KR20160020298A - Method for Preparing Osteoinductive Material Loaded Biphasic Calcium Phosphate Granule/Scaffold - Google Patents

Abstract

The present invention relates to a manufacturing method of a biphasic calcium phosphate (BCP) particle/scaffold loaded with an osteogenesis-inducing factor and, more specifically, to a scaffold or a particle in which an osteogenesis-promoting material is fixated on the surface of the BCP particle or scaffold for promoting osteogenesis, and to a manufacturing method thereof. According to the present invention, the composition is capable of continuously discharging the osteogenesis-promoting material for a long period of time due to a biphasic structure of the BCP particle or scaffold, continuously promoting the differentiation of osteoblasts, and improving the osteogenesis of the bone-defective region.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention [0001] The present invention relates to a method for preparing a calcium phosphate particle / support having a double-

The present invention relates to a method for producing a calcium phosphate particle / support having a double-pore structure on which an osteogenesis inducing factor is loaded. More particularly, the present invention relates to a method for producing a calcium phosphate particle / support having bipolar calcium phosphate (BCP) The present invention relates to a fixed particle for promoting bone formation or a support and a method for producing the same.

Bones are a characteristic feature of vertebrate animals and are one of living tissues in vivo. The bone not only protects the organs, but also acts as a hematopoietic cell that produces blood cells that transport nutrients and waste materials necessary for each tissue throughout the body, and accumulates calcium and minerals.

The cells constituting the bone include osteocytes that maintain the homeostasis of bones, osteoblasts that form bones, osteoclasts that absorb bones, and cartilage cells that play a joint function of the distal ends of bones Cells (chondrocyte) and so on. In bone growth, osteoblasts actively form bones, and after growth, bone formation and bone resorption are remodeling.

If the balance between the bone formation and the bone resorption is broken, it can cause bone disease if it is deviated to either side, which can cause serious symptoms and can result in intractable. Intractable bone disease includes non-union fractures that are not associated with osteoporosis and diabetes, osteogenic deficiency due to inherent bone defects, osteomalacia caused by lack of mineral deposits in bone matrix, And bone defects due to accidents.

Problems are also raised in the case of autografting, allografting, and artificial bone grafting, which are proposed as conventional treatment methods for such bone defects. Autogenous bone grafting has complications such as infection, pain or hematoma at the site of bone harvesting. Allogeneic bone graft has the problem of possibility of disease transmission from the donor and has a disadvantage that bone union and osteogenesis effect is poor. Artificial bone grafts do not produce fundamental bone formation, take a long time to regenerate bone, and have a problem that fusion with surrounding bone tissue is difficult.

Recently, a treatment method proposed as an alternative to the conventional treatment method is a method of implanting a biodegradable carrier implanted with osteoinductive protein at the wound site. Since the osteophilic protein, which is a protein capable of inducing bone regeneration or osteogenesis, has a short in vivo activity period, administration of a large amount of recombinant protein is required to achieve therapeutic effect. Therefore, the above-mentioned treatment has a disadvantage in that not only a safety problem such as side effects due to excessive administration of the recombinant protein, but also a therapeutic effect can not be guaranteed.

To overcome these problems, recently, methods have been developed in which osteoinductive proteins are attached to porous supporters made of collagen or calcium phosphate to implant bone defect sites (Toker, H. et al ., Oral surgery, oral medicine, oral pathology and oral radiology 114: S146,2012; US 2012/0142641), the strength of the support and the effect of continuous release of the osteogenesis inducing substance in the bone defect site are not sufficient and only the transient effect is exhibited.

Thus, the present inventors have made intensive efforts to develop a support on which a bone formation inducing substance, which continuously releases bone formation inducing substance and exhibits continuous osteogenesis inducing effect, is mounted. As a result, it has been found that calcium phosphate particles having a double- / Fixing the alendronate to the support, the alendronate exhibits a sustained release ability, and it is confirmed that the osteoblast differentiation is improved, and the present invention has been completed.

It is an object of the present invention to provide a particle or a support on which a bone formation inducing substance is continuously and slowly released.

It is another object of the present invention to provide a method for producing a particle or a support on which a bone formation inducing substance is continuously and slowly released.

In order to accomplish the above object, the present invention provides a biphasic calcium phosphate (BCP) particle having a double pore structure or a particle for promoting osteogenesis or a support having an osteogenesis promoting substance immobilized on the surface of the support.

(A) mixing and fixing calcium phosphate particles having a double pore structure or a solution containing a bone formation promoting substance to a support; And (b) a step of obtaining a calcium phosphate particle or a support having a double-pore structure in which a bone formation promoting substance is immobilized, comprising the steps of: (a) forming a biphasic calcium phosphate (BCP) Wherein the promoting substance is immobilized, and a method for producing a particle for promoting bone formation or a support.

According to the present invention, osteoporosis-promoting material can be continuously released for a long period of time owing to the double pore structure of the calcium phosphate particles or the support, thereby promoting continuous osteoblast differentiation, thereby improving osteogenesis of bone defect sites.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a scanning electron micrograph of BCP particles loaded with alendronate. FIG.
FIG. 2A shows chemical elemental analysis of the surface using an X-ray photoelectron spectrometer of BCP particles and Alendronate-fixed BCP particles < Aln (0.1, 0.5 mg) / BCP, B shows BCP and Alendronate- The results of the chemical elemental analysis of the surface using an X-ray photoelectron spectrometer of the support <Aln (0.1, 1 mg) / BCP are shown.
FIG. 3 is a graph showing drug release behavior of BCP particles (A) and support (B) loaded with alendronate over time.
FIG. 4 shows the results of measurement of cytotoxicity of BCP particles loaded with alendronate on adipose stem cells after culturing for 24 hours and 48 hours.
FIG. 5 shows the results of measurement of cell proliferative capacity of adipocyte-loaded BCP particles on adipose stem cells after culturing for 24 hours and 48 hours.
FIG. 6 shows the result of measuring the alkaline phosphatase activity of osteoblast according to the treatment time of the BCP support loaded with alendronate.
Fig. 7 shows the results of bone morphogenesis and calcium deposition of adipose stem cells according to treatment of BCP particles (A) and supporters (B) loaded with alendronate.
FIG. 8 shows the expression rate of osteogenic markers of adipose stem cells according to treatment with BCP particles loaded with alendronate.
FIG. 9 shows the expression rate of bone morphogenetic markers in adipose stem cells according to treatment with BCP support loaded with alendronate.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

In one aspect, the present invention relates to a biphasic calcium phosphate (BCP) particle having a double-pore structure, or a particle for promoting osteogenesis or a support having an osteogenesis promoting substance immobilized on the surface of the support.

Biphasic calcium phosphate (BCP, Ossgen, Korea) used in the present invention is a bone filler used in the field of dentistry and orthopedics, and has a pore structure of 200-500 m in size, It is a porous granular bone filler having a microporous structure of 5 mm. It is composed of HAp (hydroxyapatite) and TCP (tricalcium phosphate) and is similar to the natural bone mineral. It is also the most suitable support for bone tissue regeneration.

In the present invention, the BCP particle is a BCP structure having a macro-sized pore structure of 200 to 500 μm and a micro-sized pore structure of 500 nm to 5 μm, a total particle size of 0.3 to 0.5 mm, It is a BCP structure with a macro-sized pore structure of 400 μm and a micro-sized pore structure of 1 to 6 μm mixed, with a total size of 3 to 8 mm.

In the present invention, the double pore structure of the BCP particles or the support is a structure in which TCP (tricalcium phosphate) and HAp (hydroxyapatite) are mixed at a ratio of about 6: 4.

In the present invention, the calcium phosphate particles having a double-pore structure or the bone formation-promoting substance which can be fixed to the support include bisphosphonate alendronate, risedronate, zoledronate, etidronate, (BMP), angiogenic factor (VEGF), platelet-derived growth factor (VEGF), and platelet-derived growth factor (VEGF) (IGFBP), fibroblast factor (FGF), insulin-like growth factor (IGF), etc. and can be antibiotics tobramycin, gentamicin, vancomycin, etc., .

In one embodiment of the present invention, the alendronate used as the bone formation promoting substance is an amino-bisphosphonate-based drug. It inhibits osteoclast to prevent osteoporosis and has a therapeutic effect. It inhibits the formation of osteoclast by decreasing RANKL on the osteoblast surface And increase the proliferation and differentiation of osteoblasts.

In the present invention, the calcium phosphate having the double pore structure may have pores having a size of 200 to 500 mm and pores having a size of 500 nm to 5 mm.

Since the double pore structure of the particle for promoting bone formation or the support of the present invention is connected without breaking the pores of two sizes, when the bone is implanted in the bone defect site, the cells attached at the early stage do not die out, It is an easy structure and it is easy to supply nutrients of osteoblasts, so that osteoblasts are formed by the metabolic action of osteoblasts inside the granules, and at the same time, pores on the surface improve adhesion of osteoblasts. In addition, the osteogenesis promoting substance immobilized deeply inside the pores is slowly released to continuously improve the growth of osteoblasts.

In the present invention, the bone formation promoting material may be contained at a concentration of 0.01 to 10 mg / mL.

In another embodiment of the present invention, the adipose stem cells are differentiated into osteoblasts using a supporter on which BCP particles and alendronate loaded with 0.1 mg / ml and 0.5 mg / ml of alendronate are loaded at 0.1 mg / ml and 1 mg / ml As a result, expression of early osteoblast differentiation markers ALP, RUNX-2 and late osteoblast differentiation markers OCN (osteoncalcin) and OPN (osteopontin) were expressed, and expression of early osteoblast differentiation markers and expression of late osteoblast differentiation markers All of them were improved than the control group.

Therefore, it can be confirmed that the bone formation-promoting particle or support of the present invention continuously improves the differentiation ability until the late differentiation of osteoblast, and the osteogenesis promotion is continuously performed.

According to another aspect of the present invention, there is provided a method for preparing a calcium phosphate particle, comprising the steps of: (a) mixing and fixing calcium phosphate particles having a double pore structure or a solution containing a bone formation promoting substance to a support; And (b) a step of obtaining a calcium phosphate particle or a support having a double-pore structure in which a bone formation promoting substance is immobilized, a biphasic calcium phosphate (BCP) particle having a pore structure, The present invention relates to a particle for promoting bone formation or a method for producing a support.

In one embodiment of the present invention, BCP (BCP, Ossgen, Korea) particles and 0.1 mg / ml alendronate (Samjin Pharm, Korea) at a concentration of 1 mg / BCP particles were added to a 0.1 M MES buffer (pH 5.6), stirred overnight, washed with distilled water three to four times, and dried to prepare BCP particles and a support on which alendronate was mounted.

In another aspect, the present invention relates to a therapeutic composition for bone tissue regeneration containing the above bone formation promoting particles or a support.

The therapeutic composition of the present invention facilitates the supply of nutrients to the osteoblast cells attached to the inside of the double pores, thereby enhancing osteoblast differentiation and proliferation, thereby improving bone regeneration ability.

The therapeutic composition of the present invention may further comprise alginate, HA, CMC.

In the present invention, the therapeutic composition may be a transplantable preparation or an injectable preparation.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

Example 1 Immobilization of Alendronate on BCP (Biphasic calcium phosphate) Particle / Support Surface

0.1 M MES buffer containing 0.1, 0.5 and alendronate (Samjin Pharm, Korea) at a concentration of 1 mg / ml at the maximum (BCP, Ossgen, Korea) pH 5.6), stirred overnight, washed 3-4 times with distilled water, and dried to obtain BCP particles and a support on which alendronate was mounted.

Example 2: Surface observation of BCP particles / support

Fig. 1 shows the results of observation of BCP particles and BCP loaded with alendronate prepared in Example 1 by using a scanning electron microscope. Fig. 1 shows the results of (a, e) BCP particles (b, A 300-fold magnification and a 1000-fold magnification image of each of the modified BCP particles (c, g) at 0.5 mg / ml and (d, h) 1 mg / There was no difference in electron microscope between BCP and alendronate.

As shown in FIG. 2 and Tables 1 and 2, the BCP particles (Table 1) and the support (Table 2) loaded with BCP and alendronate were subjected to surface element analysis using x-ray photoelectron spectroscopy. As a result, unmodified BCP particles 0.6%, 1.5%, 0.6%, 3.3% phosphorus increase in the BCP particles / supports loaded with 0.1 mg / ml, 0.5 mg / ml and 1 mg / ml alendronate, respectively, It was confirmed that the alendronate was successfully mounted on the surface of the BCP particles.

Example 3. Drug Release Behavior of Alendronate in BCP Particles

In order to analyze the release behavior of alendronate from the alendronate-loaded BCP particles / support over time, the alendronate-loaded BCP particles / support prepared in Example 1 were placed in 1 ml of PBS buffer (pH 7.4) The mixture was stirred at 100 rpm.

The new buffer was exchanged at 1, 3, 5, 10 hours and at 1, 3, 5, 7, 14, 21 and 28 days. The released alendronate was analyzed by infrared and visible spectrophotometry (DU-530, Beckman Coulter ^TM , USA). The absorbance at 293 nm was measured and analyzed.

As a result, as shown in FIG. 3, it was confirmed that the alendronate-loaded BCP particles / support emitted alendronate slightly more rapidly in the first day, but then continuously released alendronate for 4 weeks thereafter.

Example 4. Evaluation of cytotoxicity of BCP particles and modified BCP particles

To evaluate the cytotoxicity of BCP particles loaded with BCP particles and alendronate on adipocyte stem cells, the supernatant was cultured in a cell culture medium (DMEM, high glucose, Gibco, USA) at 37 ° C for 24 hours, .

1 x 10 ⁴ mesenchymal stem cells (K-stemcell, Korea # LM081011) were inoculated into 96-well plates and cultured at 37 ° C for 24 hours.

The culture medium of the cultured adipose stem cells was removed, washed with PBS buffer, and the extracted culture medium of BCP particles loaded with alendronate was added. After osteoblasts were cultured for 24 hours and 48 hours, the extract medium was removed . For cytotoxicity, CCK-8 proliferation kit reagent was added and incubated at 37 ° C for 1 hour, and absorbance was measured at 450 nm.

As a result, as shown in FIG. 4, the BCP and the modified BCP particles showed cell viability of 90% or more even after 48 hours of culture, indicating that both the BCP and the modified BCP particles were in the osteogenic differentiated adipose stem cells This means that there is no cytotoxic effect on the cells.

Example 5. Cell proliferation of BCP particles and modified BCP particles

To evaluate the cell proliferation of BCP particles loaded with BCP particles and alendronate on adipose stem cells, 1 10 ⁵ adipose stem cells were placed in a 24-well plate and transwell (6.5 mm with 8.0 After the cells were incubated for 1 day, 3 days, and 7 days, BCP particles and allendronate-loaded BCP particles were carefully placed on the cells. And washed with PBS buffer. After washing, CCK-8 proliferation kit reagent (Cell Counting kit-8, Dojindo, Japan) was added and incubated for 1 hour.

As a result, as shown in FIG. 5, the adipose stem cells cultured in the BCP particles loaded with the BCP particles and the alendronate gradually proliferated after 1 day, 3 days, and 7 days, The difference in proliferation was confirmed to effectively increase the loading of BCP particles at a concentration of 0.5 mg / ml of alendronate at 7 days.

Example 6 Alkaline phosphatase (ALP) activity of a BCP support and a modified BCP support

By measuring and analyzing the alkaline phosphatase activity, the early differentiation marker of osteoblast, BCP particles / support and alendronate - loaded BCP scaffolds were evaluated for their osteointegration efficacy.

1 × 10 ⁵ adipose stem cells were placed in a 24-well plate, transwell (6.5 mm with 8.0 μm Pore PC Membrane Insert, 24 wells / case, Corning, Hong Kong) And alendronate-loaded BCP scaffold were carefully added and cultured for 3 days, 7 days, and 10 days.

After incubation, the cells are washed with PBS buffer, and 1RIPA buffer is added to the cells and sonicated at 0 to 110 watts for 1 minute. The lysed cells were centrifuged at 13,500 rpm for 3 minutes at 4 ° C. After centrifugation, the supernatant was incubated with the p-nitrophenylphosphate solution at 37 ° C for 30 minutes, and then the reaction was terminated with 500 ml of 1 N NaOH. The absorbance at 405 nm was then measured.

As a result, as shown in Fig. 6, the alkaline phosphatase activity on the osteoblast differentiated into osteoblasts in the alendronate-loaded BCP scaffold was increased in comparison with the BCP particles.

Example 7. Calcium deposition of BCP particles / support and modified BCP particles / support

Calcium deposition, a late differentiation marker of osteoblast, was measured to evaluate the osteoblastic effect of BCP particles / alendronate loaded BCP particles / support. 110 After ^five fat stem cells, 24-well plate division, where the fat stem cells dispensed place the transwell (6.5 mm with 8.0m Pore Membrane Insert PC, 24 wells / case, Corning, HongKong) BCP particles thereon / Support and alendronate-loaded BCP particles were carefully placed and cultured for 21 days. After incubation, cells were washed with PBS buffer and cells were carefully removed from each BCP particle / support with Tris-EDTA solution. The detached cells were centrifuged for 1 minute at 13,500 rpm, and 0.1% Triton X-100 solution was added to the cells. Ultrasonic wave is applied for about 1 minute at 0 degree to crush the cell membrane, and centrifugation is performed. The supernatant was separated and analyzed for absorbance at 612 nm using the QuantiChrom ™ Calcium Assay kit.

As a result, as shown in FIG. 7, the degree of calcium deposition of the BCP particles / supports fixed with the BCP particles / supports and the 0.1 mg / ml alendronate was similar, and statistically no difference was observed. However, the BCP particles / support immobilized with 0.5 mg / ml alendronate showed significantly higher calcium deposition than the BCP particles / support.

Example 8. Gene expression analysis of cells from BCP particle / support and modified BCP particle / support

The effect of BCP particles / support and alendronate-fixed BCP particles / support on osteoblast function was assessed by measuring and analyzing the genes that are late differentiation markers of osteoblasts.

1x10 ⁵ adipose stem cells were carefully placed on each group of BCP particles and differentiated and cultured for 21 days.

After incubation, the cells are washed with PBS buffer and carefully removed from each BCP particle / support with Tris-EDTA solution. The detached cells were centrifuged at 13,500 rpm for 1 minute, and Trizol reagent solution and chloroform were added to the cells. Ultrasonic waves were applied at 4 degrees for about 1 minute to separate the cells, followed by centrifugation. The supernatant was separated and the RNA concentration was measured on a NanoDrop 260/280 nm using an RNeasy Mini kit (RNeasy mini kit; Qiagen, Doncaster, VIC, Austraila ) . Separated RNA was analyzed by cDNA synthesis and real-time PCR using AccuPower RT-PCR PreMix (Bioneer, Korea).

As a result, as shown in FIG. 8 and FIG. 9, the expression of early and late osteogenic differentiation increases with increasing BCP particle / support and alendronate concentration, similar to the ALP activity and calcium deposition concentration tendency Respectively.

In addition, the genes for early differentiation markers (ALP, RUNX-2: Runt-related transcription factor-2) and Osteoncalcin (OPN: Osteopontin) genes were BCP particles, BCPs fixed with alendronate at 0.1 and 0.5 mg / (* P <0.001). However, in the BCP scaffolds immobilized with 0.1 and 1 mg / ml Alendronate at 21 days of differentiation and cultivation, Runx-2, an early differentiation marker, There was no significant difference when compared with the particles.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the invention is not limited thereby. It will be obvious. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (10)

Biphasic calcium phosphate (BCP) particles having a double-pore structure or an osteogenesis promoting substance immobilized on the surface of a support.
2. The method of claim 1, wherein the osteogenesis promoting material is selected from the group consisting of alendronate, risedronate, zoledronate, etidronate, choloronate, tyluronate, pamidronate, (BMP), angiogenic factor (VEGF), platelet derived growth factor (PDFG), fibroblast factor (FGF), insulin like growth factor (IGF), tobramycin, gentamicin and vancomycin Gt; and / or &lt; / RTI &gt;
The particle or support for promoting bone formation according to claim 1, wherein the calcium phosphate having a double pore structure has a pore size of 200 to 500 mm and a pore size of 500 nm to 5 mm.
2. The bone formation promoting particle or support according to claim 1, wherein the bone formation promoting substance is contained at a concentration of 0.01 to 10 mg / mL.
(BCP) particles having a double-pore structure including the following steps, or a method for producing a particle for promoting osteogenesis or a support having an osteogenesis promoting substance immobilized on the surface of the support:
(a) mixing and fixing calcium phosphate particles having a double-pore structure or a solution containing a bone formation promoting substance to a support; And
(b) obtaining calcium phosphate particles or a support having a double-pore structure in which an osteogenesis promoting substance is immobilized.
6. The method of claim 5, wherein the osteogenesis promoting material is selected from the group consisting of alendronate, risedronate, zoledronate, etidronate, coroloate, tyluronate, pamidronate, (BMP), angiogenic factor (VEGF), platelet derived growth factor (PDFG), fibroblast factor (FGF), insulin like growth factor (IGF), tobramycin, gentamicin and vancomycin &Lt; / RTI &gt;
6. The method of claim 5, wherein the calcium phosphate having a double-pore structure has a pore size of 200 to 500 mm and a pore size of 500 nm to 5 mm.
6. The method according to claim 5, wherein the concentration of the solution containing the bone formation promoting material is 0.01 to 10 mg / mL.
A therapeutic composition for bone tissue regeneration comprising the bone formation promoting particles or the support according to any one of claims 1 to 4.
The therapeutic composition according to claim 9, further comprising a substance selected from the group consisting of alginate, hydroxyapatite and CMC.

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