KR20180069664A - Method for preparing porous bone graft including coated multichannel biphasic calcium phosphate granule - Google Patents

Abstract

The present invention relates to a method for manufacturing a porous bone graft material, comprising the steps of: treating surfaces of porous multichannel biphasic calcium phosphate granules (MCG); and coating the surface-treated porous multichannel biphasic calcium phosphate granules with at least one material selected from a group comprising collagen, heparin and polar dopamine to modify the surfaces. The bone graft material is excellent in biocompatibility, cell proliferation, and bone regeneration through intercellular interactions between bone graft materials, thereby being used as an effective bone graft material.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention [0001] The present invention relates to a method for preparing porous bone graft-coated multichannel biphasic calcium phosphate granules,

The present invention relates to a method for preparing a porous bone graft material comprising a coated porous calcium phosphate granule.

Bone graft materials have been used in many clinical applications as an essential material for restoring and regenerating damaged bone. Autogenous bone, allogeneic bone, and xenogeneic bone graft are available, expensive, and have tissue rejection, infection as well as ethical problems. The ideal implantable bone graft material can be injected and formed easily into the defect and exhibits accelerated bone growth during the secretion of cells and body fluids.

Calcium phosphate-based bone graft materials such as hydroxyapatite (HAP), tricalcium phosphate (β-TCP) and biphasic calcium phosphate (BCP) Material. Such biomaterials have the advantage of significantly reducing the failure rate of implants by inhibiting inflammation, biodegradation, and other surgical complications. The HAP and β-TCP are biocompatible and osteoconductive. However, regular HAP is easily broken or relaxed due to its low resorbability, thus restricting stable bone formation. β-TCP is also limited in its use as a bone substitute due to its low mechanical properties combined with high reabsorption. Therefore, biphasic calcium phosphate (BCP) composed of HAP and β-TCP is the ideal bone graft material with the stability of HAP and the reactivity of β-TCP. It is believed that mixing with HA accelerates the formation of new bone by delaying the dissolution rate of β-TCP. BCP ceramics alleviate the disadvantages of using HAP and TCP separately, and have unique advantages such as strong mechanical strength and biodegradability do. In addition, pores interconnected with micropores of the synthetic bone can promote bone growth through fluid circulation.

On the other hand, the surface morphology of biomaterials has a great influence on the entire biocompatibility. Biochemical surface modification of biomaterials induces specific cell and tissue reactions by immobilization of various peptides, enzymes, extracellular matrix (ECM) Has been widely used. Accordingly, the present invention aims to develop a new coated bone graft material capable of promoting specific cell and tissue reaction by coating a bioactive substance, and promoting osteogenesis, bone joining, biocompatibility and natural healing process of bone tissue after transplantation .

Korean Patent No. 10-1427305

It is an object of the present invention to provide a method for the preparation of porous calcium phosphate granules, which comprises treating the surface of porous calcium phosphate granules (MCG) And coating the surface treated porous calcium phosphate dough with one or more materials selected from the group consisting of collagen, heparin and polar dopamine to modify the surface of the porous bone graft material.

The present inventors have found that after the surface of a porous calcium phosphate granule (MCG) is treated, the surface-treated porous calcium phosphate granules are coated with at least one substance selected from the group consisting of collagen, heparin and polar dopamine The porous bone graft material prepared by modifying the surface has excellent biocompatibility and has excellent cell proliferation and bone regeneration effect through intercellular interactions between bone graft materials and completed the present invention.

According to one aspect of the present invention, there is provided a method for preparing a calcium phosphate granule, comprising the steps of: treating a surface of a porous calcium phosphate granule (MCG); And coating the surface-treated porous ideal calcium phosphate granules with at least one material selected from the group consisting of collagen, heparin, and polar dopamine to modify the surface of the porous bone graft material.

The method for preparing a porous bone graft material according to the present invention includes the step of treating the surface of a porous polyphosphoric acid calcium phosphate granule (MCG).

The porous anhydrous calcium phosphate granules may be prepared by mixing hydroxyapatite (HAP), tricalcium phosphate (TCP), biphasic calcium phosphate (BCP), calcium pyrophosphate (CPC), dicalcium anhydride (DCPA), dicalcium dehydrate (DCPD) , Zirconia, and alumina.

In one embodiment of the present invention, the surface was treated with deionized water (DI), washed and dried for effective coating of the porous calcium phosphate granules surface to produce a bone graft material.

In one embodiment of the present invention, hydroxylapatite (HAP, ICDD No: 01-072-1243) and tricalcium phosphate (TCP, ICDD No: 01-072-7587) in porous BCP granules (MCG) These porous interconnected network structures have an irregular pore structure and have an average pore diameter size of about 175 μm from the volume analysis through Image J. In addition, .

The method for preparing a porous bone graft material according to the present invention includes the step of modifying the surface by coating the surface treated porous calcium phosphate granules with at least one material selected from the group consisting of collagen, heparin and polar dopamine.

Coating of bioactive materials on bone graft materials has a great effect on improving various functions such as solving the host's immune response and accelerating osseointegration. Collagen (C), the most abundant component of connective tissue, forms an extracellular matrix with low antigenicity, and collagen protein coating such as natural bone plays an important role in improving bioactivity of the implant surface. In addition, heparin (H) is a mixture of linear antigenic polysaccharides widely used in various blood contact sites as an anticoagulant. Polydopamine (PD) is a synthetic eumelanin polymer derived from dopamine. It is an excellent and flexible agent for surface coating of inorganic nanoparticles and contains high concentrations of amine groups.

In one embodiment of the present invention, the surface-treated porous abnormal calcium phosphate granules are coated with at least one material selected from the group consisting of collagen, heparin, and polar dopamine to modify the surface.

Specifically, the coating was prepared by immersing 1 g / ml of porous calcium phosphate granules (MCG) in a 17.5 mM acetic acid solution diluted with 4 mg / ml of collagen, or adding 1 g / ml of a 10% (w / v) ml of a porous calcium phosphate granule (MCG) or a 4 mg / ml dopamine hydrochloride solution was mixed with a 10 mM tris (hydroxymethyl) aminomethane solution. Calcium phosphate granules (MCG).

In a specific embodiment of the present invention, 4 mg / ml collagen was diluted in 17.5 mM acetic acid to prepare a 0.1% (w / v) collagen solution, 1 g / ml of porous BCP granules (MCG) W / V), and the mixture was shaken at room temperature for about 2 hours. Then, the BSA solution was removed. Porous BCP granules (MCG) were immersed in the collagen solution diluted in acetic acid to prepare collagen- To obtain granules (MCG).

The porous BCP granules (MCG) were immersed in EDC / NHS crosslinked solution after immersing 1 g / ml porous BCP granules (MCG) in a 10% (w / v) heparin solution and dipping them at 4 ° C for 12 hours The porous BCP granules (MCG) coated with heparin were obtained.

Further, a 4 mg / ml dopamine hydrochloride solution was mixed with a 10 mM tris (hydroxymethyl) aminomethane solution to prepare a polypodamine solution. 1 g / mL porous BCP granules (MCG) were immersed in the prepared poly , And porous BCP granules (MCG) coated with polydodamine.

In addition, in one embodiment of the present invention, SEM, EDX and FTIR spectra of the surface-modified porous abnormal calcium phosphate coated with at least one substance selected from the group consisting of collagen, heparin and polar dopamine were analyzed. As a result, It was confirmed that collagen, heparin and polar dopamine were uniformly coated on the whole surface without affecting the porous structure of BCP granule (MCG).

In addition, in one embodiment of the present invention, in the case of the porous modified calcium phosphate granules coated with one or more substances selected from the group consisting of collagen, heparin, and polar dopamine as described above, C-MCG , H-MCG, and PD-MCG, and cell viability was further increased with the lapse of time from day 1 to day 3, day 7.

In addition, in one embodiment of the present invention, bone regeneration ability of the femoral deficient part was evaluated, and it was confirmed that bone growth of collagen, heparin and polydodamine coated MCG was significantly increased after 6 weeks of transplantation.

In addition, in one embodiment of the present invention, the degree of bone ingrowth through the histological staining of five types of samples of the control group and MCG, C-MCG, H-MCG and PD- > C-MCG> H-MCG> It has excellent biocompatibility by confirming the effect of bone regeneration in the order of MCG, and can be used as an effective bone graft material because of its excellent cell proliferation and bone regeneration effect through bone graft material and cell interactions. Respectively.

The present invention relates to a method of treating a surface of a porous calcium phosphate granule (MCG); And coating the surface-treated porous calcium phosphate aphid granules with at least one material selected from the group consisting of collagen, heparin, and polar dopamine to modify the surface of the porous bone graft material. It is excellent and has excellent cell proliferation and bone regeneration effect through intercellular interactions between bone graft materials and can be used as an effective bone graft material.

Figure 1 shows the granular structure of porous BCP granules (MCG) and the morphological characteristics of EDX. 1 (a) is a SEM micrograph showing a low magnification, (b) a high magnification, and (c) a longitudinal transfer surface (high magnification). FIG. 1 (d) shows an XRD spectrum and FIG.
FIG. 2 shows morphological and chemical compositions of C-MCG, H-MCG and PD-MCG, which are surface modified porous BCP granules (MCG). Specifically, FIG. 2 shows SEM and EDX images of (a) C-MCG, (b) H-MCG and (c) PD-MCG.
FIG. 3 is an FTIR spectroscopic analysis for the chemical structure analysis of MCG, surface-modified C-MCG, H-MCG and PD-MCG surfaces. Figure 3 is a graphical representation of (a) MCG; (b) C-MCG; (c) H-MCG; (d) FTIR spectrum of PD-MCG.
Fig. 4 shows (a) cell viability after 1 day, 3 days and 7 days of MCG, C-MCG, H-MCG and PD-MCG; (b) shows a confocal microscope image after 7 days.
FIG. 5 shows a cut-out micro-CT 2D image of the femoral defect implanted with the defect (control) and MCG, C-MCG, H-MCG and PD-MCG after 3 and 6 weeks. Specifically, two-dimensional micro-CT images of MCG, C-MCG, H-MCG, and PD-MCG at 3 and 6 weeks after transplantation (red circle represents the borderline, Indicating a goal).
FIG. 6 shows the volume (BV / SV) percent of new bone formed at 3 weeks and 6 weeks after implantation calculated from the Micro-CT image of FIG.
FIG. 7 shows histological staining of five types of samples of the control and MCG, C-MCG, H-MCG, and PD-MCG to observe the degree of bone ingrowth. Histologic low-magnification images of control, MCG, C-MCG, H-MCG, and PDMCG by hematoxylin and eosin and Masson's Tricrome staining at 3 and 6 weeks after transplantation are shown.
Figure 8 shows histologically high-magnification images of MCG, C-MCG, H-MCG, and PDMCG via hematoxylin and eosin and Masson's Tricrome staining 6 weeks after transplantation.
FIG. 9 is a schematic view of C-MCG, H-MCG and PD-MCG prepared by coating porous BCP granules (MCG) and porous BCP granules (MCG) with collagen, heparin and polydodamine.
10 shows a process for producing collagen-coated porous BCP granules (MCG).
Figure 11 shows the process for preparing heparin coated porous BCP granules (MCG).
Figure 12 shows the process for preparing porous BCP granules (MCG) coated with polydodamine.

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings so that those skilled in the art can easily carry out the present invention. The present invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.

Example  1: Porous BCP granules ( multichannel  BCP granule, MCG ) Surface of  process

Surface treatment of porous BCP (biphasic calcium phosphate) granules (MCG) is very important for effective coating. The porous BCP granules (MCG) were then washed in deionized water (DI) and simultaneously shaken with a shaker at room temperature for about 2 hours. The washing step was repeated three or more times in each of the following types of coatings. The porous BCP granules (MCG) were frozen at -20 < 0 > C for 24 hours and lyophilized overnight. The lyophilized porous BCP granules (MCG) were autoclaved at 120 ° C and autoclaved porous BCP granules (MCG) were used for coating (FIG. 9).

Example  2: Porous BCP granules ( MCG ) With collagen coating

First, 4 mg / ml collagen was diluted in 17.5 mM acetic acid to prepare a 0.1% (w / v) collagen solution. Separately, a 1% (w / v) BSA solution was denatured at 80 ° C for about 2 hours. 1 g / ml of porous BCP granules (MCG) were first immersed in the denatured 1% (w / v) BSA solution and shaken at room temperature for about 2 hours in a shaker. Next, the BSA solution was removed, and porous BCP granules (MCG) were immersed in the collagen solution diluted in acetic acid. Porous BCP granules (MCG) immersed in the collagen solution were incubated at 4 占 폚 for 12 hours. To prepare the crosslinking solution, 4 mg EDC / 2.4 mg NHS was dissolved in 2 ml of 0.05 M MES buffer (pH 5.6). The collagen-coated porous BCP granules (MCG) were mixed with the dissolved EDC / NHS bridging solution. Finally, the mixed porous BCP granules (MCG) were dried and lyophilized for 12 hours to obtain collagen-coated porous BCP granules (MCG), which was named C-MCG (FIG.

Example  3: Porous BCP granules ( MCG ) With heparin coating

First, 1 g / ml porous BCP granules (MCG) were immersed in a 10% (w / v) heparin solution and immersed at 4 ° C for 12 hours. Subsequently, the porous BCP granules (MCG) coated with heparin were immersed in the EDC / NHS crosslinked solution, and then the same washing process as the previous one was performed. The washed heparin-coated porous BCP granules (MCG) were dried and lyophilized and dried for 12 hours to obtain heparin-coated porous BCP granules (MCG), which was named H-MCG (FIG. 11) .

Example  4: Porous BCP granules ( MCG )on Polydopamine  coating

A 4 mg / ml dopamine hydrochloride solution was mixed with a 10 mM tris (hydroxymethyl) aminomethane solution to prepare a polypodamine solution. 1 g / mL porous BCP granules (MCG) were introduced into the prepared polydopamine solution and mixed in a shaker for 24 hours to fix the polydodamine on the porous BCP granules (MCG). Then, the porous BCP granules (MCG) coated with washed polydodamine were dried and then lyophilized and dried for 12 hours to obtain porous BCP granules (MCG) coated with polydodamine And named PD-MCG (Fig. 12).

Experimental Example  1: Porous BCP granules ( MCG ) Analysis

Figure 1 shows the granular structure of porous BCP granules (MCG) and the morphological characteristics of EDX. 1 (a) is a SEM micrograph showing a low magnification, (b) showing a high magnification, and (c) showing a longitudinal transfer surface (high magnification). FIG. 2 shows an XRD spectrum and an EDX spectrum.

Referring to FIG. 1 (a), in a SEM at low magnification, porous BCP granules (MCG) exhibited a cylindrical porous structure with a diameter of 1 mm and a thickness of 80-120 μm. These porous interconnected network structures have irregular pore structures, and from the volume analysis through Image J, pore diameter sizes of about 175 μm on average were identified (FIG. 1A). A dense hemispherical porous structure was identified along with a distinguishable interface (G.B.) in the high magnification image of the selected area of FIG. 1A (FIG. 1b). Porous structures interconnected in the high-intensity longitudinal section of porous BCP granules (MCG) were identified (Fig. 1c). In addition, hydroxylapatite (HAP, ICDD No: 01-072-1243) and tricalcium phosphate (TCP, ICDD No: 01-072-7587) were commonly found in porous BCP granules (MCG) through X-ray diffraction patterns Fig. 1d). The presence of Ca, C, P and O elements was confirmed by analyzing the composition of the porous BCP granules (MCG) (Fig. 1e).

Experimental Example  2: Coated C- MCG , H- MCG  And PD- MCG  Surface analysis

FIG. 2 shows morphological and chemical compositions of C-MCG, H-MCG and PD-MCG, which are surface modified porous BCP granules (MCG). Specifically, FIG. 2 shows SEM and EDX images of (a) C-MCG, (b) H-MCG and (c) PD-MCG.

In Figure 2a, interconnected collagen fibers were identified at the top of the porous BCP granules (MCG). EDX analysis also confirmed Ca, N, P, C and O elements on collagen coated porous BCP granules (MCG) surface. No particular change in the surface of the porous BCP granules (MCG) due to the addition of heparin (Fig. 2b) and polydodamine (Fig. 2c) could be observed, but the surface contour after the surface modification became somewhat faint and the faded surface contour And polydopamine coatings. However, some fiber type precipitates were found on heparin (Fig. 2b) and polydopamine (Fig. 2c) coated surfaces. Quantitative analysis revealed that heparin was successfully coated as S and N elements appeared along with Ca, C, P, and O in H-MCG (Fig. 2b). In PD-MCG, it was also found that P dopamine was successfully coated as N element appeared along with Ca, C, P, and O (Fig. 2c). Surface-modified porous BCP granules (MCG), C-MCG, H-MCG and PD-MCG showed uniform and crack-free surface morphology. Thus, the coating of collagen, heparin and polydopamine did not significantly affect the porous structure of porous BCP granules (MCG). As the grain boundary interface disappears after each type of coating, it is confirmed that the coating agent is uniformly coated on the entire surface (Fig. 2a-c).

FIG. 3 is an FTIR spectroscopic analysis for the chemical structure analysis of MCG, surface-modified C-MCG, H-MCG and PD-MCG surfaces. Figure 3 is a graphical representation of (a) MCG; (b) C-MCG; (c) H-MCG; (d) FTIR spectrum of PD-MCG.

One non-nitride MCG is 960-1035 cm ^- in the range from ¹ PO ₄ ^3- coupler and 1106 cm ^-1 PO coupler (Fig. 3a). FTIR analysis of MCG showed a pattern consistent with hydroxyapatite (HAP) and tricalcium phosphate (TCP) identified in XRD of 1d. The absorbance of the collagen in the MCG was measured at 1644 cm ^-1 (amide I, C = O), 1538 cm ^-1 (amide II, NH stretching and CN deformation) and 1456 cm ^-1 (Fig. 3b). Through the FTIR spectrum of H-MCG, the S = O bond group of the saccharide group showing an absorption peak at 1240-1260 cm ^-1 and the -OH bond group at 3400 cm ^-1 were confirmed. A coating of heparin added on the MCG via some C═O and NH bond groups between 1626 cm ^-1 and 1410 cm ^{-1 was} also observed (Figure 3c). PD-MCG also clearly confirmed the coating of polydopamine through the FTIR spectrum. The high absorbance at 3379 cm ^-1 confirmed the presence of --OH and NH bonds in polydopamine and various NH bond groups in the range of 1500-1600 cm ^-1 . In addition, it was confirmed that peaks at 1627 cm ^-1 and 1296 cm ^-1 were respectively attributable to C═O and CO bond groups (FIG.

Experimental Example  3: Cell Survival  analysis

Fig. 4 shows (a) cell viability after 1 day, 3 days and 7 days of MCG, C-MCG, H-MCG and PD-MCG; (b) shows a confocal microscope image after 7 days. As a result, the cell viability was increased in C-MCG, H-MCG, and PD-MCG compared with MCG, and cell viability was further increased with the lapse of 3 days and 7 days compared with 1 day.

Experimental Example  4: Femoral defect Playability  Measure

FIG. 5 shows a cut-out micro-CT 2D image of the femoral defect implanted with the defect (control) and MCG, C-MCG, H-MCG and PD-MCG after 3 and 6 weeks. Specifically, two-dimensional micro-CT images of MCG, C-MCG, H-MCG, and PD-MCG at 3 and 6 weeks after transplantation (red circle represents the borderline, Indicating a goal).

FIG. 6 shows the volume (BV / SV) percent of new bone formed at 3 weeks and 6 weeks after implantation calculated from the Micro-CT image of FIG.

As a result, significantly improved bone formation was not observed in MCG and all samples 3 weeks after transplantation (Fig. 5). However, after 6 weeks, the space between the MCG and the new bone formed internally was clearly observed through the micro-CT image (Fig. It was confirmed that the bone regeneration ability of PD-MCG was superior to that of C-MCG and H-MCG from the new bone filled more around PD-MCG and internal space. On the other hand, H-MCG showed the lowest bone regeneration ability.

Experimental Example  5: Estimation of growth inside the bone

FIG. 7 shows histological staining of five types of samples of the control and MCG, C-MCG, H-MCG, and PD-MCG to observe the degree of bone ingrowth. Histologic low-magnification images of control, MCG, C-MCG, H-MCG, and PDMCG by hematoxylin and eosin and Masson's Tricrome staining at 3 and 6 weeks after transplantation are shown. Figure 8 shows histologically high-magnification images of MCG, C-MCG, H-MCG, and PDMCG via hematoxylin and eosin and Masson's Tricrome staining 6 weeks after transplantation.

Figure 7 shows the performance comparison for each sample transplanted for 3 weeks and 6 weeks at low magnification. The entire deficient area was marked with a rectangular border. There was no significant difference between the surface modified samples observed at low magnification but very good bone regeneration ability was observed compared with the control group. In order to compare MCG with surface modified MCG, the central zone (red border zone) was selected and cell migration to the defect and osteoconduction between the granules were observed. These results are shown in FIG. As shown in Fig. 8, bone conduction was evaluated by mature bone in the selected region (high magnification image). All results showed that the bone - healing ability of surface - modified MCG was higher than that of MCG. Primary osteogenesis at the site of the defect after 3 weeks was faster in PD-MCG than in MCG, C-MCG and H-MCG (Fig. 7). After 6 weeks, the mature bone tissue of the healing area was clearly shown at higher magnification in FIG. 8, and it was confirmed that PD-MCG showed the highest osseous fluidity among the high magnification images of the dyed samples (Fig. 8). The excellent bone formation of PD-MCG is due to effective delivery of bone induction signal. Therefore, MCG coated with polydodamine, which is an antibiotic, showed the highest potential among all the samples, which is considered to be the best candidates for bone graft materials. The bone marrow regeneration was confirmed by PD-MCG> C-MCG> H-MCG> MCG in order to synthesize iIn-vivo studies analyzed by micro-CT and histological staining.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments, Of the right.

Claims (6)

Treating the surface of the porous polycrystalline phosphate granule (MCG); And
Coating the surface-treated porous ideal calcium phosphate granules with at least one substance selected from the group consisting of collagen, heparin, and polar dopamine to modify the surface of the porous bone graft material. The method of claim 1, wherein the porous calcium phosphate granules are selected from the group consisting of hydroxyapatite, tricalcium phosphate, biphasic calcium phosphate, calcium pyrophosphate (CPC), dicalcium anhydrate (DCPA), dicalcium dehydrate (DCPD) Wherein the porous bone graft material comprises at least one selected from the group consisting of tetra calcium phosphate (TTCP), zirconia, and alumina. The method according to claim 1, wherein the porous calcium phosphate granules have a cylindrical porous structure with a diameter of 1 mm and a thickness of 80 to 120 占 퐉. The method according to claim 1, wherein the coating is prepared by immersing a porous calcium phosphate granule (MCG) having a porosity of 1 g / ml or more in a 17.5 mM acetic acid solution diluted with 4 mg / ml of collagen. The method of claim 1, wherein the coating is performed by immersing a porous calcium phosphate granule (MCG) having a porosity of 1 g / ml or more in a 10% (w / v) heparin solution. The method of claim 1, wherein the coating comprises 1 g / ml of porous calcium phosphate granules (MCG) in a polydodamine solution prepared by mixing a 4 mg / ml dopamine hydrochloride solution with a 10 mM tris (hydroxymethyl) Wherein the porous bone graft material is immersed in the porous bone graft material.

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