FORAMINIFERA-DERIVED BONE GRAFT MATERIAL

Abstract

The present disclosure relates to a foraminifera-derived bone graft material. A foraminifera-derived bone graft material according to an aspect of the present disclosure has remarkable abilities in terms of cell proliferation, cell adhesion, and osteoblast differentiation, and includes a structure that can support newly formed bones. Accordingly, the foraminifera-derived bone graft material may be effectively utilized as a bone graft material.

Claims

1. A bone graft material comprising hydroxyapatite, wherein the hydroxyapatite comprises a plurality of chambers separated by partition walls, and the partition walls comprise a plurality of pores.

2. The bone graft material of claim 1, wherein the hydroxyapatite is derived from foraminifera.

3. The bone graft material of claim 2, wherein the foraminifera is Baculogypsina sphaerulata, Baculogypsina bonarellii, Baculogypsina gallowayi, Baculogypsina lenticulate, Baculogypsina meneghinii, Baculogypsina saoneki, or Baculogypsina sphaerica.

4. The bone graft material of claim 1, wherein a plurality of the hydroxyapatite comprises particles, and the size of the particles is 100 um to 4,000 um.

5. The bone graft material of claim 1, wherein the chamber has a diameter in a range of 10 um to 80 um, the partition wall has a thickness in a range of 5 um to 15 um, or the pore has a size in a range of 0.1 um to 3 um.

6. The bone graft material of claim 1, wherein the bone graft material has 20,000 to 100,000 uniform chambers per 1 cm.sup.2 of the hydroxyapatite surface.

7. The bone graft material of claim 1, wherein the particle of the hydroxyapatite comprises 0.5 weight % to 10 weight % of magnesium ions, 0.2 weight % to 10 weight % of silicon ions, or 0.1 weight % to 5 weight % of strontium ions.

8. The bone graft material of claim 1, wherein the hydroxyapatite is prepared by performing a hydrothermal reaction on a pretreated exoskeleton of foraminifera at 30° C. to 600° C. for 2 hours to 40 hours, or by treating a pretreated exoskeleton of foraminifera with a microwave in a wavelength range of 300 MHz to 300 GHz in an amount of 100 to 1500 W for 0.5 minute to 48 hours.

9. The bone graft material of claim 1, wherein the bone graft material is for treating a bone defect.

10. A method of preparing the bone graft material of claim 1, the method comprising: pretreating foraminifera, and preparing hydroxyapatite by performing a hydrothermal reaction on the pretreated exoskeleton of foraminifera at 30° C. to 600° C. for 2 hours to 40 hours, or by treating the pretreated exoskeleton of foraminifera with a microwave in a wavelength range of 300 MHz to 300 GHz in an amount of 100 to 1500 W for 0.5 minute to 48 hours; and sintering the hydroxyapatite.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0048] FIG. 1 shows scanning electron microscopy images of a structure of hydroxyapatite particles according to an embodiment; A: 1,000 X, 10,000 X, B: 100 X, 1,000 X, 3,000 X .

[0049] FIG. 2 shows scanning electron microscopy images showing the measured size of the chambers and pores of hydroxyapatite particles according to an embodiment.

[0050] FIG. 3 shows comparison results of the XRD analysis of hydroxyapatite particles according to an embodiment with a control group HAp.

[0051] FIG. 4 is an image showing the cytotoxicity results measured from hydroxyapatite according to an embodiment.

[0052] FIG. 5 is a graph showing the cell proliferation results measured from hydroxyapatite according to an embodiment.

[0053] FIG. 6 is a scanning electron microscopy image showing the cell adhesion and infiltration results measured from hydroxyapatite according to an embodiment.

[0054] FIG. 7 is a graph showing the ALP activity analysis results measured from hydroxyapatite according to an embodiment.

[0055] FIG. 8 is a graph showing the expression levels of osteoblast marker genes in hydroxyapatite according to an embodiment.

[0056] FIG. 9 shows a figure (A) showing the micro-CT results at the 8th week of in vivo graft of hydroxyapatite according to an embodiment and a graph (B) showing quantified data thereof.

[0057] FIG. 10 shows the H&E staining results showing in vivo osteoanagenesis effects of hydroxyapatite according to an embodiment.

[0058] FIG. 11 shows the Goldner's Masson trichrome staining results showing in vivo osteoanagenesis effects of hydroxyapatite according to an embodiment.

MODE OF DISCLOSURE

[0059] Hereinafter, the present disclosure will be described in detail with reference to Examples below. However, these Examples are provided for illustrative purposes only, and the scope of the present disclosure is not limited thereto.

REFERENCE EXAMPLE 1

Cell Culture

[0060] The biocompatibility of foraminifera-derived hydroxyapatite (HAp) (also, referred to as foraminifera-HAp) was evaluated by using human mesenchymal stem cells (hMSCs). In detail, cells were cultured under conditions of 5% CO.sub.2 and 37° C. in a-MEM (Gibco-BRL, MD, Gaithersburg, Md.) supplemented with 10% fetal bovine serum (FBS; Gibco-BRL), wherein the cells subcultured from passage 4 to passage 6 were used for in vitro experiments.

[0061] To induce osteoblasts differentiation, the cells were cultured in a medium supplemented with an osteogenic stimulator (0.1 mM of dexamethasone, 0.1 M of 6-glycerophosphate, and 50 μg/mL of ascorbic acid). All compounds used in the medium supplemented with the osteogenic stimulator were cell culture-grade reagents (Sigma Aldrich, St. Louis, Mo., USA). A normal growth medium and the medium supplemented with the osteogenic stimulator were replaced with fresh media every 2 days during the experiments.

[0062] Before the cell culture experiments, HAp particles were first sterilized with 70% alcohol and washed with a phosphate buffer solution (PBS) three times for 10 minutes. After the sterilization, 20 mg of the sterilized pure HAp particles and foraminifera-derived HAp particles were added to a 48-well culture plate. The cells were seeded (at a concentration of 5×10.sup.4 cells/well) and cultured for 2 hours so that the initial cells were attached to each HAp particle. The HAp particles to which the cells were attached were transferred to a new culture plate, and then cultured for in vitro experiments.

REFERENCE EXAMPLE 2

Cell Proliferation

[0063] The cell proliferation was evaluated according to mitochondrial activity-based analysis using CellTiter96® Aqueous One solution (MTS assay, Invitrogen, Carlsbad, Calif., USA). As described above, the hMSCs were seeded and cultured for 1 day, 3 days, 6 days, 9 days, and 12 days. At a predetermined point, 50 μL of a CellTiter96® reagent solution was mixed with 250 μL of the normal medium, and the mixture was added to each well. After 4 hours of the cell culture, the supernatant was collected to measure absorbance at 490 nm by using an ELISA plate reader (SpectraMAX M3; Molecular Devices, Sunnyvale, Calif.).

REFERENCE EXAMPLE 3

Cell Viability and Cytotoxicity

[0064] The cell viability and cytotoxicity were evaluated according to fluorescence staining by using Live/Dead® and a viability/cytotoxicity kit (Invitrogen, Carlsbad, Calif., USA). According to the preparation protocol, the cultured HAp particles were washed with a PBS for 30 minutes. Subsequently, the cells were stained by using calcein acetoxymethyl ester (Calcein AM) and ethidium homodimer-1(EthD-1) of the kit, and then observed with an inverted fluorescence microscope (DM IL LED Flu( ) Leica Microsystems, Wetzlar, Germany).

REFERENCE EXAMPLE 4

In Vitro Evaluation of Cell Adhesion and Cell Infiltration

[0065] To observe the cell adhesion on the surface and the cell infiltration to each HAp particle, the cells were cultured for 5 days. A specimen was washed with a PBS, fixed in 2.5% glutaraldehyde solution at 4° C. for 2 hours, and post-fixed with 0.1% osmium tetroxide solution. Next, the specimen was dehydrated via graded ethanol series (30%, 50%, 75%, 85%, 95%, and 100%, each for 10 minutes). Then, the resulting specimen was sputter-coated with gold, and then observed with an electronic microscope (EM; EM-30). To observe the cell infiltration, the HAp particles were sliced with a sharp lancet to expose cross sections of the surface, followed by SEM analysis.

REFERENCE EXAMPLE 5

Measurement of Alkaline Phosphatase (ALP) Activity

[0066] The osteoblast differentiation was evaluated by ALP activity analysis. The ALP activity analysis was performed by using p-nitrophenylphosphate (p-NPP) as a substrate. In detail, the hMSCs were seeded on the particles, and then cultured in the medium supplemented with the osteogenic stimulator. After 5 days of the cell culture, adhesive cells were lysed by sonication in 1% Triton X-100/PBS solution under an icebox condition. To remove the particles and residues, the sample was centrifuged at 4° C. at a speed of 12,000 rpm. The supernatant was used for the ALP activity analysis and the protein concentration analysis. In the present study, the ALP activity was normalized to total protein contents.

reference example 6

Real-Time Polymerase Chain Reaction

[0067] To measure the osteoblast differentiation of the hMSCs cultured on the HAp particles, several osteogenesis marker genes, such as ALP, collagen type Iα1 (CoI1αI), osteocalcin (OCN), and bone sialoprotein (BSP), were measured by quantitative real-time polymerase chain reaction (RPCR). After the cells were cultured for 7 days in the medium supplemented with the osteogenic stimulator, total mRNAs were isolated from the cells, and cDNAs were transcribed with a reverse transcriptase (Invitrogen) and oligo (dT) primers. Then, the cDNAs were amplified with TaqMan Universal PCR Master mix(Applied Biosystem) and primers and TaqMan probe sets for ALP (Hs01029144_m1), Colla) (Hs00164004_m1), OCN (Hs01587814_g1), BSP (Hs00173720_m1), and 18S (Hsscience). All TaqMan PCRs were performed by using a StepOne Plus RPCR system (Applied Biosystems, Foster City, Calif., USA), and 18S rRNA gene was co-amplified as an internal standard.

REFERENCE EXAMPLE 7

Animal Model

[0068] 4 adult male New Zealand white rabbits (age >3 months or more, 2.5 kg to 3.0 kg) were anesthetized with intramuscular doses of ketamine (35 mg/kg; Yuhan Corporation, Seoul) and xylazine (5 mg/kg; Bayer Korea, Seoul). Then, 2% lidocaine solution was used for local anesthesia. After tissue excision, three isolated circular cranial defects were made by using trephine with an outer diameter of 6 mm. One defect was used as a control group, another defect was filled with pure HAp particles, and the other defect was filled with foraminifera-derived HAp particles. The healing process was observed 8 weeks after the graft, and the defects and the surrounding bones thereof were incised from host bones. Then, before performing further analysis, the specimen was removed and placed in a fixing solution (phosphate buffered 4% paraformaldehyde solution, pH 7.2) at 4° C. for 7 days.

REFERENCE EXAMPLE 8

Bone Analysis by Micro-CT

[0069] The newly formed bones were evaluated pathologically by micro-CT (Sky-Scan 1172TM, Skyscan, Kontich, Belgium). At the 8th week after the graft, the sample was analyzed by using an aluminum filter (0.5 mm) with X-ray set at a voltage of 60 kV and a current of 167 pA. Based on the micro-CT results, the qualitative images of the defects were observed with 3D reconstruction images based on a 3D software (CTVol, Skyscan), and the percent bone volume (BV) was calculated as follows by using an image analysis software (CT-analyzer TM, Skyscan).

BV(%)=(volume of new bones)-(volume of remaining graft material)/total defect volume

REFERENCE EXAMPLE 9

Histological Analysis

[0070] The histological analysis was performed at the 8th week after the graft. The fixed specimen of a rat cranium was treated with 8% formic acid/8% HCl to remove calcium deposit, and then dehydrated with graded alcohol series (70% to 100%). Finally, the specimen was placed in paraffin. Each specimen was cut into 5 pm sections by using a rotary microtone (HM 325™, Microm, Walldorf, Germany). Five sections from the center of each sample were stained with hematoxylin-eosin (HE) and Goldner's masson trichromand (MT). Then, the samples were randomly selected, and the formation of new bones was observed under a microscope (DMR, Leica, Nussloch, Germany).

REFERENCE EXAMPLE 10

Statistical Analysis

[0071] The numerical values are expressed as mean ±standard deviation (SD), and the statistical analysis was performed by one-way analysis of variance (ANOVA). Then, the Dunnett's post-hoc test was performed by using GraphPad Prism version 5.3 (GraphPad Software, SanDiego, Calif., USA), wherein P<0.05 was considered statistically significant.

EXAMPLES

Preparation and Analysis of Hydroxyapatite Particles

[0072] 1. Preparation of Hydroxyapatite (HAp) Particles by Using Foraminifera

[0073] The hydroxyapatite by using foraminifera were prepared as follows.

[0074] First, foraminifer (Baculogypsina sphaerulata, Okinawa, Japan) was purchased in the market. To remove residual contaminants and organic ingredients, the sample was boiled with 4% sodium perchlorate (NaClO.sub.4) and washed with distilled water. In detail, the sample was added to an ammonium phosphate monobasic ((NH.sub.4)H.sub.2PO.sub.4) aqueous solution, wherein a molar ratio of Ca:P was 10:6.

[0075] Subsequently, the sample was added to a Teflon-lined stainless autoclave, and heated at 200° C. for 24 hours. The transformed sample was washed with boiling water, and dried at 60° C. Then, HAp particles were chopped with a lancet, and HAp particles having a diameter in a range of 200 pm to 500 pm were separated by filtration through a stainless mesh. Afterwards, the crystallization was performed on the separated particles by a sintering process. The sintering was performed in an electric furnace (Muffle Furnace, SH-FU-4MH), wherein, after the temperature was raised to 600° C. at a heating rate of 5° C./min, the same temperature was maintained for 2 hours. Afterwards, the temperature was raised again to 800° C. at a heating rate of 5° C./min, and the same temperature was maintained for 4 hours. Then, the temperature was lowered to room temperature, thereby completing the sintering progress.

[0076] Here, as a control group, HAp particles (pure HAp particles having a particle size in a range of 200 μm to 500 μm) that were stoichiometrically synthesized were obtained from Dio Implant Inc. (Busan, Korea) and used.

[0077] 2. Chemical and Morphological Characteristics

[0078] To analyze compositions of the foraminifera-derived HAp of Example 1, the X-ray diffraction analysis (XRD; D8, Bruker AXS, Karlsruhe, Germany) using CuKa radiation was performed under conditions of a scanning rate of 0.02°/minute, voltage of 50 kV, and currency of 30 mA in a range of 10° to 80°. The ionic compositions of the sample was evaluated by X-ray fluorescence (XRF, Bruker) spectroscopy. The microstructure and surface morphology of the foraminifer-derived HAp particles were observed under vacuum by using a scanning electron microscope (SEM; EM-30, COXEM, Daejeon, Korea).

[0079] The SEM images obtained by observation are shown in FIGS. 1 and 2, and the XRD analysis results are shown in FIG. 3. The ionic compositions of the sample are shown in Table 1.

TABLE-US-00001 TABLE 1 Element Amount (%) Mg 2.98% Si 1.53% to 2.5% Sr 0.52% to 1.11%

[0080] FIG. 1 shows the SEM images of the structure of hydroxyapatite particles according to an embodiment. FIG. 2 shows the SEM images showing the measured size of the chambers and pores of hydroxyapatite particles according to an embodiment.

[0081] FIG. 3 shows comparison results of the XRD analysis of hydroxyapatite particles according to an embodiment with a control group HAp.

[0082] As shown in FIGS. 1 and 2, the SEM images of the pure HAp particles show densely packed HAp blocks and surface morphology with mostly non-micron-sized porosity. Meanwhile, hydroxyapatite according to an embodiment had a plurality of chambers separated by partition walls having pores, and thus had a macro-sized pore structure divided by the plurality of chambers that were interconnected inside. In addition, it was confirmed that the pores were uniformly distributed on the surface of the particles. In addition, no significant morphological change was found, and such morphological characteristics were maintained during the transformation into HAp.

[0083] In addition, as shown in FIG. 2, the HAp had about 53,300 uniform chambers per 1 cm.sup.2, and the size of the chamber was about 50 um*25 um. In addition, the size of the pore of the partition wall was ˜2 um.

[0084] As shown in FIG. 3, it was confirmed that the peak patterns after the hydrothermal reaction were consistent with hydroxyapatite according to an embodiment and commercially available pure HAp. These results indicate that foraminifera was successfully transformed into HAp by a hydrothermal reaction.

EXPERIMENTAL EXAMPLE 1

Cytotoxicity Measurement

[0085] To evaluate cytotoxicity of the foraminifera-derived HAp particles, the live/dead staining analysis was performed as described in Reference Examples, and results are shown in FIG. 4.

[0086] FIG. 4 shows the images of the cytotoxicity results measured from hydroxyapatite according to an embodiment.

[0087] As shown in FIG. 4, the survived hMSCs were stained green so that most of the cells present on the particles were stained green, whereas dead cells that were stained red were not observed. These results indicate that the foraminifera-derived HAp particles did not exhibit cytotoxicity.

EXPERIMENTAL EXAMPLE 2

Evaluation of Cell Proliferation

[0088] As described in Reference Examples, the cell proliferation of hMSCs in foraminifera-derived HAp was measured by using MTS mitochondrial activity-based assay. Results are shown in FIG. 5.

[0089] FIG. 5 is a graph showing the cell proliferation results measured from hydroxyapatite according to an embodiment.

[0090] As shown in FIG. 5, the hMSCs grew well over time in both pure HAp and foraminifera-derived HAp. In particular, after 1 day of the cell culture, the hMSCs cultured in foraminifera-derived HAp had an optical density value (0.25±0.10) that was significantly higher than an optical density value (0.07±0.01) of the hMSCs cultured in pure HAp. These results indicate that foraminifera-derived HAp may be more advantageous for initial cell adhesion than pure HAp. In addition, the optical density of the hMSCs with respect to the foraminifera-derived HAp particles was significantly higher than the optical density of pure HAp at all experimental time points.

EXPERIMENTAL EXAMPLE 3

Observation of Cell Adhesion and Cell Infiltration

[0091] By using the methods described in Reference Examples, the cell adhesion and infiltration of the hMSCs with respect to the pure HAp particles and the foraminifera-derived HAp particles were observed by using SEM on the 5th day of the cell culture, and results are shown in FIG. 6.

[0092] FIG. 6 is the SEM image showing the cell adhesion and infiltration results of hydroxyapatite according to an embodiment.

[0093] As shown in FIG. 6, the hMSCs were attached infrequently to the surface of the pure HAp particles only, and grew thereon, whereas a number of the hMSCs were attached to the surface of the foraminifera-derived HAp particles, compared to the hMSCs attached to the surface of the pure HAp particles. The infiltrated and attached hMSCs were also observed inside the chambers of the foraminifera-derived HAp particles. These results indicate that the particle structure of hydroxyapatite according to an embodiment provides a favorable environment for the cell adhesion.

EXPERIMENTAL EXAMPLE 4

Analysis of Osteoblast Differentiation Ability

[0094] To analyze the osteoblast differentiation ability, the ALP activity and qRT-PCR were performed as described in Reference Examples to detect osteoblast marker genes, such as ALP, Colla1, OCN, and BSP. Results are shown in FIGS. 7 and 8.

[0095] FIG. 7 is a graph showing the ALP activity analysis results of hydroxyapatite according to an embodiment.

[0096] FIG. 8 is a graph showing the expression levels of osteoblast marker genes in hydroxyapatite according to an embodiment.

[0097] As shown in FIG. 7, when the cells were cultured in the medium supplemented with the osteogenic stimulator, the ALP activity was measured with 19.59 ±3.06 nmol/mg of proteins in the foraminifera-derived HAp particles. This measurement was found to be approximately 3.4 times higher than the ALP activity of the cells cultured in pure HAp (measured with 5.69±0.68 nmol/mg of proteins).

[0098] Also, as shown in FIG. 8, the mRNA expression levels of ALP, Colla1, OCN, and BSP in the foraminifera-derived HAp particles were about 6.6 times, about 10.5 times, about 2.6 times, and about 16.5 times higher, respectively, than the mRNA expression levels thereof in the pure HAp particles.

[0099] These results suggest that hydroxyapatite according to an embodiment had significant osteoblast differentiation ability compared to pure hydroxyapatite.

EXPERIMENTAL EXAMPLE 5

Analysis of In Vivo Osteoanagenesis Ability

[0100] To evaluate in vivo osteoanagenesis ability of foraminifera-derived HAp prepared according to Examples above, the Micro-CT evaluation and histological evaluation were performed.

[0101] In detail, as described in Reference Examples, 3D micro-CT images were obtained at the 8th week after the graft, and results are shown in FIG. 9. On the 8th week after the graft, the bone defect site was stained according to the H&E staining and Goldner's Masson trichrome staining methods, and results are respectively shown in FIGS. 10 and 11.

[0102] FIG. 9 shows a figure (A) showing the micro-CT results at the 8th week of in vivo graft of hydroxyapatite according to an embodiment and a graph (B) showing quantified data thereof.

[0103] FIG. 10 shows the H&E staining results showing in vivo osteoanagenesis effects of hydroxyapatite according to an embodiment.

[0104] FIG. 11 shows the Goldner's Masson trichrome staining results showing in vivo osteoanagenesis effects of hydroxyapatite according to an embodiment.

[0105] As shown in FIG. 9A, in a control group (also referred to as an empty group which does not undergo graft), new bones were regenerated at the edge of the defect site. However, in groups where pure HAp and foraminifera-derived HAp were grafted, the generation of new bones was observed not only at the edge of the defect site but also inside the grafted HAp particles. In particular, the newly formed bones in the foraminifera-derived HAp-grafted group covered the surface of the defect site. In addition, as shown in FIG. 9B, the percent bone volume of foraminifera-derived HAp (31.54±3.61%) was significantly higher than that of the control group (or empty group) (8.43±0.52%) and pure HAp (21.18±2.61%).

[0106] Also, as shown in FIG. 10, it was observed that, in all the control group (also referred to as an empty group which does not undergo graft), the pure HAp-grafted group, and the foraminifera-derived HAp-grafted group, new bones were formed at the edge of the defect site. Here, in all the grafted groups, the newly formed bones and the defects tended to be merged. In particular, it was observed that, in the pure HAp-grafted group and the foraminifera-derived HAp-grafted group, new bones were formed at the edge of the defect site, and that the new bones were formed over the outer surface of the HAp particles, resulting in bone marrow cavity-like morphology. It was also found that the mature new bones were formed at a substantial thickness in the foraminifera-derived HAp-grafted group.

[0107] As shown in FIG. 11, it was observed that, in the pure HAp-grafted group and the foraminifera-derived HAp-grafted group, new formed bones were formed not only in the peripheral region, but also in the central region of the defect site. Meanwhile, in the control group (also referred to as an empty group which does not undergo graft), only loose connective tissues were observed in the central region of the defect site. When compared with other groups, the foraminifera-derived HAp-grafted group showed a significantly large amount of mature newly formed bones. Also, only in the foraminifera-derived HAp-grafted group, it was found that new bones were formed in a similar manner with ingrowth tissues inside the porous chamber. These results indicate that the foraminifera-derived HAp particles not only stimulated the formation of new bones, but also supported the infiltration of newly formed bones inside the chamber structures.