Bone graft material derived from foraminifera

Abstract

To provide a bone graft material which has a characteristic structure, has remarkable ability of cell proliferation, cell adhesion, and osteoblast differentiation, and includes a structure that can support newly formed bones.SOLUTION: A bone graft material comprises hydroxyapatite, where the hydroxyapatite comprises a plurality of chambers separated by partition walls, and the partition walls comprise a plurality of pores. Preferably, the hydroxyapatite is derived from foraminifera.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a bone graft material derived from foraminifera.

Background Art

[0002] Generally, when bone tissue is damaged due to trauma, deformity, physiological phenomena, etc., a guided bone regeneration (GBR) procedure is performed, in which a bone graft material is filled into the site, covered with a barrier membrane to prevent the intervention of soft tissue, and new bone is generated. The most common GBR methods for the recovery of such bone defects include an autograft method of harvesting and transplanting a part of the patient's own bone from another site, an allograft method of chemically treating and transplanting bone from another person, and a xenograft method of chemically treating and transplanting animal bone.

[0003] Generally, in the case of an autogenous cancellous bone (ACB) used as a bone graft material for the autograft method of harvesting and transplanting a part of the patient's own bone, there are advantages such as almost no immune rejection reaction, almost no bone resorption, and good osteoconductivity and osteoinductivity. In the case of an allograft bone graft material and a xenograft bone graft material used for the allograft method and the xenograft method, which utilize bone from another person or another animal, respectively, there is an advantage that no secondary surgery is required at a site other than the bone loss site of the graft recipient (the patient).

[0004] Allografts are obtained from corpses or other living donors. They are prepared by freezing or freeze-drying the graft material, demineralizing it, freeze-drying it, and then irradiating it to remove its antigenicity. While this process requires a longer time for bone formation and produces less new bone, it has the advantage of allowing for the use of any amount needed at any time without creating additional surgical sites. Types of allografts include non-demineralized freeze-dried bone allografts (DFDBA), demineralized bone allografts (FDBA), and irradiated cancellous bone (ICB).

[0005] Xenografts are graft materials that utilize bone harvested from animals such as cows and pigs, undergoing various processes to weaken the immune response and promote bone conduction. They have the advantage of not requiring further surgical sites and allowing for the use of the desired amount. However, because absorption and replacement take a long time, they are currently understood to rely more on bone conduction than bone induction. Examples of such xenografts include Bio-Oss, ABM / P-15, and BioCera. TM These are some examples.

[0006] Synthetic bone, being artificially synthesized rather than actual bone, is of the lowest quality compared to other bone graft materials and requires a longer bone formation period, but it has the advantage of being inexpensive. Examples include HAp (non-porous hydroxyapatite), hydroxyapatite cement, porous hydroxyapatite, and beta-type triphosphate. Calcium (beta tricalcium phosphate), PMMA (polymethlymethacrylate), H EMA (hydroxyethylmethacrylate) polymers and bioactive glass are used clinically. HA, PMMA, and HEMA polymers are non-absorbent, while tricalcium phosphate and bioactive materials are absorbent. Examples of such synthetic bones include tricalcium phosphate, hard tissue polymers, and viable glass ceramics. Hydroxyapatite (HAp) has been widely used as a bone substitute in bone grafts and dental devices. HAp possesses biocompatibility with osteoconductive properties, and its chemical composition in bioactive materials is similar to that of natural bone tissue. HAp is a type of calcium phosphate bioceramic and can be synthesized using chemicals containing calcium ions and phosphate ions as raw materials. Several reports have been published concerning the production of HAp ceramics from marine algae via hydrothermal reactions.

[0007] Therefore, in order to use them as bone graft materials, there is a need to develop materials with even higher cell proliferation and adhesion capabilities. [Overview of the project] [Problems that the invention aims to solve]

[0008] The problem that the present invention aims to solve is to provide a bone graft material that has a unique structure, possesses remarkable cell proliferation ability, cell adhesion ability, and osteoblast differentiation ability, and includes a structure that can support newly formed bone.

[0009] Another problem that the present invention aims to solve is to provide a method for manufacturing the bone graft material. [Means for solving the problem]

[0010] One embodiment provides a bone graft material containing hydroxyapatite, wherein the hydroxyapatite comprises a plurality of chambers separated by septa, and the septa contain a large number of pores.

[0011] The aforementioned hydroxyapatite is also derived from foraminifera (for example, from the exoskeleton of foraminifera).

[0012] The term "foraminifera" may refer to protozoa of the rhizopod class that possess a shell. Examples of the foraminifera may include the genera Globigerina, Camerina, Elphidium, Miyogipsina, or Baculogypsina. More specifically, the genus Baculogypsina may also include Baculogypsina sphaerulata, Baculogypsina bonarellii, Baculogypsina gallowayi, Baculogypsina lenticulate, Baculogypsina meneghinii, Baculogypsina saoneki, or Baculogypsina sphaerica.

[0013] As used herein, the term "bone graft" may refer to a material used for preserving bone defects, stimulating bone formation, joint fusion, joint stabilization, and preventing dislocation, and may also refer to a material used in bone grafting. Therefore, in this specification, the bone graft material is also used for the treatment of bone defects.

[0014] In this specification, bone graft material refers to, for example, the ethmoid bone, frontal bone, nasal bone, occipital bone, parietal bone, temporal bone, mandible, maxilla, zygomatic bone, cervical vertebra, thoracic vertebra, lumbar vertebra, sacrum, rib, sternum, clavicle, scapula, humerus, radius, ulna, carpal bones, and metacarpal bones. It can be applied to bones such as phalanges, ilium, ischium, pubis, femur, tibia, fibula, patella, calcaneus, tarsal bones, and metatarsal bones.

[0015] Furthermore, in this specification, bone graft material is also used in dentistry (dental implants), plastic surgery, or orthopedic surgery.

[0016] As used herein, the term "osteogenesis" refers to the function of inducing or promoting bone matrix formation and bone regeneration (osteoanagensis) by osteoblasts, and includes cartilaginous osteogenesis, connective tissue osteogenesis, and roll formation osteogenesis.

[0017] As used herein, the term "osteoconductive ability" refers to the function of attracting osteoblasts and inducing or promoting osteoblast formation of the bone matrix.

[0018] The term "bone induction ability" as used in this invention refers to the function of inducing regeneration in desired bone tissue by bringing only cells and substances useful for bone tissue regeneration close to the defect.

[0019] The terms “administer,” “introduce,” and “transplant” are used interchangeably and may mean the placement of a particular composition into an organism by a method or route that results in at least partial localization of the composition to a desired site. Administration may also occur by any suitable route that delivers at least a portion of the cells or cellular components of a particular composition to a desired site within a living organism.

[0020] In one specific example, the bone graft material also contains a plurality of hydroxyapatite particles. The size of the particles may be 50 to 4,000 μm, 50 to 3,000 μm, 50 to 2,000 μm, 80 to 2,000 μm, 100 to 4,000 μm, 100 to 2,000 μm, 100 to 1,500 μm, 100 to 1,200 μm, 100 to 1,000 μm, 100 to 700 μm, or 120 to 600 μm.

[0021] In one specific example, the diameter of the chamber may be 5 to 200 μm, 5 to 180 μm, 5 to 150 μm, 5 to 120 μm, 10 to 100 μm, 10 to 80 μm, 10 to 60 μm, or 15 to 60 μm. The cross-sectional area of ​​the chamber may be elliptical, and the diameter may refer to either the shorter or longer diameter of the ellipse.

[0022] In one specific example, the diameter of the pore may be 0.05 to 5 μm, 0.05 to 4.5 μm, 0.05 to 4 μm, 0.08 to 3 μm, 0.08 to 2 μm, 0.1 to 3 μm, 0.1 to 2 μm, or 0.2 to 2 μm.

[0023] In other specific examples, the thickness of the partition wall may be 1 to 50 μm, 1 to 45 μm, 1 to 40 μm, 2 to 40 μm, 4 to 40 μm, 4 to 30 μm, 5 to 30 μm, 5 to 20 μm, or 5 to 15 μm.

[0024] In other specific examples, 1 cm of the hydroxyapatite surface2 It may also have 20,000 to 100,000, 20,000 to 80,000, 25,000 to 80,000, 30,000 to 80,000, 30,000 to 750,000, 30,000 to 70,000, or 40,000 to 60,000 uniform chambers per hit.

[0025] In other specific examples, the hydroxyapatite particles may also contain magnesium ions at 0.5 to 10 weight% (atomic%), silicon ions at 0.2 to 10 weight% (at%), or strontium ions at 0.1 to 5 weight% (at%). In this specification, weight% indicates weight percentage, typically expressed as a weight percentage of the total weight. In one specific example, the hydroxyapatite is also produced by subjecting the exoskeleton of foraminifera to a hydrothermal reaction.

[0026] The term "hydrothermal synthesis" used in this specification also refers to a hydrothermal reaction and means a synthesis reaction of a substance carried out in the presence of high-temperature water or high-temperature and high-pressure water.

[0027] In the bone graft material according to this specification, the hydroxyapatite is also produced by subjecting the pretreated exoskeleton of foraminifera to a hydrothermal reaction at at least 30 °C, 50 °C, 80 °C or 100 °C or higher, for example, at 30 to 600 °C, 80 to 600 °C, 100 to 600 °C, 100 to 500 °C, 100 to 400 °C, or 100 to 300 °C for 2 to 40 hours, 2 to 30 hours, 10 to 40 hours, or 12 to 36 hours.

[0028] In other specific examples, in the bone graft material according to this specification, the hydroxyapatite is also produced by treating the pretreated exoskeleton of foraminifera with microwaves.

[0029] In one specific example, the bone graft material may or may not contain TCP (tricalcium phosphate).

[0030] The aforementioned "does not include" means "does not include in substance," and in this context, "in substance" means that it is included in an amount that does not affect the activity of the bone graft material.

[0031] In other specific examples, the bone graft material may also possess cell proliferation ability, cell adhesion ability, or osteoblast differentiation ability.

[0032] In one specific example, the transplant material may further include cells, such as somatic cells or stem cells.

[0033] In this specification, the term "stem cell" may mean an undifferentiated cell capable of differentiating into other cells. The stem cell may also be an embryonic stem cell, an adult stem cell, an induced pluripotent stem cell, or a mesenchymal stem cell. The mesenchymal stem cell may also be isolated from people of diverse tissues, diverse races, or diverse ages. For example, the mesenchymal stem cell may be derived from adipose tissue, placenta, umbilical cord blood, muscle tissue, corneal tissue, or bone marrow. Alternatively, for example, the mesenchymal stem cell may also be an adipose stem cell, a bone marrow stem cell, an umbilical cord blood stem cell, a neural stem cell, a placental stem cell, or an umbilical cord blood stem cell.

[0034] The bone graft material according to this specification may further contain a binding agent. In the present invention, the binding agent may mean a material that physically fixes, binds, or aggregates the hydroxyapatite, thereby preventing the bone graft material from immediately detaching from the bone defect when applied to the bone defect. Examples of such binding agents may include known biocompatible substances such as dental resin cement; glass ionomer cement; collagen-based glues; phosphate-based cements such as zinc phosphate and magnesium phosphate; zinc carboxylate; fibrin glues; and protein-based binders such as mussel-derived adhesive proteins.

[0035] The bone graft materials of this specification may further contain additives. These additives are also medically acceptable additional components added to prevent infection of the bone defect or to further enhance the bone formation, osteoconductivity, or osteoinduction capabilities of the bone graft material. Examples of such additives include drugs such as antiviral agents, antibacterial agents, antibiotics, anticancer agents, and angiogenic drugs; Polysaccharide aqueous solutions; amino acids; peptides; vitamins; cofactors involved in protein synthesis; growth hormones such as somatotropin; endocrine tissue fragments; enzymes such as collagenase, peptidases, and oxidases; living cells such as cartilage fragments, chondrocytes, and bone marrow cells; immunosuppressants; fatty acid esters; and nucleic acids; are included but not limited to these. A person skilled in the art can select one or more suitable additives depending on the site of the bone defect and the condition of the target.

[0036] The bone graft materials described herein are formulated by conventional methods into powders, suspensions, emulsions, ointments, or injections, and are applied to the bone defect site or surrounding area. In this specification, “dosage” means an amount sufficient to induce or promote bone formation or regeneration when applied to the target site where it is needed. The dosage of the bone graft material according to the present invention may vary depending on the patient’s weight, age, sex, health condition, and the extent of the bone defect, and can be appropriately selected by those skilled in the art.

[0037] Furthermore, this specification provides compositions for manufacturing bone graft materials.

[0038] The bone graft material composition according to this specification may further contain one or more selected from the group consisting of water, saline solution, sterile water, Ringer's solution, buffered saline, cyclodextrin, dextrose solution, maltodextrin solution, glycerol, ethanol, liposomes, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, polyvinylpyrrolidone, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, calcium carbonate, dextrin, propylene glycol, and liquid paraffin.

[0039] Another embodiment provides a method for manufacturing the bone graft material.

[0040] The method may include the steps of: pre-treating foraminifera; hydrothermally reacting the pre-treated exoskeleton at 120 to 400°C for 2 to 40 hours to produce hydroxyapite / or treating the pre-treated exoskeleton with microwaves to produce hydroxyapatite; and / or sintering the hydroxyapatite.

[0041] The pretreatment step may include washing the foraminifera; and / or adding the washed foraminifera to an aqueous solution containing a compound that can provide phosphoric acid.

[0042] Examples of compounds that can provide the aforementioned phosphoric acid include (NH4)H2PO4, H3PO4, Na3PO4, or Na2HPO4.

[0043] Furthermore, the step of carrying out the hydrothermal reaction to produce hydroxyapatite may include carrying out the hydrothermal reaction at a temperature of at least 30°C, 50°C, 80°C, or 100°C or higher, for example, 30 to 600°C, 80 to 600°C, 100 to 600°C, 100 to 500°C, 100 to 400°C, or 100 to 300°C for 2 to 40 hours, 2 to 30 hours, 10 to 40 hours, or 12 to 36 hours.

[0044] Furthermore, the step of processing with microwaves to produce hydroxyapatite may include processing with microwaves in the wavelength range of 300 MHz to 300 GHz at a power of 100 to 1,500 W for 0.5 minutes to 48 hours.

[0045] Furthermore, the sintering step may include a step of raising the temperature of the manufactured hydroxyapatite to 200 to 1,500°C; and / or a step of cooling. The heating step may also be performed at least multiple times. For example, the heating step may include raising the temperature to 400 to 800°C at a rate of 1 to 20°C / min, maintaining it for a certain period of time (e.g., 1 to 8 hours), and then further raising the temperature to 600 to 1,500°C at a rate of 1 to 20°C / min. The cooling may also proceed to at least room temperature. [Effects of the Invention]

[0046] According to one embodiment of a bone graft material derived from foraminifera, it possesses remarkable cell proliferation ability, cell adhesion ability, and osteoblast differentiation ability, and contains a structure that can support newly formed bone, thus having the effect of being useful as a bone graft material. [Brief explanation of the drawing]

[0047] [Figure 1A] These are scanning electron microscope images showing the structure of hydroxyapatite particles in one specific example: 1,000X, 10,000X. [Figure 1B]Scanning electron microscope images at 100X, 1,000X, and 3,000X showing the structure of a specific example of hydroxyapatite particles. [Figure 2] This is a scanning electron microscope image showing the size of the chamber and pore of hydroxyapatite particles in one specific example. [Figure 3] This figure shows the results of XRD diffraction analysis of hydroxyapatite particles using a specific example, compared to the control group HAp. [Figure 4] This image shows the results of a cytotoxicity measurement of hydroxyapatite using a specific example. [Figure 5] This graph shows the results of cell proliferation measurements using hydroxyapatite in a specific example. [Figure 6] This is a scanning electron microscope image showing the results of cell adhesion and cell invasion by hydroxyapatite in one specific example. [Figure 7] This graph shows the results of ALP activity analysis of hydroxyapatite using a specific example. [Figure 8] This graph shows the gene expression levels of hydroxyapatite, a specific example of a marker for osteoblastic cells. [Figure 9A] This diagram shows the results of a micro-CT scan 8 weeks after in vivo transplantation of hydroxyapatite, as an example. [Figure 9B] This graph quantifies the results of a micro-CT scan performed eight weeks after in vivo transplantation of hydroxyapatite, using one specific example. [Figure 10] This is a H&E staining result demonstrating the in vivo bone regeneration effect of hydroxyapatite in one specific example. [Figure 11] This is a Goldner's Masson trichrome staining result demonstrating the in vivo bone regeneration effect of hydroxyapatite in a specific example. [Modes for carrying out the invention]

[0048] The present invention will be described in more detail below with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

[0049] Reference example 1. Cell culture The biocompatibility of foraminifera-derived HAp was evaluated using human mesenchymal stem cells (hMSCs). Specifically, the cells were cultured in α-MEM (Gibco-BRL, MD; Gaithersburg, MD) containing 10% fetal bovine serum (FBS, Gibco-BRL) under conditions of 5% CO2 and 37°C. Then, in vitro experiments were performed using hMSCs that had been subcultured in 4 to 6 stages.

[0050] To induce osteoblast differentiation, cells were cultured in a medium supplemented with osteogenic stimulants (0.1 mM dexamethasone, 0.1 M β-glycerophosphate, and 50 μg / mL ascorbic acid). All compounds used in the medium containing the osteogenic stimulants were cell culture grade reagents (Sigma Aldrich, St. Louis, MO, USA). The normal growth medium and the bone formation stimulant-containing medium were replaced every two days during the experiment.

[0051] Before the cell culture experiment, HAp particles were first sterilized with 70% alcohol for 30 minutes and then washed three times with phosphate buffer solution (PBS) for 10 minutes each. After sterilization, 20 mg of sterile pure HAp and foraminifera-derived HAp particles were placed in a 48-well culture plate. Cells (5x10) 4 The cells were seeded (at a cell / well concentration) and incubated for 2 hours to allow the initial cells to attach to each HAp particle. The HAp particles with the attached cells were then transferred to new culture plate wells and cultured for in vitro experiments.

[0052] Reference Example 2. Cell Proliferation Cell proliferation was evaluated by mitochondrial activity-based analysis using CellTiter96® Aqueous One solution (MTS assay, Invitrogen, Carlsbad, CA, USA). As previously described, hMSCs were seeded and cultured for 1, 3, 6, 9, and 12 days. At predetermined time points, 50 μL of CellTiter96® reagent solution was mixed with 250 μL of normal medium and added to each well. After 4 hours of cell culture, the supernatant was collected and absorbance was measured at 490 nm using an ELISA plate reader (SpectraMAX M3, Molecular Devices, Sunnyvale, CA).

[0053] Reference Example 3. Cell viability and cytotoxicity Cell viability and cytotoxicity are measured using the Live / Dead® and Viability / Cytotoxicity kits. Evaluation was performed using a fluorescence staining method with Invitrogen (Carlsbad, CA, USA). According to the manufacturing protocol, cell-cultured HAp particles were washed with PBS buffer for 30 minutes. Next, the cells were stained with the kit's calcein AM (calcein acetoxymethyl ester) and EthD-1 (ethidium homodimer-1), and then observed with an inverse fluorescence microscope (DM IL LED Fluo, Leica Microsystems, Wetzlar, Germany).

[0054] Reference Example 4. In vitro evaluation of cell adhesion and cell invasion. Cells were cultured for 5 days to observe cell adhesion on the surface and cell infiltration into each HAp particle. The samples were washed with PBS buffer, fixed in 2.5% glutaraldehyde solution at 4°C for 2 hours, and then post-fixed with 0.1% osmium tetroxide solution. Next, the samples were dehydrated using a graded ethanol series (30%, 50%, 75%, 85%, 95%, 100%, each for 10 minutes). Then, they were sputter-coated with gold and observed with EM (EM-30). To observe cell infiltration, HAp particles were sliced ​​with a sharp lancet, the cross-sections were exposed, and then SEM analysis was performed.

[0055] Reference Example 5. Measurement of ALP (alkaline phosphatase) activity Osteocyte differentiation was evaluated by ALP (alkaline phosphatase) activity analysis. ALP activity analysis was performed using p-NPP (p-nitrophenylphosphate) as a substrate. Specifically, hMSCs were seeded onto particles and then cultured in a medium containing a bone formation stimulant. After 5 days of cell culture, adherent cells were dissolved by sonication in 1% Triton X-100 / PBS solution for 10 minutes under icebox conditions. To remove particles and debris, the sample was centrifuged at 12,000 rpm at 4°C. The supernatant was used for ALP activity analysis and protein concentration analysis. In this study, ALP activity was normalized by total protein content.

[0056] Reference Example 6. Real-time polymerization chain reaction To measure osteoblast differentiation in hMSCs cultured on HAp particles, several osteogenic marker genes, such as ALP, Col1αI (collagen type Iα1), OCN (osteocalcin), and BSP (bone sialoprotein), were measured using quantitative real-time polymerase chain reaction (RPCR). After culturing in a medium containing an osteogenic stimulant for 7 days, total mRNA was isolated from the cells, and cDNA was transcribed onto reverse transcriptase (Invitrogen) and oligo(dT) primers. The cDNA was amplified using TaqMan Universal PCR Master mix (Applied Biosystem) and primer & TaqMan probe sets associated with ALP (Hs01029144_m1), Col1αI (Hs00164004_m1), OCN (Hs01587814_g1), BSP (Hs00173720_m1), and 18S (Hsscience). All TaqMan PCRs were performed using the StepOne Plus RPCR system (Applied Biosystems, Foster City, CA, USA) and co-amplified with the 18S rRNA gene as an internal standard.

[0057] Reference Example 7. Animal Models Four adult male New Zealand white rabbits (2.5-3.0 kg), aged >3 months or more, were anesthetized using intramuscular doses of ketamine (35 mg / kg, Yu Han Yanghaang, Seoul) and xylazine (5 mg / kg, Bayer Korea, Seoul). Local anesthesia was then administered using a 2% lidocaine solution. After tissue excision, three isolated circular skull defects were created using trephine with an outer diameter of 6 mm. One defect was used as a control group, another was filled with pure HAp particles, and the third defect was filled with foraminifera-derived HAp particles. Eight weeks after transplantation, the healing process was observed, and the defects were incised from the host bone along with the surrounding bone. Before further analysis, the specimens were removed and placed in fixative (phosphate-buffered 4% paraformaldehyde solution, pH 7.2) at 4°C for 7 days.

[0058] Reference Example 8. Micro-CT Bone Analysis The newly formed bone was pathologically evaluated by microcomputed tomography (micro-CT) (Sky-Scan 1172™, Skyscan, Kontich, Belgium). Eight weeks after transplantation, the samples were analyzed using X-rays set up with a 0.5 mm aluminum filter, a voltage of 60 kV, and a current of 167 μA. Based on the micro-CT results, 3D reconstructed images were created using 3D software (CTVol, Skyscan). Observe the qualitative image of the defect and use image analysis software (CT-analyzer TM Using Skyscan, the bone volume ratio (BV) was calculated as follows.

[0059] BV(%) = (New bone volume) - (Residual graft volume) / Total defect volume Reference example 9. Histological analysis Histological analysis was performed 8 weeks after transplantation. Fixed rat skull specimens were decalcified with 8% formic acid / 8% HCl, followed by dehydration with a graded alcohol series (70-100%). Finally, the specimens were placed in paraffin. Rotary microtome (HM 325) was used. TM Using a Microm (Walldorf, Germany) microscope, samples were cut into 5 μm sections. Five sections from the center of each sample were stained with HE (hematoxylin-eosin) and Goldner's MT (Masson trichrom). Samples were then randomly selected and observed under a microscope for new bone formation (DMR, Leica, Nussloch, Germany).

[0060] Reference example 10. Statistical analysis Numerical values ​​are expressed as mean ± standard deviation (SD). Statistical analysis was performed using one-way analysis of variance (ANOVA), followed by Dunnett's post-hoc test using GraphPadPrism version 5.3 (GraphPad Software, San Diego, CA, USA); P<0.05 was considered statistically significant.

[0061] Examples: Production and analysis of hydroxyapatite particles 1. Production of hydroxyapatite (HAp) particles using foraminifera Hydroxyapatite was produced using foraminifera as follows.

[0062] First, the foraminifera (Baculogypsina sphaerulata, Okinawa) was purchased from the market. Residual contaminants To remove chemical and organic components, the samples were boiled in 4% sodium perchlorate (NaClO4) and then washed with distilled water. Specifically, the samples were added to an aqueous solution of monoammonium phosphate ((NH4)H2PO4) to achieve a Ca:P molar ratio of 10:6.

[0063] Next, the sample was placed in a Teflon-lined stainless steel pressure vessel and heated at 200°C for 24 hours. The converted sample was washed with boiling water and dried at 60°C. Then, the HAp particles were finely chopped with a lancet, and then particles in the 200-500 μm range were separated using a stainless steel sieve. Subsequently, the particles were subjected to a sintering process to promote crystallization. The sintering was carried out in an electric furnace (Muffle furnace, SH-FU-4MH), where the temperature was raised to 600°C at a heating rate of 5°C / min and maintained at 600°C for 2 hours. After that, the temperature was raised further to 800°C at a heating rate of 5°C / min and maintained at 800°C for 4 hours. After that, the temperature was lowered to room temperature and the sintering process continued.

[0064] For the control group, stoichiometrically synthesized HAp particles (pure HAp, particle size in the range of 200-500 μm) were obtained from Dio Implant Inc. (Busan, South Korea) and used.

[0065] 2. Chemical and morphological characteristics To analyze the foraminifera-derived HAp composition produced in Example 1 described above, X-ray diffraction analysis using CuKα radiation (XRD, D8, Bruker AXS, Karlsruhe, Germany) was performed under conditions of a scan speed of 0.02° / min, 50kV, and a depth of 30mÅ in the range of 10-80°. The ion composition of the sample was evaluated by X-ray fluorescence spectroscopy (XRF, Bruker). The microstructure and surface morphology of the foraminifera-derived HAp particles were observed under vacuum using a scanning electron microscope (SEM) (EM-30, COXEM, Daejeon, South Korea).

[0066] The scanning electron microscope images are shown in Figures 1 and 2, the XRD diffraction analysis results are shown in Figure 3, and the ion composition of the sample is shown in Table 1 below.

[0067] [Table 1]

[0068] Figure 1 is a scanning electron microscope image showing the structure of a hydroxyapatite particle in one specific example. Figure 2 is a scanning electron microscope image showing the measured chamber and pore sizes of a hydroxyapatite particle in one specific example.

[0069] Figure 3 shows the results of XRD diffraction analysis of hydroxyapatite particles using a specific example, compared with the control group HAp.

[0070] As can be seen from Figures 1 and 2, the SEM images of pure HAp particles show a tightly compressed HAp block and a surface morphology that is mostly non-micron sized porosity. On the other hand, hydroxyapatite, as a specific example, has multiple chambers separated by pore-containing septa, and possesses a macro-sized pore structure divided into numerous interconnected chambers. Furthermore, it was found that pores are uniformly distributed on the particle surface. In addition, no significant morphological changes were observed, and such morphological characteristics were maintained during the conversion to HAp.

[0071] Also, as can be seen from Figure 2, 1 cm 2 Each chamber had approximately 53,300 uniform chambers, each measuring approximately 50 μm x 25 μm. The pore size of the septa was approximately 2 μm.

[0072] As can be seen from Figure 3, the peak pattern after the hydrothermal reaction was found to be identical for hydroxyapatite from one specific example and for commercially available pure HAp. This means that the foraminifera was successfully transformed into HAp by the hydrothermal reaction.

[0073] Experimental Example 1: Measurement of Cytotoxicity To evaluate the cytotoxicity of foraminifera-derived HAp particles, live / dead staining analysis was performed as described in the reference example above, and the results are shown in Figure 4.

[0074] Figure 4 is an image illustrating the results of cytotoxicity measurements of hydroxyapatite using a specific example.

[0075] As can be seen in Figure 4, viable hMSCs are stained green, but most cells present on the particles are stained green, and no dead cells stained red are observed. Such results indicate that foraminifera-derived HAp particles do not exhibit cytotoxicity.

[0076] Experimental Example 2. Evaluation of Cell Proliferation As described in the reference example above, the mitochondrial activity-based assay (MTS) was used to measure the cell proliferation of hMSCs in foraminifera-derived HAp. The results are shown in Figure 5.

[0077] Figure 5 is a graph showing the results of cell proliferation measurements using hydroxyapatite in one specific example.

[0078] As can be seen in Figure 5, hMSC cells grow well over time in both pure HAp and foraminifera-derived HAp. In particular, after 1 day of culture, the optical density (0.25±0.10) of cells cultured with foraminifera-derived HAp was significantly higher than that of cells cultured with pure HAp (0.07±0.01). Such results suggest that foraminifera-derived HAp is even more advantageous than pure HAp for initial cell adhesion. Furthermore, the optical density of hMSCs associated with foraminifera-derived HAp particles was significantly higher than that of cells cultured with pure HAp at all experimental time points.

[0079] Experimental Example 3: Observation of Cell Adhesion and Cell Invasion Using the method described in the aforementioned reference example, cell adhesion and cell infiltration of hMSCs to pure HAp particles and foraminifera-derived HAp particles were observed by SEM on day 5 of cell culture, and the results are shown in Figure 6.

[0080] Figure 6 shows the results of cell adhesion and cell invasion by hydroxyapatite in one specific example. This is a scanning electron microscope image.

[0081] As shown in Figure 6, hMSCs rarely attach and grow on the surface of pure HAp. In contrast, a larger number of cells attach to the surface of foraminifera-derived HAp than to pure HAp. Furthermore, infiltrated and attached hMSCs were also observed within the chamber of foraminifera-derived HAp. Such results suggest that the particulate structure of hydroxyapatite, as exemplified by this example, provides a favorable environment for cell adhesion.

[0082] Experimental Example 4. Analysis of Osteocyte Differentiation Potential To analyze the osteoblast differentiation potential, ALP activity and qRT-PCR were performed as described in the reference example above, and osteoblast marker genes such as ALP, ColIa1, OCN, and BSP were detected. The results are shown in Figures 7 and 8.

[0083] Figure 7 is a graph showing the results of ALP activity analysis of hydroxyapatite in one specific example.

[0084] Figure 8 is a graph showing the expression levels of osteoblast marker genes for hydroxyapatite in one specific example.

[0085] As can be seen in Figure 7, when cells were cultured in a medium containing a bone formation stimulant, ALP activity was measured as 19.59 ± 3.06 nmol / mg of protein on foraminifera-derived HAp particles, which was approximately 3.4 times higher than that of cells cultured on pure HAp (5.69 ± 0.68 nmol / mg of protein).

[0086] Furthermore, as can be seen in Figure 8, the mRNA expression levels of ALP, ColIa1, OCN, and BSP on foraminifera-derived HAp particles were approximately 6.6 times, 10.5 times, 2.6 times, and 16.5 times higher, respectively, compared to pure HAp particles.

[0087] Such results mean that hydroxyapatite, as a specific example, possesses significantly greater osteoblast differentiation potential compared to pure hydroxyapatite.

[0088] Experimental Example 5. Analysis of In Vivo Bone Regeneration Potential To analyze the in vivo bone regeneration capacity of foraminifera-derived HAp produced in the aforementioned examples, micro-CT evaluation and histological evaluation were performed.

[0089] Specifically, as described in the reference example above, a three-dimensional micro-CT image was obtained 8 weeks post-transplant, and the results are shown in Figure 9. Also 8 weeks post-transplant, H&E staining and Goldner's Masson trichrome staining were performed on the bone defect site, and the results are shown in Figures 10 and 10, respectively. This is shown in 11.

[0090] Figure 9 shows a diagram (Figure 9A) and a graph (Figure 9B) illustrating the results of a micro-CT scan 8 weeks after in vivo transplantation of hydroxyapatite, as a specific example.

[0091] Figure 10 shows the H&E staining results illustrating the in vivo bone regeneration effect of hydroxyapatite in one specific example.

[0092] Figure 11 shows the in vivo bone regeneration effect of hydroxyapatite as a specific example, according to Goldner. This is the result of Masson trichrome staining.

[0093] As can be seen from Figure 9A, in the control group (empty group; no transplant), the deletion The figure shows the regeneration of new bone at the site margin. However, in the groups transplanted with pure HAp and foraminifera-derived HAp, new bone regeneration was observed not only at the defect margin but also within the transplanted HAp particles. In particular, in the foraminifera-derived HAp transplant group, the newly formed bone covered the surface of the foraminifera defect site. Furthermore, as can be seen from Figure 9B, the bone volume percentage of the foraminifera-derived HAp group (31.54±3.61%) was significantly higher than that of the control group (empty group) (8.43±0.52%) and the pure HAp group (21.18±2.61%).

[0094] Furthermore, as can be seen from Figure 10, in all groups—the control group (empty; no transplant), the group that received pure HAp transplants, and the group that received foraminifera-derived HAp transplants—new bone formation was observed at the edges of the defect sites, and in all transplanted groups, there was a tendency for the newly formed bone to merge with the defect. In particular, in the groups that received pure HAp and foraminifera-derived HAp transplants, new bone was observed to form at the edge of the defect site, and new bone was observed on the outer surface of the HAp particles, exhibiting a morphology similar to bone marrow cavity-like morphology. The matured new bone was found to be much thicker in the foraminifera-transplanted group.

[0095] Furthermore, as can be seen from Figure 11, in the groups to which pure HAp and foraminifera-derived HAp were transplanted, newly formed bone was observed not only in the peripheral areas but also in the central region. On the other hand, in the central region of the control group (empty group; no transplantation), loose bone was observed. Only connective tissue was shown. Furthermore, compared to other groups, the foraminifera-derived HAp transplantation group showed significantly greater and more mature bone formation. In addition, it was found that only in the foraminifera-derived HAp transplantation group did new bone form in a manner similar to ingrowth tissue within porous chambers. Such results indicate that foraminifera-derived HAp particles not only stimulate the formation of new bone but also support the infiltration of newly formed bone within these chamber structures.

Claims

1. A method for producing a bone graft material containing hydroxyapatite, wherein the hydroxyapatite comprises a plurality of chambers separated by partitions, the partitions comprising a plurality of pores, and the production method is The stage of pre-treating foraminifera, The process involves the steps of producing hydroxyapatite by hydrothermally reacting the pre-treated foraminiferal exoskeleton at 30 to 600°C for 2 to 40 hours, or by treating it with microwaves in the wavelength range of 300 MHz to 300 GHz at a power of 100 to 1,500 W for 0.5 minutes to 48 hours, The steps include sintering the hydroxyapatite and Includes, The sintering step is as follows: The bone graft material contains a plurality of hydroxyapatite particles, the size of the particles is 100 to 4,000 μm, the diameter of the chamber is 15 to 60 μm, the thickness of the partition is 5 to 15 μm, the diameter of the pore is 0.2 to 2 μm, and the bone graft material has a hydroxyapatite surface of 1 cm ２ The process includes the steps of: heating the hydroxyapatite to 400 to 600°C at a rate of 1 to 20°C / min so that it has 40,000 to 60,000 chambers per unit, and then maintaining the temperature for 1 to 8 hours; then heating the hydroxyapatite to over 600°C and up to 800°C at a rate of 1 to 20°C / min, and then maintaining the temperature; and then cooling it to room temperature. A method for manufacturing bone graft material.

2. The method for producing bone graft material according to claim 1, wherein the foraminifer is Baculogypsina sphaerulata, Baculogypsina bonarellii, Baculogypsina gallowayi, Baculogypsina lenticulate, Baculogypsina meneghinii, Baculogypsina saoneki, or Baculogypsina sphaerica.

3. The method for producing a bone graft material according to claim 1, wherein the hydroxyapatite particles contain 0.5 to 10% by weight of magnesium ions, 0.2 to 10% by weight of silicon ions, or 0.1 to 5% by weight of strontium ions.

4. The method for manufacturing a bone graft material according to claim 1, wherein the bone graft material is for the treatment of bone defects.

Images