic solution; And measuring the content of phosphate freed from the hydrogel composition. The acidic solution may be a hydrogen chloride solution, a nitric acid solution, a sulfuric acid solution, or an acetic acid solution.

In one embodiment, prior to dissolving the hydrogel composition in an acidic solution, it may include the step of bioprinting the hydrogel composition. After dissolving the structure prepared by bioprinting in an acidic solution, calcium phosphate can be quantified by measuring the content of phosphate liberated from the hydrogel composition.

In another embodiment, the hydrogel composition may be a hydrogel bead composition. That is, since the hydrogel bead composition contains a certain amount of calcium phosphate, calcium phosphate can be quantified by measuring the content of phosphate liberated from the hydrogel bead composition.

The method for preparing a hydrogel composition according to one aspect is that the content of the phosphate is constant by crosslinking the hydrogel by adjusting the pH instantaneously, and the calcium phosphate or calcium phosphate contained in the hydrogel composition is measured by measuring the calcium salt or phosphate free from calcium phosphate. Can be quantitatively confirmed. In addition, unlike a composition containing a conventional calcium phosphate compound, which requires a high-temperature sintering and lyophilization process to improve physical properties, it is possible to prepare a bio-friendly composition by a normal temperature process, and thus, human bone tissue and chemically and physically It can be used as a fabrication system for osteoporosis tissues using a similar hydrogel composition and an evaluation system for osteoporosis treatments using the same.

1 is a diagram of a hydrogel composition according to an aspect and a method of manufacturing beads comprising the composition.
Figure 2a is a photograph confirming the calcium contained in the hydrogel after dyeing the hydrogel according to one aspect with alizarin Red S solution.
Figure 2b is a graph measuring the amount of calcium contained in the hydrogel according to one aspect.
2C is a graph for quantifying the amount of calcium contained in a hydrogel according to an aspect.
3A is a photograph of a hydrogel according to an aspect made of beads having a size of 3 ± 0.5 mm.
Figure 3b is a graph measuring the content of phosphate contained in a hydrogel bead according to one aspect.
Figure 4 is a standard curve graph measured using a phosphate standard solution for the content of phosphate contained in a hydrogel bead according to one aspect.
5 is a graph measuring the content of phosphate contained in a hydrogel bead according to one aspect over time.

Hereinafter, preferred embodiments are provided to help understanding of the present invention. However, the following examples are only provided to more easily understand the present invention, and the contents of the present invention are not limited by the following examples.

[Example]

Example 1. Preparation of a hydrogel composition comprising chitosan and calcium phosphate

0.15 g of gelatin (sigma, G2500) was added to 8.5 ml of 0.1M HCl, and the mixture was stirred at 120 ° C. at 120 rpm for 10 to 15 minutes. Thereafter, 0.15 g of chitosan (sigma, 448869) was added, followed by bathing and stirring at 120 ° C for 300 to 300 rpm for 3 to 4 hours to prepare a first mixture. Chitosan exhibits high solubility in acidic environments below pH 6, while it has a gelling property in environments above pH 6. Using these properties, a mixture was prepared using HCL 0.1M, a strongly acidic solvent having a pH of 1, as a solvent of the first mixture.

Next, 1.5 ml of 0.1M HCl was added to a 5 ml tube, and 0.147 g of CaCl ₂ (wako, 031-00435) was added, followed by vortexing sufficiently. Thereafter, 0.2 g of tricalcium phosphate (TC) (samchun, C0196) was added to the solution, followed by sonication for 10 minutes to prepare a second mixture. The second mixture was slowly added dropwise to the first mixture, and stirred at 120 ° C. for 1 hour to prepare a hydrogel composition. Subsequently, the hydrogel beads were prepared by dropping 10 µl of the composition in a 1N NaOH solution as a strong alkaline aqueous solution at room temperature.

1 is a diagram of a hydrogel composition according to an aspect and a method of manufacturing beads comprising the composition. In the case of a typical calcium phosphate compound, physical properties are improved through a high temperature sintering and freeze-drying process. However, in the case of the hydro composition, chitosan is prepared by crosslinking the calcium phosphate hydrogel by instantaneously adjusting the pH using the property that the physical properties vary depending on the pH. That is, when a hydrogel composition in which chitosan and calcium phosphate dissolved in a strong acidic solution is uniformly dispersed is injected into a strong alkali solution, the chitosan instantly gels while surrounding the surrounding calcium phosphate. Therefore, there is an advantage in that the content of calcium phosphate contained in the beads can be constantly controlled.

[Experimental Example]

Experimental Example 1. Detection of calcium contained in hydrogel composition

Calcium phosphate-based materials have been studied a lot due to their biocompatibility and chemical and physical similarities with human bone tissue. Among them, TCP is composed of calcium and phosphoric acid, which are chemical components similar to actual bone, and releases calcium and phosphoric acid through a biodegradation process. Therefore, it was intended to quantify by indirectly measuring calcium phosphate combined with the calcium salt contained in the hydrogel. In order to detect calcium contained in the hydrogel, an alizarin red S (ARS) assay was performed by a conventional method. First, the hydrogel composition prepared in Example 1 was 3D printed in two layers using a 26G needle, and then treated with 1M NaOH for 5 minutes and washed with D.W to prepare a hydrogel scaffold. Thereafter, the scaffold was incubated with 1% w / v alizarin red S solution (pH 4.1-4.3) at room temperature. The solution was removed, the scaffold was washed with distilled water and dried. Thereafter, the stained scaffold was visualized using a digital camera. Thereafter, the scaffold stained with alizarin red S was incubated for 1 hour in 10% w / v cetylpyridinium chloride (CPC) in 10 mM sodium phosphate solution (pH 7) to elute alizarin red S. Thereafter, the absorbance of the elution solution was measured at 562 nm using a microplate reader.

Figure 2a is a photograph confirming the calcium contained in the hydrogel after dyeing the hydrogel according to one aspect with alizarin Red S solution. As shown in Figure 2a, when the amount of the TCP contained in the hydrogel composition was changed to a range of 0 to 5%, it was confirmed that the degree of staining of the hydrogel was visually classified in proportion to the amount of TCP. .

2B is a graph measuring absorbance after eluting alizarin red S in which the amount of calcium contained in a hydrogel according to an aspect is combined with a calcium salt. As shown in Figure 2b, the amount of TCP contained in the hydrogel composition is changed to a range of 0 to 5%, and when the dyeing time is treated with 10 seconds, the difference in absorbance value is directly proportional to 3% of the TCP content. I could confirm that.

2C is a graph for quantifying the amount of alizarin red S eluted from a hydrogel according to one aspect. As shown in Fig. 2c, up to 3% of the TCP content included in the hydrogel composition, the R-squared value was close to 1 (0.99), and it was confirmed that the difference in absorbance value was directly proportional. However, when the dyeing time was 10 seconds or more, a difference in absorbance values could not be confirmed due to overstaining. That is, the hydrogel composition according to one aspect may indirectly quantify the calcium salt through alizarin red S staining, which was used in a conventional calcium qualitative analysis method.

Experimental Example 2. Detection of phosphate contained in hydrogel composition

Since TCP is composed of calcium and phosphoric acid, which are chemical components similar to actual bone, it is possible to measure the TCP content by measuring free calcium salt or phosphate. Therefore, it was intended to measure the TCP content by measuring the free phosphate from TCP using the Malachite Green Phosphate Assay method based on the quantitation of the green complex formed between malachite green, molybdate and free phosphate. First, the hydrogel composition prepared in Example 1 was pretreated by washing three times for 10 minutes at 37 ° C. D.W. Thereafter, the pretreated hydrogel beads were transferred to 1.5 ml tubes one by one, 1 ml of 1N HCl was added, and then dissolved at 60 to 70 ° C. for 1 hour. Thereafter, the solution in which the hydrogel beads were dissolved was diluted 1/100, and 50 µl was loaded into 96 wells with a standard solution (1-50 mM). Thereafter, 10 µl of Malachite Green Reagent A was added to each well and reacted for 10 minutes at room temperature, and 10 µl of Malachite Green Reagent B was further added to each well and reacted for 20 minutes at room temperature. Thereafter, absorbance was measured at 620 nm.

Figure 3a is a photograph of a hydrogel composition according to an aspect made of beads of 3 ± 0.5㎜ size.

3B is a graph measuring the content of TCP contained in a hydrogel bead according to one aspect.

As shown in Figure 3a, it can be seen that when the hydrogel is made of beads, the size is almost the same as about 3 mm. In addition, it can be confirmed that the TCP content in the hydrogel beads is constant at 100 ± 15 μg / piece (FIG. 3B). That is, it can be seen that the manufacturing method according to one aspect is capable of producing hydrogel beads of the same size through instantaneous pH adjustment, and the phosphate contained in the hydrogel beads is uniform.

Experimental Example 3. Confirmation of quantitative analysis method through detection of phosphate contained in hydrogel composition

Through the detection of the phosphate contained in the hydrogel composition was confirmed a method for quantitative analysis of the content of TCP. Specifically, hydrogel beads were prepared by adding the contents of TCP in the hydrogel composition prepared in Example 1 to be 0, 0.3, 0.6, and 1%. Then, the amount of phosphate contained in the hydrogel beads was quantified in the same manner as in Experimental Example 2.

Figure 4 is a standard curve graph measured using a phosphate standard solution for the content of phosphate contained in a hydrogel bead according to one aspect. Specifically, 100 μM phosphate solution was continuously diluted to 50, 25, 12.5 and 6.25 μM, and absorbance of the solution was measured. Subsequently, using the measured series of values, the main power line was created and the R squared value was calculated to confirm the proportionality. As a result, the R-squared value is 0.9999, and by substituting the OD value of the sample in the standard curve graph, it is possible to accurately check the amount of PO ₄ dissolved.

5 is a graph measuring the content of phosphate contained in a hydrogel bead according to one aspect over time.

As shown in FIG. 5, as the content of the TCP contained in the hydrogel beads was changed, the content of the phosphate also showed a direct change (R ² > 0.99 or more). That is, the hydrogel composition according to one aspect can measure the content of TCP through the quantitation of calcium salt and phosphate, and thus can be used as an evaluation system for evaluating osteoporotic tissue and confirming the efficacy of a therapeutic agent for osteoporosis.

Claims (12)

Preparing a first composition by dissolving chitosan in an acidic solution containing gelatin;
Adding calcium phosphate to an acidic solution containing calcium chloride, followed by sound treatment to prepare a second composition;
Preparing a mixed solution by mixing the first composition and the second composition; And
A method of preparing a hydrogel composition, comprising dropping the mixed solution into a basic solution. The method of claim 1, wherein the hydrogel composition is a hydrogel bead composition. The method according to claim 1, wherein the chitosan is a method for producing a hydrogel that is contained in 0.1 to 10% by weight of the first composition. The method of claim 1, wherein the calcium phosphate is contained in 0.1 to 10% by weight of the second composition. delete The method of claim 1, wherein the pH of the basic solution is 8 to 11. The method of claim 6, wherein the basic solution is selected from the group consisting of sodium hydroxide solution, calcium hydroxide solution, and ammonia solution. Calcium phosphate hydrogel composition prepared by the method of claim 1. The hydrogel composition according to claim 8, which comprises 0.1 to 10% by weight of calcium phosphate in the hydrogel composition. Dissolving the hydrogel composition of claim 8 in an acidic solution; And
Method for quantifying calcium phosphate comprising the step of measuring the content of phosphate liberated from the hydrogel composition. The method of claim 10, wherein the acidic solution is a method for quantifying calcium phosphate selected from the group consisting of a hydrogen chloride solution, a nitric acid solution, a sulfuric acid solution, and an acetic acid solution. The method of claim 10, wherein the hydrogel composition is a bead composition.

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