WO2009048270A1

WO2009048270A1 - Drug-incorporated calcium phosphate hybrid thin film

Info

Publication number: WO2009048270A1
Application number: PCT/KR2008/005925
Authority: WO
Inventors: Woo-Kul Lee; Jae Young Jung
Original assignee: Industry-Academy Cooperation Foundation, Dankook University
Priority date: 2007-10-09
Filing date: 2008-10-09
Publication date: 2009-04-16
Also published as: KR20090036201A; KR100977214B1

Abstract

The present invention provides a method for preparing a drug-incorporated calcium phosphate hybrid thin film comprising the steps of: (a) preparing a drug solution; (b) preparing a calcium ion solution and a phosphate solution; (c) mixing the drug solution, the calcium ion solution and the phosphate solution; and (d) fabricating a drug-incorporated calcium phosphate hybrid thin film by applying the mixture solution of the step (c) to a solid substrate. The present invention develops a drug-incorporated porous coating layer with a three-dimensional structure with low crystallinity on the solid surface. As results, the present invention will be beneficially applied to modify the surface of artificial substitute materials or medical implants for incorporating into human body with excellent biocompatibility, cell compatibility and osteoconductivity.

Description

DRUG-INCORPORATED CALCIUM PHOSPHATE HYBRID THIN FILM

BACKGROUND OF THE INVENTION FIELD OF THE INVENTION The present invention relates to a drug-incorporated calcium phosphate hybrid thin film.

BACKGROUND OF TECHNIQUE

Currently, researches for various artificial synthetic materials to substitute for damaged hard tissue have been intensively studied and particularly calcium phosphate-based materials such as hydroxyapatite similar to chemical structure of bone in which calcium and phosphate are present as main components, were applied (J.C. Elliott, Structure and chemistry of the apatites and other calcium orthophosphates, Elsvier, New York, 1994; H. Aoki, Medical Applications of hydroxyapatite, Ishiyaku EuroAmerica, Inc., Tokyo, 1994).

Calcium phosphate {e.g., hydroxyapatite) in which calcium and phosphate are present as main components is not appropriate to be used as alternatives in sites being subject to weight due to its low mechanical strength. To overcome the defects, the formation of coating layer on the surface of solid support such as a metal or a ceramic has been proposed to provide hard tissue alternatives which posses improved biological function as well as mechanical properties. Therefore, various methods to form a calcium phosphate-coating layer on the surface of solid substrates have been studied (Ha et al, J. Mater. Sd.-Mater. M., 8:881(1997); Weng and Baptista, J. Mater. Sd.-Mater. M., 9:159(1998); Xu and Lu, J. Mater. Sd.-Mater. M., 10: 243(1999); and Pham et al, J. Mater. Sd.-Mater. M., 11:383(2000)).

A multitude of methods has been reported to form a calcium phosphate- coating layer. However, most of them are carried out under conditions significantly different from those for forming hard tissues in vivo. These methods are performed under conditions significantly different from biological environments, for example high temperature and vacuum, resulting in generation of problems in which physiochemical properties of the resulting coating layer are much more different from biological hard tissues. In addition, the methods are likely to form a coating layer only on the outer surface of materials; they are unlikely to form a coating layer on the inner surface of pores in porous materials. To make matters worse, where theses preparation process are used, organic compounds as drugs could not be incorporated into a synthetic calcium phosphate hybrid film. The cost for synthesis becomes much higher because high-cost equipments are required to form the coating layer. In these contexts, it could be appreciated that conventional methods have very limited applicability and ineffective economy.

Throughout this application, various publications and patents are referred and citations are provided in parentheses. The disclosures of these publications and patents in their entities are hereby incorporated by references into this application in order to fully describe this invention and the state of the art to which this invention pertains.

DETAILED DESCRIPTION OF THE INVENTION The present inventors have made intensive studies to develop a drug- incorporated calcium phosphate matrix with an excellent biocompatibility, particularly a novel approach in which a body-implanted artificial substitute material has desirable therapeutic efficacies by modifying the surface of artificial substitute materials or implants incorporated into body to strongly stimulate in vivo osteogenesis. As results, we have discovered that the bone-formation is much more effectively accelerated where a drug particularly a bone morphogenic stimulant is incorporated into a calcium phosphate hybrid thin film.

Accordingly, it is an object of this invention to provide a method for preparing a drug-incorporated calcium phosphate hybrid thin film.

It is another object of this invention to provide a calcium phosphate hybrid thin film for stimulating a bone-formation comprising its stimulant.

It is still another object of this invention to provide an artificial substitute material or an implant coated with the calcium phosphate hybrid thin film for stimulating a bone-formation.

Other objects and advantages of the present invention will become apparent from the following detailed description together with the appended claims and drawings.

In one aspect of this invention, there is provided a method for preparing a drug-incorporated calcium phosphate hybrid thin film comprising the steps of:

(a) preparing a drug solution; (b) preparing a calcium ion solution and a phosphate solution;

(c) mixing the drug solution, the calcium ion solution and the phosphate solution; and

(d) preparing the drug-incorporated calcium phosphate hybrid thin film to apply the mixture solution of step (c) to a solid substrate.

In another aspect of this invention, there is provided a calcium phosphate hybrid thin film for stimulating a bone-formation comprising its stimulant to be prepared according to the present method.

In still another aspect of this invention, there is provided an artificial substitute material or an implant to be coated with the calcium phosphate hybrid thin film for stimulating a bone-formation according to the present method. The present inventors have made intensive studies to develop a drug- incorporated calcium phosphate matrix with an excellent biocompatibility, particularly a novel approach in which a body-implanted artificial substitute material has desirable therapeutic efficacies by modifying the surface of artificial substitute materials or implants incorporated into body to strongly stimulate in vivo osteogenesis. As results, we have discovered that the bone-formation is much more effectively accelerated where a drug particularly a bone morphogenic stimulant is incorporated into a calcium phosphate hybrid thin film.

The present invention relates to a drug-incorporated calcium phosphate hybrid thin film.

The present method will be described in more detail as follows: Step (a): Preparation of a Drug Solution

According to this invention, the drug to be incorporated into the calcium phosphate hybrid thin film is not particularly limited and preferably comprises a solubilizable drug.

According to a preferable embodiment, the drug to be incorporated into the calcium phosphate hybrid thin film includes a protein, a peptide, a nucleic acid molecule or a compound. More preferably, the drug used comprises a chondrocyte proliferant, a cytokine, an anticancer agent or a bone morphogenic stimulant. Much more preferably, the drug is the bone morphogenic stimulant including Vitamin D, Vitamin D₃, Vitamin C, lanthanium (III) compounds [lanthanium salts (lanthanium carbonate, lanthanium carbonate hydrate, lanthanium chloride, etc.) lanthanium chelate, lanthanium resin, lanthanium absorbent, etc.], calcitonin, tamoxipen, biphosphonate, statin, sodium fluoride, oxysterol (20S-hydroxycholesterol, 22S-hydroxycholesteroll, 22R-hydroxycholesterol, 25-hydroxycholesterol, pregnanolone, etc.), BMP (bone morphogenic protein: BMP2, BMP7, BMP14, etc.), parathyroid hormone, IGF-I (insulin-like growth factor-1), TGF-β (transforming growth factor-beta), calcium or combinations thereof. Still much more preferably, the bone morphogenic stimulant comprises Vitamin D or Vitamin D₃, and most preferably Vitamin D₃.

The present inventors have discovered that the present calcium phosphate hybrid thin film is particularly suitable to incorporate Vitamin D₃ and notably promotes the bone-formation. As described in Examples, a tertiary structure of the Vitamin D₃-incorporated calcium phosphate hybrid thin film shows no significant difference as compared with that of the calcium phosphate hybrid thin film and the Vitamin D₃-incorporated calcium phosphate hybrid thin film exhibits more excellent effects on cell adhesion stimulation, cell differentiation acceleration and bone nodule formation as compared to any of conventional calcium phosphate hybrid thin films.

The drug preferably has water- or alcohol (preferably, ethanol)-solubility in the senses that the present thin film is implanted into body. Such solubility of the drug contributes to improvement of a biocompatibility.

Step (b): Preparation of a Calcium Ion and a Phosphate Solution The process to prepare ionic solutions is carried out as previously suggested by the present inventors (see Korean Pat. No. 10-0675573).

For the preparation of a calcium ion and a phosphate solution, preferably water, more preferably distilled water and much more preferably distilled deionized water are used as a solvent. The calcium ion solution is prepared by dissolving various calcium sources in water. The calcium source used is preferably

Ca(NO₃)₂ ^• 4H₂O, CaCI₂ or CaHPO₄. The phosphate ion solution is prepared by dissolving diverse phosphate sources in water. The present phosphate source is preferably (NH₄)₂HPO₄ or NH₄H₂PO₄. According to a preferable embodiment, water is used as a solvent to prepare the calcium ion solution and the phosphate ion solution.

The utilization of water as solvents makes the preparation and handling process much easier. In addition, the presence of ionic components in buffer solutions reacted with calcium or phosphate ions may be greatly prevented in the calcium phosphate thin film by the use of water.

Step (c): Preparation of a Mixture Solution of a Drug, a Calcium Ion and a Phosphate Solution

The mixture solution of the drug (e.g., a bone morphogenic stimulant), the calcium ion and the phosphate solution is prepared. The mixture solution is prepared by simple mixing of three solutions. Preferably, the mixture solution is an aqueous solution. It is preferable to adequately adjust the concentrations of the drug, the calcium ion and the phosphate ion in preparation of the mixture solution.

Because calcium and phosphate ions are easily precipitated in solution due to their higher reactivity, it is preferable that concentrations of calcium and phosphate ions are properly adjusted for the prevention of homogeneous precipitation reactions to form a precipitate and for the promotion of heterogeneous precipitation reactions to allow ions forming a coating layer on the surface of solid materials.

According to a preferable embodiment, the concentration of calcium ions in the mixture solution is in a range of 0.1-10 mM, more preferably 1-10 mM, much more preferably 1-5 mM and most preferably 2-4 mM. According to a preferable embodiment, the concentration of phosphate ions in the mixture solution is in a range of 0.1-20 mM, more preferably 2-15 mM, much more preferably 2-10 mM and most preferably 3-6 mM.

According to a preferable embodiment, the concentration of the drug, particularly the bone morphogenic stimulant in the mixture solution is in a range of 1 nM-10 mM, more preferably 100 nM-500 μM and most preferably 100 nM-50 μM.

Preferably, the mixture solution prepared by step (c) is an aqueous solution.

According to a preferable embodiment, the present process further comprises the step to filter the mixture solution before step (d) and after step (c) using a filter with a pore size of not more than 1 μm, more preferably 0.5 μm and most preferably 0.25 μm.

Although the concentrations of calcium and phosphate ions are adequately adjusted, the precipitation by homogeneous precipitation reactions may occur due to their high reactivity. Therefore, it is preferable that the present process further comprises the step to remove the precipitated crystals using a filter with a suitable pore size.

Step (d): Fabrication of a Drug-Incorporated Calcium Phosphate Hybrid Thin Rim

The drug {e.g., a bone morphogenic stimulant)-incorporated calcium phosphate hybrid thin film is prepared by applying the mixture solution to the solid substrate. In other words, the surface of solid substrates is modified with the drug- incorporated calcium phosphate hybrid thin film. The solid substrate used may be any of substrates known in the art, including metals, ceramic products, glasses and organic materials comprising polymers and diverse biological tissues from human, plant and animal such as body-incorporated artificial substitute materials, medical implants and bone cements for dental implant.

According to a preferable embodiment, the step (d) is performed by applying the mixture solution to the solid substrate and developing nuclei for 1 min-24 hrs at

1-20⁰C for processing the thin film on the solid substrate. Afterwards, the solid substrate is kept to stand for more than 5 min at 1-6O⁰C to prepare the calcium phosphate hybrid thin film.

The nucleation process is carried out more preferably at 4-10⁰C and most preferably at 4°C. The nucleation is carried out more preferably for 5 min-24 hrs, much more preferably for 10 min-10 hrs, still much more preferably 30 min-3 hrs and most preferably for 30 min-2 hrs.

The step to form the thin film is carried out more preferably at 10-50⁰C, much more preferably at 25-40⁰C and most preferably at 30-40⁰C. The thin film formation is carried out more preferably for 10 min-24 hrs, much more preferably 30 min-10 hrs and most preferably for 30 min-2 hrs.

The solubility of ions is reduced in proportion to increase of temperature. Particularly, calcium and phosphate ions are likely to be precipitated by homogeneous reactions. It is necessary to prevent the homogeneous reaction by maintaining much lower temperature conditions during the preparation process to form the coating layer. It is so preferable to carry out the preparation process under conditions to be maintained at 1-2O⁰C as low as possible. Unlikely, the present preparation process could be carried out in a broad temperature range of 4-6O⁰C so long as concentrations of ions are adequately controlled. Most preferably, the present invention enables the calcium phosphate hybrid thin film to be fabricated at 37°C {i.e., body temperature); therefore it may be called as a biomimetic preparation process. The present invention can be considered as a biomimetic preparation process in the senses that the formation of the calcium phosphate coating layer is carried out under conditions that pH and temperature of solution are adjusted to be similar to body. Another superiority of this invention is a shorter time for formation of the calcium phosphate hybrid thin film. It is possible to prepare the hybrid thin film for a short period of time, for example, 5 min-24 hrs and most preferably 30 min-2 hrs.

The Ca/P molar ratio of the present calcium phosphate hybrid thin film is in a range of preferably 1.0-2.2, more preferably 1.5-2.0 and most preferably 1.7-2.0.

As demonstrated in Examples below, the present drug-incorporated calcium phosphate hybrid thin film has an much more excellent effects on the bone-like nodule formation compared with the drug-unincorporated calcium phosphate hybrid thin film, because differentiation of osteoblasts is stimulated by incorporated drug

(particularly, Vitamin D₃) resulting in rapid development of bone-like nodules. The present invention relates to a method for modifying the surface properties of hard tissue substitute materials or organs for human body implantation to provide the surface with excellent biocompatibility, cell compatibility and osteoconductivity. The surface modification method of this invention may be applied to various forms of substances comprising metals, ceramic products, glasses, organic materials, inorganic materials and bone cements, and diverse samples such as biological tissues of human, animal and plant. It is also possible to form the calcium phosphate hybrid thin film on the interior surface of pores present in porous materials. The concentrations of drug incorporated may be adequately controlled.

The DDW (distilled deionized water) may be used as solvents, enabling the present invention to become more convenient. The present invention may be useful as various implants which are utilized in conventional dental services and orthopedics.

The present invention develops a drug-incorporated porous coating layer with a three-dimensional structure with low crystallinity on the solid surface. As results, the present invention will be beneficially applied to modify the surface of artificial substitute materials or medical implants for incorporating into human body with excellent biocompatibility, cell compatibility and osteoconductivity.

According to this invention, there is provided a preparation method of the new-concept calcium phosphate organic/inorganic hybrid thin film which the calcium phosphate hybrid thin film is formed on the surface of artificial organs in an aqueous solution and its biological functionality is remarkably improved using the functional drug simultaneously incorporated into the thin film in an easy manner. The formation of the calcium phosphate hybrid thin film is carried out under conditions of temperature and pH of solution adjusted to be similar to physiological environments, resulting in the preparation of a biomimetric calcium phosphate. Since the calcium phosphate hybrid thin film is fabricated in an aqueous solution, it is another feature of this invention that the coating layer is formed without reference to the surface shape of materials for the hybrid thin film. Furthermore, it is of great advantage that the coating layer is formed on the interior surface of pore present in porous materials. The new-concept calcium phosphate hybrid thin film in which drug- incorporated organic compounds and inorganic materials are complexed, possesses excellent effects on the bone-formation stimulation compared with other types of the thin films known to date. Therefore, the present invention ensures the development of hard tissue substituent materials, materials with treatment potentials and drug delivery systems.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 represents FE-SEM image to the calcium phosphate hybrid thin film incorporating lα,25-dihydroxyvitamin D₃ as a drug stimulating activity of osteoblast on cell culture dish of polystyrene. The concentrations of lα,25-dihydroxyvitamin D₃ are (a) 0 M, (b) 1 x 10^"12 M and (c) 1 x 10^"6 M. As shown in Fig. 1, it could be observed that all calcium phosphate hybrid thin films formed on the surface of polystyrene had three-dimensional porous structures. The porosity is resulted in increased surface area for adsorbing various proteins of body, influencing significantly on cell adhesion and cell activation. However, the morphology of the surface dependent on concentrations of lα,25-dihydroxyvitamin D₃ was not changed. Fig. 2 represents the ratios of elements contained in the present drug- incorporated calcium phosphate hybrid thin film analyzed by EDX (energy dispersive X-ray analysis). The measurement clearly shows that the calcium phosphate hybrid thin film containing calcium and phosphorus is formed. Carbon and oxygen elements are derived from the thin film, drug and culture dish: concentrations of lα,25- dihydroxyvitamin D₃ are (a) 0 M, (b) 1 x 10^"12 M and (c) 1 x 10^"6 M. Fig. 3 is a histogram representing adhesion of MG63 cells (a type of osteoblast cell lines) on the drug-incorporated calcium phosphate hybrid thin film of this invention. MG63 cells were seeded on the surface of the thin film and cultured for 4 hrs and 12 hrs. The cell adhesion on the lα,25-dihydroxyvitamin D₃- incorporated calcium phosphate hybrid thin film occurred about 2-fold higher than in the control (CTL) at all time points. Accordingly, it could be appreciated that the present calcium phosphate hybrid thin film remarkably induces cell adhesion. The filled bars correspond to samples cultured for 4 hrs and hatched bars to those cultured for 12 hrs. Fig. 4 represents measurement results of specific activities of alkaline phosphatase (ALP specific activity) after MG63 cell lines were cultured on the calcium phosphate hybrid thin films synthesized with lα,25-dihydroxyvitamin D₃ of 1 x 10^"12 M and 1 x 10^"6 M for 2 weeks and 3 weeks, respectively. The symbol "CTRL" denotes a control. The filled bars correspond to samples cultured for 2 weeks and hatched bars to those cultured for 3 weeks.

Fig. 5 is a graph to show measurement results of the number of bone-like nodules formed on the calcium phosphate hybrid thin films synthesized with lα,25- dihydroxyvitamin D₃ of 1 x 10 ^~12 M and 1 x 10^"6 M after MG63 cell lines were grown on the surface of the thin films for 1 week. The symbol "CTRL" denotes a control. Fig. 6 represents photographs of bone-like nodules under an optical microscope which were formed on the calcium phosphate hybrid thin film synthesized with 1 x 10^"6 M lα,25-dihydroxyvitamin D₃ after MG63 cell lines were grown on the surface of the thin films for 1 week: magnification, (a) x 40, (b) x 100.

The present invention will now be described in further detail by examples. It would be obvious to those skilled in the art that these examples are intended to be more concretely illustrative and the scope of the present invention as set forth in the appended claims is not limited to or by the examples. EXAMPLES

EXAMPLE 1: Preparation of Supersaturated Ionic Solution of Calcium and Phosphorus Containing lα,25-dihydroxyvitamin D₃ A calcium ion solution containing 299 mM calcium ions was prepared by completely dissolving 8.85 g of Ca(NO₃)₂ ^• 4H₂O in 125 mL of DDW (distilled deionized water). A phosphorous ion solution containing 605.6 mM phosphorous ions was prepared by dissolving 20 g of (NH₄)₂ ^• HPO₄ in 250 mL of DDW. One mg of lα,25-dihydroxyvitamin D₃ (Sigma, D1530) as powder was dissolved in 200 μL of 99.9% ethanol with high purity (CARLO ERBA, 414577). The lα,25-dihydroxyvitamin D₃ solution prepared at 4⁰C was dissolved in DDW, generating lα,25- dihydroxyvitamin D₃ solutions of final concentrations of 1 x 10^"12 M and 1 x 10^"6 M. Each calcium and phosphorous ion solution was added to the lα,25- dihydroxyvitamin D₃ solution at final concentrations of 2.39 mM and 4.84 mM and mixed. The mixture solutions were adjusted to pH 7.2.

To form a calcium phosphate hybrid thin film on a substrate surface, the mixture of lα,25-dihydroxyvitamin D₃ solution and the ion solution prepared above were sufficiently agitated, followed by eliminating precipitates generated by heterogeneous precipitation reactions using a filter with a pore size of 0.2 μm. The mixture solutions prepared thus were injected to a polystyrene culture dish and incubated for 1 hr at 4⁰C to form nuclei for generation of hybrid thin films. The resultants were additionally incubated for 90 min at 37°C, finally giving the calcium phosphate hybrid thin films on substrates.

EXAMPLE 2: Physiochemical Characteristics of lα,25-dihydroxyvitamϊn D₃-Incorporated Calcium Phosphate Hybrid Thin Film <2-l> SEM (Scanning Electron Microscope) Analysis

The surface shapes of the calcium phosphate hybrid thin films synthesized in the presence or absence of lα,25-dihydroxyvitamin D₃ incorporated were observed using SEM (JSM-5800, Jeol, Japan). The SEM images were represented in Fig. 1.

As shown in Rg. 1, it could be demonstrated that there were only slight differences between control and samples by comparing SEM images of control with those of samples containing lα,25-dihydroxyvitamin D₃ at concentrations of 1 x 10^"12

M and 1 x 10^"6 M, suggesting that evident changes do not occur in physical shape of calcium phosphate hybrid thin films finally be formed due to lower concentrations of lα,25-dihydroxyvitamin D₃. It could be appreciated that lα,25-dihydroxyvitamin D₃ had not considerable effects on three-dimensional structure of the calcium phosphate hybrid thin film.

<2-2> EDX Analysis (Energy Dispersive X-ray Analysis)

The chemical state of lα,25-dihydroxyvitamin D₃-incorporated calcium phosphate hybrid thin film was analyzed by EDX (FE-SEM; Inca program; Oxford Co.), yielding a value of Ca/P ratio. The EDX spectrum was represented in Fig. 2.

As shown in Fig. 2, the calcium phosphate hybrid thin film without incorporated lα,25-dihydroxyvitamin D₃ as a control was revealed to have a Ca/P value of 2.20 and the calcium phosphate hybrid thin film to incorporate lα,25- dihydroxyvitamin D₃ at a concentration of 1 x 10^"12 M was analyzed to have a Ca/P value of 1.934 which is similar to control. In contrast, the Ca/P value was decreased to 1.769 in the calcium phosphate hybrid thin film containing lα,25-dihydroxyvitamin D₃ at a concentration of 1 x 10^'6 M.

It could be appreciated that the incorporation of lα,25-dihydroxyvitamin D₃ significantly affected the chemical state of the thin film not that of its outer surface morphology. In particular, there were generated the changes of chemical compositions in which the Ca/P values are reduced in proportion to increase in concentrations of lα,25-dihydroxyvitamin D₃. The carbon proportion was measured at not less than 70%, suggesting that X-ray was penetrated to polystyrene beyond the calcium phosphate hybrid thin film.

EXAMPLE 3: Effect of lα,25-Dihydroxyvitamin D₃-Incorporated Calcium Phosphate Hybrid Thin Film on Cell Adhesion, Differentiation and Bone- like Nodule Formation

<3-l> Cell Culture Analysis

To analyze cell response to lα,25-dihydroxyvitamin D₃-incorporated calcium phosphate hybrid thin film, MG63 cell line (ATCC) similar to osteoblast was used. For cell experiment, MG63 cells were cultured in DMEM (Dulbeco's modified eagle medium) containing 10% FBS (fetal bovine serum) and 0.5% penicillin/streptomycin at 37°C in 5% CO₂ incubator. Subculture was carried out after cells were grown on culture dish until about 80% confluency. Culture solution was regularly replaced.

<3-2> Cell Adhesion Assay Cell adhesion assay was performed on lα,25-dihydroxyvitamin D₃- incorporated calcium phosphate hybrid thin film prepared according to Example 1 procedure. MG63 cell line cultured in <3-l> was prepared at a concentration of 1 x 10³ cells/mL and seeded to untreated culture dish and the calcium phosphate hybrid thin films synthesized involving lα,25-dihydroxyvitamin D₃ at concentrations of 1 x 10^"12 M and 1 x 10^"6 M. Each MG63 cell line was incubated at 37°C under the condition of 5% CO₂ for 4 hrs and 12 hrs. After predetermined culture periods, cell samples were sufficiently washed with PBS (phosphate buffered saline) and fixed for 10 min at room temperature using cold 70% ethanol. After re-washing with PBS, cell samples were stained with crystal violet stock solution (Sigma C-6158) for 10 min at room temperature and then washed with DDW. 1% SDS solution was added to surface samples and agitated for 10 min. OD (optical density) was measured at 750 nm using a microplate reader (Fig. 3).

As shown in Fig. 3, cell adhesion of MG63 cell line on the surface of lα,25- dihydroxyvitamin D₃-incorporated calcium phosphate hybrid thin film was observed much more significantly than that of MG63 cell line in a culture dish with no thin film after MG63 cell line was adhered for 4 hrs and 12 hrs. Particularly, cell adhesion was increased by about 100% in the thin film containing lα,25-dihydroxyvitamin D₃ at a concentration of 1 x 10^"6 M compared with a generally purchasable culture dish. Accordingly, it could be understood that the properties of the lα,25-dihydroxyvitamin D₃-incorporated surface improve its cell adhesion potential.

<3-3> Cell Differentiation Assay The induction of differentiation of MG63 cell line by the properties of the film surface was analyzed using an ALP kit (alkaline phosphatase activity kit, Sigma 104). For differentiation assay, MG63 cell line was seeded on the film surface and continuously cultured for 2 weeks or 3 weeks, measuring their alkaline phosphatase activities. After predetermined culture periods, 200 mL of lysis buffer was added to samples and incubated for 10 min at 4°C. 0.5 mL of each alkaline buffer and stock substrate solution was supplemented and stored at 37°C water bath for 1 hr.

10 mL of 0.05 N NaOH was then added to each sample and the alkaline phosphatase activity was determined by measuring their OD value at 400 nm (Fig. 4).

As demonstrated in Fig. 4, the culture results for 2 weeks and 3 weeks clearly showed that the alkaline phosphatase activity was increased in the lα,25- dihydroxyvitamin D₃-incorporated calcium phosphate hybrid thin film compared with the control. The enhancement of the alkaline phosphatase-specific activity was revealed to show a similar tendency to that indicated in the patent document filed by the present inventors (Korean Pat. No. 10-0675573). In the drug-unincorporated calcium phosphate, its alkaline phosphatase activity was measured higher than in culture dish as a control. However, it could be observed that the alkaline phosphatase activity became much higher in the lα,25-dihydroxyvitamin Da- incorporated calcium phosphate. Given alkaline phosphatase, a marker protein expressed in an early stage of osteoblast differentiation, these results urge us to reason that the differentiation of MG63 cell line could be rapidly induced in the lα,25-dihydroxyvitamin D₃-incorporated calcium phosphate.

<3-4> Bone-like Nodule Formation

The bone-like nodule formation by MG63 cell in the thin film was analyzed to investigate a bone tissue regeneration potential by the lα,25-dihydroxyvitamin Da- incorporated calcium phosphate hybrid thin film (Figs. 5-6). Using a culture dish as a control, the present inventors examined whether the bone-like nodule formation was induced by culturing at different concentrations of lα,25-dihydroxyvitamin D₃. MG63 cells were grown on the lα,25-dihydroxyvitamin D₃-incorporated calcium phosphate hybrid thin film for 1 week and then the bone-like nodule formation was observed, which are represented in Figs. 5 and 6. Where osteoblast cell line, MG63 cells were grown for 1 week on commercial cell culture dishes as control and the calcium phosphate hybrid thin films synthesized in the presence of 1 x 10^"12 M or 1 x IfJ⁶ M lα,25-dihydroxyvitamin D₃, the bone-like nodules were developed rapidly on the lα,25-dihydroxyvitamin D₃-incorporated calcium phosphate hybrid thin film but not on commercial cell culture dishes. At extremely lower concentrations of lα,25- dihydroxyvitamin D₃, the bone-like formation occurred similar to the drug- unincorporated calcium phosphate. However, the bone-like nodule formation was surprisingly enhanced where the concentration of lα,25-dihydroxyvitamin D₃ became elevated to 1 x 10^"6 M. This result was represented in Fig. 5. In Fig. 5, the number of the bone-like nodule formed dependent on concentrations of lα,25-dihydroxyvitamin D₃ was counted after MG63 cells were cultured on the film surface for 1 week. As shown in Fig. 5, the bone nodule was not formed in the commercial cell culture dish and about 10 bone nodules were generated on the hybrid thin film synthesized at 1 x 10^"12 M lα,25-dihydroxyvitamin D₃. Moreover, more than 120 bone nodules were formed on the hybrid thin film synthesized at 1 x 10^"6 M lα,25-dihydroxyvitamin D₃. The bone nodule formed at 1 x 10^"6 M lα,25-dihydroxyvitamin D₃ was observed under an optical microscope (Fig. 6). It could be observed that the bone nodules were remarkably produced on the lα,25-dihydroxyvitamin D₃-incorporated calcium phosphate hybrid thin films. The bone-like nodule formation was never generated in the commercial culture dish. It has been generally reported that the bone-like nodule is formed only under conditions of 4-week cell culture and a drug- induced stimulation. Interestingly, the bone-like nodule formation occurred by even 1-week cell culture on the calcium phosphate hybrid thin film prepared according to the present method, which are considered as exceptional and notable results. Accordingly, it could be appreciated that the drug-incorporated calcium phosphate hybrid thin film synthesized by the present invention has excellent functions and its capability of forming bone-like nodules is prominent by continuously stimulating osteoblasts in local regions by lα,25-dihydroxyvitamin D₃ incorporation into the thin film.

Having described a preferred embodiment of the present invention, it is to be understood that variants and modifications thereof falling within the spirit of the invention may become apparent to those skilled in this art, and the scope of this invention is to be determined by appended claims and their equivalents.

Claims

What is claimed is:

1. A method for preparing a drug-incorporated calcium phosphate hybrid thin film comprising the steps of:

(d) fabricating the drug-incorporated calcium phosphate hybrid thin film by applying the mixture solution of the step (c) to a solid substrate.

2. The method according to claim 1, wherein the drug comprises a protein, a peptide, a nucleic acid molecule or a compound.

3. The method according to claim 1, wherein the drug comprises a chondrocyte proliferant, a cytokine, an anti-cancer agent or a bone morphogenic stimulant.

4. The method according to claim 3, wherein the drug comprises the bone morphogenic stimulant selected from the group consisting of Vitamin D, Vitamin D₃, Vitamin C, lanthanium (III) compound, calcitonin, tamoxipen, biphosphonate, statin, sodium fluoride, oxysterol, BMP (bone morphogenic protein), parathyroid hormone, IGF-I (insulin-like growth factor-1), TGF-β (transforming growth factor-beta), calcium and combinations thereof.

5. The method according to claim 4, wherein the bone morphogenic stimulant comprises Vitamin D₃.

6. The method according to claim 4, wherein the bone morphogenic stimulant in the mixture solution of step (c) is present in a concentration of 1 nM-10 mM.

7. The method according to claim 1, wherein the calcium ion in the mixture solution of step (c) is present in a concentration of 1-10 mM.

8. The method according to claim 1, wherein the phosphate ion in the mixture solution of step (c) is present in a concentration of 1-10 mM.

9. The method according to claim 1, wherein the step (d) is carried out by applying the mixture solution to the solid substrate for 1 min-24 hrs at 1-20⁰C to form a nucleus for the formation of a thin film on the solid substrate and keeping the solid substrate to stand for more than 5 min at 1-6O⁰C to form a calcium phosphate hybrid thin film on the solid substrate.

10. The method according to claim 1, wherein the method further comprises, prior to the step (d), filtering the mixture solution through a filter with a pore size of not more than 0.5 μm.

11. The method according to claim 1, wherein the calcium phosphate hybrid thin film prepared by the step (d) comprises the Ca/P molar ratio of 1.5-2.0.

12. A calcium phosphate hybrid thin film for promoting osteogenesis, characterized in that it is prepared by the method according to any one of claims 1-11 and comprises the bone morphogenic stimulant as a drug.

13. The calcium phosphate hybrid thin film according to claim 12, wherein the calcium phosphate hybrid thin film comprises the Ca/P molar ratio of 1.5-2.0.

14. The calcium phosphate hybrid thin film according to claim 12, wherein the bone morphogenic stimulant comprises Vitamin D₃.

15. An artificial substitute material or implant coated with the calcium phosphate hybrid thin film of claim 12 for promoting osteogenesis.