JP2003176111A - Controlled hydroxyapatite and method for synthesizing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide controlled hydroxyapatite having controlled crystallinity, solubility, a wide range of application and achieving desired functions, and to provide a method for synthesizing the hydroxyapatite. <P>SOLUTION: The hydroxyapatite is synthesized by a wet method, in the presence of amino acids such as glycine, serine, aspartic acid or glutamic acid. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to reconstructive medicine, regenerative medicine, tissue engineering, DDS (Drug Delivery System) in the field of medicine and dentistry.
stem) etc., not only supports and base materials but also genetic engineering, utilization technology of biological components, technology of remodeling and mimicking living body and its components, etc. The present invention relates to a controlled hydroxyapatite in which characteristics such as shape and properties are controlled, and a method for synthesizing the same.

[0002]

2. Description of the Related Art Hydroxyapatite (hereinafter referred to as HAp
Abbreviated) is the main component of biological hard tissue (bone tissue),
Since it has high biocompatibility, it is widely used in the field of biomaterials such as bone substitutes and bone substitutes.

[0003] Such a biomaterial is sintered to impart strength, and is used only as a material for reconstructing living tissue. Once implanted, the biomaterial remains in the body semipermanently and is metabolized in the body. It is rarely done.

However, as a treatment for reconstructing living tissue,
A tissue regeneration material capable of restoring not only the function of the tissue to be reconstructed but also both the function and the tissue is desirable.

[0005] Further, studies on gene delivery using a drug delivery system or a vector for the purpose of specific treatment in a living body have been advanced mainly on polymer materials. HAp has also been investigated for its possibility, but at present, drug release and plasmid DNA release control can hardly be performed.

[0006] If the solubility of HAp can be controlled, when a molded article made of the above HAp is transplanted into a living body as a biomaterial, the time remaining in the body can be controlled, and the biomaterial disappears due to bone regeneration. . At that time, the eluted calcium ions and phosphate ions are effective in forming new bone.

Furthermore, by setting the release of the drug or plasmid DNA to depend on the dissolution of HAp, the drug release time and gene transfer time can be controlled.

The control of crystal size is an important factor in controlling the amount of drug and plasmid DNA adsorbed. If the crystal size of HAp can be controlled, it becomes possible to control the amount of drug or DNA adsorbed, and in combination with the above solubility control, true control of drug or gene release becomes possible.

[0009]

Conventionally, the solubility of HAp is controlled by lowering the synthesis temperature and substituting various ions such as magnesium, iron and carbonic acid. However, lowering the synthesis temperature alone does not lead to a marked improvement in solubility, and there is a concern that these ions, which are eluted in the bone regeneration site, may weaken the bone quality of the regenerated bone by substitution of various ions.

The crystal size of HAp is controlled by controlling its synthesis temperature, pressure and time. In that case, it was easy to make a crystal having a large crystal size, but it was difficult to make a crystal having a smaller crystal size. there were.

Therefore, the object of the present invention is to obtain the crystal size,
It is an object of the present invention to establish a controlled HAp with controlled solubility and a method for synthesizing the controlled HAp, and to provide a biomaterial effective for human hard tissue regeneration and tissue healing promotion.

[0012]

[Means for Solving the Problems] The present inventors inhibit the crystal growth of HAp by the presence (coexistence) of acidic amino acids such as aspartic acid and glutamic acid, and neutral amino acids such as glycine and serine during HAp synthesis. I found it.

By changing the abundance of these amino acids, we succeeded in obtaining a controlled HAp in which the crystal size and solubility of HAp are controlled.

That is, the controlled HAp of the present invention is characterized in that it is obtained by being synthesized in the coexistence of an amino acid in order to solve the above problems.

In the above controlled HAp, it is preferable that at least one of the shape and properties of the obtained crystal is controlled by an amino acid.

According to the above-mentioned constitution, since it is obtained by synthesizing in the coexistence of amino acid, at least one of the shape and properties of the obtained crystal is controlled, and the solubility and the protein adsorption ability are controlled. , Controllable.

As a result, the above-mentioned structure is obtained by using the biomaterial and D
Since it is possible to impart effective functionality as a DS carrier to a desired purpose, it becomes possible to make it more suitable as a biomaterial or a material for a DDS carrier.

The method for synthesizing a regulated HAp of the present invention is characterized in that it is produced in the coexistence of an amino acid in order to solve the above problems.

In the above synthetic method, when the amino acid is produced in the presence of an amino acid, the amino acid is contained in, included in, or adsorbed to the crystal to control at least one of the crystal shape and properties of the obtained controlled HAp. Preferably.

According to the above method, a controlled HAp more suitable as a biomaterial or a material for a carrier of DDS can be obtained by a simple method of coexistence of amino acids, so that the production of a controlled HAp having an excellent function is simplified. Can be converted.

In the above-mentioned regulated HAp and the method for synthesizing it, it is desirable that the amino acid is at least one of acidic amino acid and neutral amino acid.

In the above-mentioned regulated HAp and the method for synthesizing the same, the amino acid is preferably at least one amino acid selected from the group consisting of aspartic acid, glutamic acid, glycine, and serine.

In the above structure and method, by using the above amino acid, a regulated HAp having an excellent function can be obtained more reliably.

[0024]

BEST MODE FOR CARRYING OUT THE INVENTION Control H according to an embodiment of the present invention
As a method of synthesizing Ap, a wet method of synthesizing HAp is used. The above-mentioned wet synthesis method is a method that is very simple and can synthesize HAp inexpensively and in large quantities.

In the wet synthesis method, specifically, a calcium solution and a phosphoric acid solution are mixed at a pH of 10: 6 at a molar concentration ratio.
Is sequentially dropped into a buffer solution maintained at a predetermined value of 7.4 or more for a long period of time to precipitate HAp in the buffer solution and collect the precipitated HAp. The HAp may be carbonate apatite or octacalcium phosphate.

The synthesis temperature is not particularly limited, but is preferably in the range of 0 ° C to 100 ° C, more preferably 30 ° C to 90 ° C.

In the present invention, at least one selected from the amino acid group consisting of aspartic acid, glutamic acid, glycine and serine is allowed to exist (coexist) in the buffer during the above synthesis. The concentration of the amino acid may be in the range of 0.005 mol / L to saturation concentration, but more preferably 0.01 mol / L to 0.5 mol.
/ L.

It has been found that when the synthesis is carried out in the presence of an amino acid in this manner, the crystal growth of HAp is suppressed and a controlled HAp having a small crystal size and high solubility can be synthesized.

The crystal size and the solubility depend on the amount of the amino acid present in the buffer solution. At this time, the most notable effect is aspartic acid, and glutamic acid, serine and glycine become less effective in this order. I found out.

Next, a specific example of the above synthesis will be described. As shown in Fig. 1, first, 100 ml of an aqueous solution of calcium acetate monohydrate with a concentration of 100 mmol / L and a concentration of 6
100 ml of 0 mmol / L monoammonium phosphate aqueous solution is stirred at the same speed, and the concentration is 1.3 mmol / L.
In 200 ml of the acetic acid buffer solution (buffer solution) of, the tube pump 1 was sequentially added dropwise to precipitate (generate) HAp.
The precipitate was collected and synthesized.

The synthesis temperature was maintained at 60 ° C. ± 1 ° C. by the constant temperature bath 2. The buffer solution was monitored by the pH meter 3, and the ammonium aqueous solution was maintained by the tube pump 4 controlled by the above monitoring result so that the pH was 7.4 ± 0.1.

At the time of this synthesis, 12 kinds of amino acids were added in advance to the buffer solution so that the concentration of each of them was 0.5 mol / L. Twelve amino acids are glycine (Gl
y), alanine (Ala), proline (Pro), hydroxyproline (Hyp), serine (Ser), valine (Val), threonine (Th
r), methionine (Met), aspartic acid (Asp), glutamic acid (Glu), arginine (Arg), and histidine (His).

The HAp thus synthesized in the presence (in the coexistence) of the amino acid is the controlled HAp of the present invention whose crystallinity and solubility are controlled as described below, and washed with distilled water. It was dried in a thermostat at 60 ° C. for 72 hours. Further, as a comparative example, a comparative HAp (HAp-con) synthesized without adding an amino acid in the buffer solution was separately prepared.

In order to evaluate the crystallinity of the control HAp and the comparative HAp thus obtained, they were subjected to X-ray diffractometry (apparatus used: Rigaku Ultima +, 40 kV and 30 mA, C
X-ray diffractograms were obtained using uKα). The results are shown in FIGS. 2 and 3. In addition, based on those X-ray diffraction patterns, the values of (3
The reciprocal of the half-width was calculated from the (00) and (002) reflections, respectively. The results are shown in Table 1. In Table 1, those marked with * indicate that the average value has a significant difference with respect to HAp-con at less than 5%. Also. Unmeasurable (Imme
Asurable) means that the peak was too broad to be calculated.

[0035]

[Table 1]

From the above X-ray analysis, the control HAp synthesized in the presence of amino acid was compared with the comparative HAp as shown in FIG.
As shown in FIG. 3 and Table 1, it was found that the peaks were broad and therefore the crystallinity was different, and the shift of each peak was not observed.
It turns out that it is p.

In particular, control HA synthesized in the presence of glycine, serine, aspartic acid, or glutamic acid
p (hereinafter, HAp-Gly, HAp-Ser, HAp-Asp, HAp
-Glu) has a significant difference of less than 5% compared to the comparative HAp, and has significantly low crystallinity (large crystal strain).
You can see that

As a result, the controlled HAp of the present invention has a significantly low crystallinity (large crystal strain), and thus controls the adsorption ability of an ionic substance, for example, a protein, as will be described later. Can be improved).

From the peak of the above X-ray diffraction pattern, HAp-
The crystal sizes of con, HAp-Gly, HAp-Ser, HAp-Asp, and HAp-Glu were determined by the following formula (Scherrer's formula).

D = 0.9λ / β cos θ Here, λ is the wavelength, β is the half width, and θ is the incident angle. The respective crystal sizes are shown in Table 2 below.

[0041]

[Table 2]

In the controlled HAp and the method for synthesizing the same according to the present invention, the larger the amount of the amino acid present, the smaller the crystal size, and it is possible to reliably, easily and stably realize the minimum size of 200 nm.

Next, the compositions of the control HAp and the comparative HAp were examined. First, a 10 mg sample is added to 10 ml of 0.1
It was dissolved in N hydrochloric acid aqueous solution. Using the atomic absorption method (used equipment: Shimadzu AA6400F) from the solution, the calcium (Ca) concentration in the sample was measured, and the UV spectrophotometric method was used.
The concentration of phosphoric acid (P) in each sample was measured using (apparatus used: Shimadzu UV-150-02). The results are shown in Table 3.
It was shown to. In Table 3, those marked with * indicate that the average value has a significant difference of less than 5% with respect to HAp-con.

[0044]

[Table 3]

From the above results, it can be seen that the control HAp synthesized in the presence of HAp-Asp and HAp-Glu has significantly lower composition components of calcium ion and phosphate ion than the comparative HAp. I understand.

Subsequently, in order to identify the above-mentioned control HAp and comparative HAp and to investigate the molecular structure, K was determined using Fourier transform infrared spectrophotometry (apparatus used: Shimadzu FT-IR 8300M).
The infrared absorption of the sample in Br pellet (sample concentration 10 mg / 400 mg KBr) was examined. The results are shown in FIG.

From the above results, HAp-Gly, HAp-Ser, HAp-As
For p and HAp-Glu, a unique peak was observed in the range of 1600 to 1700 (1 / cm). This peak is due to the carbonyl (C = O) of the carboxyl group of the amino acid, and even in the control HAp after washing, the amino acid (at least glycine, serine, aspartic acid,
It can be seen that glutamic acid) is contained in at least one of inclusion, adsorption and inclusion in the HAp crystal.

Further, the addition concentration is 0.05, 0.15,
Each HAp-Asp was prepared by adding 0.25 and 0.5 mol / L, respectively. The results are shown in FIG.

From the above results, it can be seen that the peak showing the carbonyl (C = O) of the carboxyl group increases depending on the concentration added, and that the amino acid in the controlled HAp can be quantified.

For each precipitate of the control HAp and the comparative HAp, a scanning electron microscope (apparatus used: Hitachi S-50
00) was used to observe their crystal size and morphology.

From the observation result, HAp-Asp has a flake-like shape with a smaller thickness than those of other control HAp.
However, the boundary of each crystal was unclear. HAp-Gly, HAp-Ser, and HAp-Glu are plate-shaped (plate-li
The crystal size was smaller than that of the comparative HAp.

Finally, the solubilities of the control HAp and comparative HAp were examined. First, 10 mg of each sample,
10 ml acetate buffer (pH 5.0, concentration 0.1 mol /
A mixed solution mixed with L) was prepared and left standing in a 37 ° C. incubator for 1 week. From the supernatant of the above mixed solution, the calcium concentration in the sample was measured using the above-mentioned atomic absorption method (apparatus used: Shimadzu AA6400F), and the solubility was investigated. The results are shown in FIG.

From the above results, HAp-Ser, HAp-Asp, and HA
p-Glu has a significantly low crystallinity (large crystal strain), and thus has a significant difference of less than 5% compared to HAp-con, and has an apparently high solubility. It can be seen that there are differences depending on each amino acid used. Their solubilities are HAp-Asp>HAp-Ser>HAp-Glu> H.
It was Ap-Gly. It was considered that these differences were due to the difference in affinity between HAp and each amino acid.

In the present embodiment, the quantitative test was repeated four times. The average value and standard deviation value of those results were calculated, and the average value was used as the result value. The above test data is
Statistical analysis was performed by factor analysis of variance (ANOVA). In the test results, Scheff's F-test method was used as a method for testing a significant difference of 95% or more between each average value using the above average value and standard deviation value.

Next, HAp-con and HAp-Asp were synthesized at the respective synthesis temperatures (80 ° C. and 40 ° C. in addition to 60 ° C.) and the amino acid concentration (0.25 mol / L), and the others were the same as above. Each was similarly prepared. The results of examining each of them by the above-mentioned X-ray diffraction method are shown in FIGS. 7 and 8, respectively. The above results show that HAp synthesis in the presence of amino acids is effective for controlling HAp crystallinity.

In addition, the acetic acid solution (pH of HAp-con and HAp-Asp synthesized above at each synthesis temperature)
The results of examining the elution amount of calcium (Ca) in 5) are shown in FIG. 9. From the above results, HAp-Asp
It can be seen that the elution amount (release amount) of Ca is significantly larger than that of HAp-con.

Further, FIG. 10 shows the results of examining the specific surface areas of the above-mentioned HAp-con and HAp-Asp synthesized at each synthesis temperature. From the above results, HAp-
It can be seen that Asp has a significantly larger specific surface area (adsorption capacity) than HAp-con.

Next, the results of examining the basic protein adsorption ability of the above-described HAp-con and HAp-Asp synthesized at each synthesis temperature will be described.

First, the above HA was added to an aqueous solution of cytochrome c (basic protein, pI = 10, MW = 10,000).
After immersing p-con and HAp-Asp in each (37 ° C., 4 hours) and centrifuging, the amount of residual protein in the supernatant solution was quantified and the adsorption amount was examined. The results are shown in FIG. From the above results, HAp-Asp is HAp-c
It can be seen that the adsorption capacity of basic protein is significantly larger than that of on.

From the results shown in FIGS. 10 and 11, HAp-
It can be seen that con and HAp-Asp have a good positive correlation between the specific surface area and the adsorption capacity.

Further, when HAp-Asp having the protein immobilized as described above was subjected to an in vitro dissolution experiment, Hp-Asp was
Upon the dissolution of Ap, release of the immobilized protein into the solution was confirmed, and it was confirmed that the release was sustained until HAp was completely dissolved.

Thus, in the presence of amino acids (in the coexistence)
Control HA obtained by generating, crystallization, and synthesizing HAp
Depending on the type and concentration of the amino acid, at least one of the crystal morphology (needle shape, petal (flake) shape, plate shape, amorphous shape, etc.), crystal size, and solubility (water solubility) of the obtained control HAp is determined by p. It can be controlled to suit the desired purpose.

Therefore, the above-mentioned regulated HAp can purposely have various solubilizing ability and protein adsorbing ability which are different from each other, and thus, it has a function of reconstructive medicine, regenerative medicine, tissue engineering, DDS (Drug). Not only the support (carrier) and base material in the Delivery System), but also genetic engineering, technology for utilizing biological components, technology for modifying and mimicking living organisms and their components, etc.
It is suitable for a wide variety of so-called biotechnology.

An example of application of the controlled HAp of the present invention is to use it as a carrier for growth factors. The above-mentioned growth factor is a protein that is said to have a function of controlling the differentiation and growth of various cells, and many attempts have been made to make good use of it in regenerative medicine. Recently, spray using bFGF released from Kaken Pharmaceutical and administration of HGF at Osaka University can be mentioned.

Proteins are classified into acidic, neutral and basic proteins according to the types of their constituent amino acids. Many proteins such as BMP, bFGF, and TGF-β that are effective for hard tissue regeneration are basic proteins, and a small amount of growth factor is very expensive (1 mg is several hundred thousand yen). Since the control HAp has a large basic protein adsorption ability, it can facilitate adjustment of the release amount of the growth factor and is extremely effective as a carrier for the growth factor.

Other application examples include a drug delivery system (DDS) and a control release (Contro
lled release). The drug (drug) moves in the body after administration and reaches the site of action to exert a therapeutic effect.
DDS is a system designed to deliver a drug to a required site in a required amount.

Control release means controlling the drug so that the drug can be released in a required amount at a required time. By using DDS, control release, or a combination of them, it becomes possible to exert effective drug effects locally and suppress side effects.

Controlled HA of the Invention as Carrier of Drug
p can control the release of the drug by regulating its metabolic properties (dissolution by dissolution), so it is possible to release the retained drug sequentially when necessary and to eliminate it after the completion of treatment. Can be set to, which makes it a suitable carrier for DDS and control releases.

By the way, HA which is the main component of living hard tissue
p is 97% or more for enamel and 70 for dentin and bone
%, It is widely used as a biomaterial for hard tissues due to its high biocompatibility and osteosynthesis.

The control HAp of the present invention has the same biocompatibility and osteosynthesis as the above HAp, and it is clear from the above X-ray diffraction results that the bone defect (both wide area and narrow area). In this case, it is suitable for use as a bone filling material, which is an artificial material that can have the function of bone by being embedded in a bone defect.

Further, as another application example, a GTR (Guide which is embedded in a living body and disappears as the living tissue is regenerated) is used.
d Tissue Regeneration) membrane, substrate for cell culture and carrier for cells, vector for gene therapy incorporating plasmid DNA or RNA, column packing material for liquid chromatography and the like.

As the GTR membrane, a type I collagen (porcine skin) solution and a controlled HAp (carbonate apatite, C / P ratio = 10) NaOH solution (concentration: 2.5 m) of the present invention were used.
(g / ml or less) and gelled by mixing with each other. Such a GTR membrane can be adjusted in its absorbability in vivo by adjusting the controlled HAp content and the collagen concentration.

When used for repairing a damaged part of a living body, the GTR film is bioabsorbable and does not require a secondary operation.
Due to the elution of the controlled HAp, the local Ca ion and P ion concentrations increase, and it can be expected to promote local calcification.

[0074]

The control HAp of the present invention is, as described above,
It is a composition obtained by being synthesized in the coexistence of an amino acid.

Therefore, the above-mentioned structure is applicable to biomaterials and DD.
Since the functional properties effective as the carrier of S can be added according to the desired purpose, there is an effect that it can be made more suitable as a biomaterial or a material of the carrier of DDS.

[Brief description of drawings]

FIG. 1 is a schematic configuration diagram of a manufacturing apparatus in a control HAp of the present invention.

FIG. 2 is an X-ray diffraction diagram of a part of the control HAp and a comparative HAp.

FIG. 3 is an X-ray diffractogram of the rest of the control HAp.

FIG. 4 Part of the above-mentioned regulated HAp (HAp-Asp, HAp-Ser, HA
3 is a graph showing IR measurement results of p-Glu, HAp-Gly) and comparative HAp.

FIG. 5 is a graph showing changes in IR measurement results corresponding to the difference in the concentration of addition in the above HAp-Asp.

FIG. 6 is a part of the above-mentioned regulated HAp (HAp-Asp, HAp-Ser, HA
Fig. 3 is a graph showing the solubility of p-Glu, HAp-Gly) and comparative HAp.

FIG. 7 is an X-ray diffraction diagram at each synthesis temperature of the comparative HAp.

FIG. 8 is an X-ray diffraction pattern of the above HAp-Asp at each synthesis temperature.

FIG. 9 Ca in the above HAp-Asp and the above comparative HAp
It is a graph which shows the amount of release.

FIG. 10 shows the HAp-Asp and the comparative HAp,
It is a graph which shows a specific surface area.

FIG. 11 shows the HAp-Asp and the comparative HAp,
It is a graph which shows a protein adsorption amount.

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Masayuki Okazaki             3-7-10 Ushida-Waseda, Higashi-ku, Hiroshima City, Hiroshima Prefecture             −305 F term (reference) 4C076 DD26 FF31                 4C081 AB03 BA12 BA13 BA16 BB01                       CF031 EA11                 4C084 AA13 MA67 NA12

Claims (8)

[Claims] 1. A controlled hydroxyapatite which is obtained by being synthesized in the presence of an amino acid. 2. The controlled hydroxyapatite according to claim 1, wherein at least one of crystal shape and properties of the hydroxyapatite body is controlled by the amino acid. 3. The controlled hydroxyapatite according to claim 1, wherein the amino acid is at least one of an acidic amino acid and a neutral amino acid. 4. The controlled hydroxyapatite according to claim 1, wherein the amino acid is at least one amino acid selected from the group consisting of aspartic acid, glutamic acid, glycine, and serine. 5. A method for synthesizing controlled hydroxyapatite, which is produced in the coexistence of an amino acid. 6. When produced in the coexistence of an amino acid, at least one of the crystal shape and the properties of the obtained controlled hydroxyapatite crystal is controlled by including, including or adsorbing the amino acid in the crystal. 6. The method for synthesizing controlled hydroxyapatite according to claim 5. 7. The method for synthesizing controlled hydroxyapatite according to claim 5, wherein the coexisting amino acid is at least one of an acidic amino acid and a neutral amino acid. 8. The controlled hydroxyapatite according to claim 5, wherein the coexisting amino acid is at least one amino acid selected from the group consisting of aspartic acid, glutamic acid, glycine, and serine. Synthesis method.