WO2014116000A1

WO2014116000A1 - Method for producing collagen scaffold coated with apatite

Info

Publication number: WO2014116000A1
Application number: PCT/KR2014/000546
Authority: WO
Inventors: 김병수; 노승서; 강서경
Original assignee: 서울대학교산학협력단
Priority date: 2013-01-22
Filing date: 2014-01-20
Publication date: 2014-07-31
Also published as: KR101449237B1; KR20140094305A

Abstract

The present invention relates to a method for producing a collagen scaffold coated with apatite by processing a surface of the collagen scaffold with a simulated body fluid of pH 8-13. The collagen scaffold coated by apatite, according to the present invention, is effectively produced in a short period of time and is modified so as to have osteanagenesis effects including excellent biocompatibility, cell compatibility, osteoconductive properties, and osteogenic protein sustained-release properties, and is thus useful as a medical graft material.

Description

Method for preparing collagen support coated with apatite

The present invention relates to a method for producing a medical support, more specifically, a collagen support coated with apatite having excellent bone regeneration effect.

Bones are composed of bone tissue, which serves to support and protect the organs of the body. What constitutes bone tissue is bone tissue (osseous tissue), which constitutes the rigid part of the bone organs and forms the skeletal system and thus is classified as an important supportive connective tissue of the body.

Such bone tissue is an organic / inorganic hybrid biocomposite material composed of various organic components such as proteins and cells and inorganic components such as apatite. For example, the important chemical and mechanical properties of natural bones include the formation of hydroxyapatite (Ca ₁₀ (PO ₄ ) ₆ (OH) ₂ ), a calcium phosphate (CaP) crystal, that is spatially arranged between collagen fibrils. It comes from a hierarchical structure.

In general, cracking or splitting of bones may occur due to physical trauma, or may be caused by certain medical conditions that weaken bones such as osteoporosis. Synthetic biomaterials, such as artificial bones, are used to heal fractures or damaged bones, and various methods have been tried to further improve the biocompatibility of such synthetic biomass.

As a specific example, a method of coating a biocompatible calcium phosphate thin film on the surface of a target material by using plasma spraying is known (J. Biomed. Mater. Res. 48, 741 (1999)). However, this method has a disadvantage in that it is difficult to uniformly coat a thin film of calcium phosphate on the inner surface of the material, and that the polymer support may be deformed by being exposed to high temperature during plasma emission.

On the other hand, various documents are known in the literature to immerse a support body in a simulated body fluid (SBF) of pH 7 to coat the surface of the support with apatite crystals, the main component of calcium and phosphorus (see J. Biomed. Mater. Res. 24, 721 (1990); Tissue Eng Part A. 17 (17-18): 2153-64 (2011); J. Biomater. Sci. 23 1659-1671 (2012)). Japanese Patent Application Laid-Open No. 2004-275533 also discloses a method of coating an apatite on the surface of the support by immersing the support in a simulated solution of pH 6 or 7. However, according to the above methods, there is a problem in that the process of surface coating the apatite with a sufficient content takes a considerable time, thereby lowering economic efficiency.

Therefore, as the necessity of various medical implants increases, there is a need for a technology for more efficiently manufacturing a support having a superior bone regeneration effect.

The problem to be solved by the present invention is to provide a method for more efficiently producing a medical support having excellent bone regeneration efficacy.

In order to achieve the above object, the present invention,

Preparing a mimetic fluid comprising at least one salt of hydrochloride, carbonate, phosphate and sulfate;

Adjusting the pH of the mimetic solution to 8 to 13; And

It provides a method for producing an apatite-coated collagen support comprising a; immersing a collagen support in the mimetic fluid to coat apatite on the surface of the support.

According to one embodiment, the salt may be used at least one of NaCl, NaHCO ₃ , KCl, K ₂ HPO ₄ , MgCl ₂ , CaCl ₂ , and Na ₂ SO ₄ .

According to one embodiment, the mimetic fluid may have a concentration of 1 to 20 times based on the concentration of the body fluid.

According to one embodiment, the pH of the mimetic liquid may have a range of 8 to 11.

According to one embodiment, the apatite may include hydroxyapitite.

According to one embodiment, a collagen sponge may be used as the collagen support.

According to one embodiment, the mimetic liquid in the coating step may have a temperature range of about 15 to about 35 ℃.

According to one embodiment, the mimetic liquid in the coating step may have a temperature range of about 15 to about 25 ℃.

The present invention provides a method for efficiently coating the collagen support with an apatite, an inorganic component of bone at an optimal pH. According to this method, it becomes possible to coat more content of apatite on the collagen support more simply and efficiently within a shorter time. In particular, when using the mimetic liquid having a predetermined temperature range within the optimum pH range, the coating content of the apatite can be maximized.

The support for bone regeneration obtained by the above method exhibits excellent biocompatibility and bone conductivity, and can release bone morphogenetic protein, a growth factor protein that promotes bone regeneration, in a sustained release manner, thereby treating bone-related diseases. It can be effectively used as a carrier for a medical implant or bone forming protein for bone regeneration.

1 is a schematic diagram schematically showing a coating process according to the pH change of the mimetic fluid.

Figure 2 is a graph showing the result of quantitatively comparing the amount of apatite coated on the surface of the collagen support according to the pH and temperature change according to the Examples and Comparative Examples, respectively.

Figure 3 shows a SEM photograph of the apatite coated on the collagen support surface as the pH is changed in the mimetic fluid of 20.

Figure 4 is a SEM photograph of the apatite coated on the surface of the collagen support as the temperature is changed in the simulated solution of pH = 10.

Hereinafter, the present invention will be described in detail. The terms or words used in this specification and claims are not to be construed as limiting in their usual or dictionary meanings, and the inventors may appropriately define the concept of terms in order to best explain their invention in the best way possible. It should be interpreted as meaning and concept corresponding to the technical idea of the present invention based on the principle that the present invention.

According to one embodiment of the present invention, a method of preparing an apatite-coated collagen support includes preparing a mimetic fluid including at least one salt of hydrochloride, carbonate, phosphate and sulfate; Adjusting the pH of the mimetic solution to 8 to 13; And coating an apatite on the surface of the support by immersing the collagen support in the mimetic fluid.

The manufacturing method provides a method for efficiently coating the collagen support with the apatite, which is an inorganic component of bone, using a mimetic fluid having optimal conditions, for example, a predetermined pH range and / or a predetermined temperature range.

As described above, when the pH of the mimetic fluid is changed, the charge distribution is changed on the target collagen support surface, and the change of the charge distribution changes the concentration of the positively charged metal ions on the support surface. This can affect the combination of. A schematic diagram schematically showing the change in the distribution of metal ions on the collagen support surface as the pH of the mimetic fluid changes is shown in FIG. 1.

The method for preparing collagen support of the present invention essentially includes adjusting the pH of the simulated solution in which various salts are dissolved to 8 or more, which is the isoelectric point of the collagen support, and the pH of collagen isoelectric point (PI) as shown in FIG. 1. When the pH of the mimetic fluid is lower than the isoelectric point, for example, pH 6 or less, the surface of the collagen becomes positively charged, which makes it difficult to adsorb calcium ions, the main component of apatite, to the surface of the collagen. Thus, the coating content of apatite is reduced.

However, when the pH of the mimetic fluid is higher than the isoelectric point, pH = 8, since the surface charge of the collagen has a lot of negative charges, metal ions such as calcium ions having positive charges bind relatively well to collagen due to electrostatic attraction. Therefore, as the pH of the mimetic fluid is higher than 8, the efficiency of apatite coating on the collagen surface can be maximized.

According to one embodiment, a collagen sponge may be used as the collagen support. As such a collagen sponge, any structure having a cavity or void in collagen can be used without particular limitation. For example, a collagen solution or a dispersion prepared by lyophilization can be used. There is no particular limitation on the lyophilization method.

When the apatite coating is applied to the surface of the collagen support, it is preferable that the collagen is not dissolved in the mimetic fluid, so that it is necessary to crosslink or insolubilize if necessary. For example, physical and / or chemical crosslinking may be used as an example of the crosslinking, UV treatment or the like may be used as such a physical crosslinking process, and aldehydes such as glutaraldehyde, formalin, and dialdehyde starch may be used as the chemical crosslinking process. Crosslinking process using a compound, water-soluble polyepoxy compounds, such as polyethyleneglycol diglycidyl ether, and isocyanate compounds, such as hexamethylene diisocyanate, etc. can be used. In such a physical or chemical crosslinking process, the crosslinking agent and the crosslinked product may be one which is not cytotoxic and excellent in biocompatibility.

According to one embodiment of the present invention, the surface of the collagen support is coated with apatite, the main component of calcium and phosphorus dissolved in the mimetic fluid. The surface may include the surface of the cavity and / or voids present in the support, in addition to the surface of the collagen support, eg, all and / or a portion of the collagen sponge. At this time, the apatite may be coated on the entire surface, or may be partially coated on the surface. Although a coating means is not specifically limited, The method of immersing a collagen support body in a mimic liquid can be illustrated.

According to one embodiment, the mimetic liquid may be a solution containing one or more salts in water, for example distilled water or deionized water, and one or more of hydrochloride, carbonate, phosphate and sulfate may be used as such salts. And, these salts may include alkali metals and / or alkaline earth metals as metal ions. Examples of the alkali metal include sodium, potassium, and the like, and magnesium, calcium, and the like may be used as the alkaline earth metal.

According to one embodiment, the hydrochloride may exemplify a compound in which an alkali metal or alkaline earth metal is combined with chlorine, and for example, NaCl, KCl, MgCl ₂ , CaCl ₂ , and the like may be used.

According to one embodiment, the carbonate is a secondary carbonate in which all of the hydrogen of carbonic acid (H ₂ CO ₃ ) is substituted with an alkali metal or alkaline earth metal, or a primary carbonate (bicarbonate) in which one hydrogen of carbonate is substituted with an alkali metal Can be used.

According to one embodiment, the phosphate includes a primary phosphate (dihydrogen phosphate) in which one hydrogen of phosphoric acid (H ₃ PO ₄ ) is substituted, and a secondary phosphate (hydrogen phosphate) in which two hydrogens of phosphoric acid are substituted. For example, a tertiary phosphate in which hydrogen of phosphoric acid is three substituted may be exemplified, and examples of the metal which may be substituted may include an alkali metal or an alkaline earth metal. Examples of these phosphates and the like can be given _{_{_{K 2 HPO 4, Na 2 HPO}}} 4, CaHPO 4, MgHPO 4, NaH 2 PO 4, KH 2 PO 4.

According to one embodiment, the sulfate is a primary sulfate (bisulfate) in which hydrogen of sulfuric acid (H ₂ SO ₄ ) is substituted with an alkali metal, secondary hydrogen in which all of the hydrogen of the sulfuric acid is substituted with an alkali metal or alkaline earth metal sulfate and the like can be exemplified, for example, may be used, such as _{_{_{Na 2 SO 4, K 2 SO}}} 4, NaHSO 4, KHSO 4, MgSO 4, CaSO 4.

According to one embodiment, the mimetic fluid may include at least one of NaCl, NaHCO ₃ , KCl, K ₂ HPO ₄ , MgCl ₂ , CaCl ₂ and Na ₂ SO ₄ as a salt component, for example NaCl, One or more of NaHCO ₃ , K ₂ HPO ₄ , and CaCl ₂ .

According to one embodiment, these salt components can be dissolved in water, for example distilled water or deionized water, and used to form a simulated body fluid, wherein the salt concentration of the simulated body fluid is of a normal healthy body, for example, a healthy human body or an animal body. It may have a range of 1 to 20 times, for example 1 to 15 times, or 1 to 10 times based on the body fluid concentration. As the concentration of the mimetic fluid is higher, the coating content of the coated apatite increases, and the coating time may also decrease.

According to one embodiment, the collagen support is immersed in the mimetic extract as described above to coat the apatite component on the surface of the support. In this coating step, the immersion process is performed for 1 hour or more, for example 5 hours. It can be carried out for more than one day, or more than one day to less than 10 days, the longer the immersion time is able to coat a large amount of apatite, but if the immersion time is too long there is a risk of collagen degeneration is caused. .

In the present invention, by adjusting the pH range of the mimetic liquid to an optimum range, for example, pH = 8 to 13, a sufficient amount of apatite coating can be achieved in a shorter time at the same concentration of the mimetic liquid. Such a short time coating has an advantage of minimizing collagen denaturation.

According to one embodiment, the apatite coating content may vary depending on the temperature of the mimetic solution in which the immersion process is performed, and the temperature of the mimetic solution in which the collagen support is immersed is, for example, 15 to 35 ° C, or 15 to 15. A range of 25 ° C. can be used. The coating time can be shortened as the temperature of the mimetic fluid increases, but it is preferable to control the collagen in a predetermined range since there is a risk of degeneration of collagen.

Collagen support coated with apatite, for example, hydroxyapatite according to the present invention is efficiently prepared in a short time in a predetermined pH range, modified to have a bone regeneration effect such as excellent biocompatibility, cell compatibility and osteoconductivity (osteoconductivity) It can be usefully used as a medical implant.

Hereinafter, the present invention will be described in detail with reference to Examples. However, embodiments according to the present invention can be modified in many different forms, the scope of the present invention should not be construed as limited to the embodiments described below. The embodiments of the present invention are provided to more completely explain the present invention to those skilled in the art.

Examples and Comparative Examples

A collagen sponge composed of bovine-derived collagen type 1 was prepared as a support.

NaCl (8.0035 g / L), NaHCO ₃ (0.355 g / L), KCl (0.225 g / L), K ₂ HPO ₄₂ O (0.231 g / L), CaCl ₂ (0.292 g / L), Na ₂ SO ₄ (0.072 g / L), and MgCl ₂₂ O (0.311 g / L) were dissolved in distilled water to prepare a mimic liquid.

NaOH or HCl was added to the mimetic liquid The pH range was set to

pH

4, 5, 6, 7, 8, 9, 10, 11. pH 4, 5, 6, 7 is a comparative example, and

pH

8, 9, 10, 11 is an Example.

The collagen support was immersed in each of the mimetic fluids at different temperature conditions of 4 ° C., 20 ° C. and 40 ° C. for 24 hours to obtain a collagen support coated with hydroxyapatite. The collagen support without any treatment was prepared for comparison.

Experimental Example

The hydroxyapatite coating content of the collagen support treated by changing the pH of the mimetic fluid from 4 to 11 at 4 ° C., 20 ° C. and 40 ° C. temperature is shown in FIG. 2.

The coating content is a value indicated by calculating the amount of calcium in the mass increase after measuring the change in the content of the collagen support before and after coating.

As can be seen in Figure 2, it can be seen that the coating content of the hydroxyapatite coated on the surface of the support at 20 ℃ and pH 8 or more the most. That is, the highest coating content was found in the simulated body fluid at 20 ° C., and the coating content was increased by about 20% compared to the case where 4 ° C. and 40 ° C. body fluid were used. In addition, as the pH of the mimetic fluid increases, the surface charge of the collagen support becomes stronger and negatively charged. This tendency can be clearly seen at the simulated body fluid temperature of 20 ° C., and the trend is similar at 4 ° C., but it is clear that this trend is somewhat unclear at 40 ° C.

FIG. 3 is an SEM image showing the degree of coating of hydroxyapatite coated on the collagen surface at 20 ° C. simulated liquid. When using the mimic body solution of 20 ℃ from the SEM picture it can be seen that the content of hydroxyapatite coated on the surface of the collagen support significantly increased. FIG. 4 shows the coating degree when using 4 ° C., 20 ° C. and 40 ° C. simulated liquid at the same pH value (pH = 10), and shows the highest coating content when using 20 ° C. simulated liquid. Able to know.

Claims

Preparing a mimetic fluid comprising at least one salt of hydrochloride, carbonate, phosphate and sulfate;

Adjusting the pH of the mimetic solution to 8 to 13; And

Coating an apatite on the surface of the support by immersing a collagen support in the mimetic fluid.
The method of claim 1,

Method for producing an apatite-coated collagen support, characterized in that the salt is at least one of NaCl, NaHCO 3 , KCl, K 2 HPO 4 , MgCl 2 , CaCl 2 , and Na 2 SO 4 .
The method of claim 1,

The method of producing apatite-coated collagen support, characterized in that the mimetic fluid has a concentration of 1 to 20 times the concentration of the body fluid.
The method of claim 1,

A method of producing an apatite-coated collagen support, characterized in that the pH of the mimetic solution is 8 to 11.
The method of claim 1,

Method for producing an apatite-coated collagen support, characterized in that the apatite is hydroxyapatite.
The method of claim 1,

Method for producing an apatite-coated collagen support, characterized in that the collagen support is a collagen sponge.
The method of claim 1,

The method of producing an apatite coated collagen support, characterized in that the temperature of the mimetic liquid in the coating step is about 15 to about 35 ℃.
The method of claim 1,

The apatite-coated collagen support, characterized in that the temperature of the mimetic liquid in the coating step is about 15 to about 25 ℃.