WO2016163612A1 - Bone regenerating scaffold coated with extracellular matrix - Google Patents

Abstract

The present invention relates to a bone regenerating scaffold comprising: a first layer comprising polycaprolactone (PCL), poly(D,L-lactic-co-glycolic acid) (PLGA) and β-tricalcium phosphate (TCP); and a second layer comprising a demineralised and decellularised bone extracellular matrix (bdECM), wherein the second layer surrounds the first layer; and to a production method for same. The bone regenerating scaffold coated with extracellular matrix of the present invention allows scaffold production in a shape that accurately matches an affected area where bone damage has been incurred by means of a 3D printer, and, as compared with existing scaffolds, can be expected to have the advantage of relatively rapid bone regeneration through more rapid fusing to the affected area and better attachment, growth and bone differentiation of surrounding bone tissue cells. Also, because there is no need for steps such as cell transplantation, bone-damaged patients can be expected to recover relatively rapidly by implanting the scaffold within a short time.

Description

Support for bone regeneration coated with extracellular matrix

The present invention comprises a first layer comprising polycaprolactone (PCL), poly (D, L-lactic-co-glycolic acid) (PLGA), and tricalcium phosphate (β-TCP); And a second layer comprising a demineralized and decellularized bone extracellular matrix (bdECM), the second layer surrounding the first layer, and a support for bone regeneration and a method of manufacturing the same.

Bone tissue is an important tissue that maintains the human skeleton, and bone graft materials for bone tissue replacement and regeneration of various materials and forms have been researched and developed for bone tissue regeneration. Bone grafts can be classified into bone forming materials, bone conducting materials, and bone inducible materials according to the bone healing mechanism, and autografts, allografts, and other types of bone grafts can be classified according to the implants used for bone transplantation or implantation. Transplantation and other methods are mainly used.

Among them, the method of minimizing the immune response is an autograft, and when transplanted to a bone injury site using autologous bone, the immune response is minimized, and thus stable bone tissue regeneration is possible. However, there are inconveniences such as secondary bone loss and long recovery period due to the extraction of autologous bone, and the amount of autologous bone that can be collected is very limited. There are other types of transplants that use other people's bones to compensate for this, but unlike autologous bones, they cause a lot of immune reactions, and the price is very expensive. Therefore, a lot of bone tissue engineering researches to manufacture and transplant synthetic bone to be used by many people while minimizing the immune response.

Scaffolds for application in bone tissue engineering must meet several key conditions for tissue formation optimized for host tissue. These conditions include cell affinity, adequate porosity for nutrient and oxygen permeation, interfacial activity to promote cell adhesion and differentiation, and the like. In addition, ideally the support should be continuously degraded and replaced by the host cell. Synthetic degradable plastics, such as polycarprolactone (PCL), poly (D, L-lactic-co-glycolic acid) (PLGA), and copolymers thereof, are more accurate and more economical than clinically occurring materials and are more economical There is an advantage that it can.

In particular, with the development of 3D printing technology, it is possible to produce an implant that exactly matches the affected part of the patient. In addition, these 3D printing techniques have repeatable reproducibility. However, biodegradable polymers used in the production of implants known to date have disadvantages of poor fusion with surrounding bone tissue-derived cells, low cell adhesion, and induction of bone differentiation of stem cells. This is due to the surface properties of the polymer, so that if the fusion with the bone tissue around the affected area does not occur immediately, the biodegradable polymer may be degraded and the bone regeneration of the intended form may not occur.

On the other hand, the present inventors mixed the conventional β-TCP and PCL / PLGA support (PCL / PLGA / β-TCP) applied to the rabbit skull injury model, the PCL / PLGA / β-TCP support is somewhat effective in bone regeneration However, the scaffold had poor cell adhesion rate and did not obtain satisfactory effects on new bone formation and bone density (Tissue Eng. Part A 2012, 19, 317).

As a result of our diligent efforts to develop a scaffold with excellent bone regeneration effect, the present inventors have prepared a porous scaffold (PCL / PLGA / β-TCP) including PCL, PLGA, and β-TCP, and produced ECM derived from actual bone tissue. Demineralized bone matrix (DBM) was extracted and subjected to decellularization (bdECM, demineralized and decellularized bone extracellular matrix) in order to suppress the immune response during transplantation. The present invention was completed by confirming that the bdECM-coated support promotes bone regeneration by improving adhesion and differentiation of bone cells.

One object of the present invention is a first layer comprising polycaprolactone (PCL), poly (D, L-lactic-co-glycolic acid) (PLGA), and tricalcium phosphate (β-TCP); And a second layer comprising a demineralized and decellularized bone extracellular matrix (bdECM), wherein the second layer surrounds the first layer.

Another object of the present invention is to prepare a support by mixing (a) polycaprolactone (PCL), poly (D, L-lactic-co-glycolic acid) (PLGA), and tricalcium phosphate (β-TCP); And (b) to provide a support for bone regeneration support comprising the step of coating the support with a demineralized and decellularized bone extracellular matrix (bdECM).

The support for bone regeneration coated with the extracellular matrix of the present invention is capable of producing a support that has a shape exactly matched to a damaged bone through a 3D printer. Proliferation and bone differentiation are improved, so a faster bone regeneration effect can be expected. In addition, since a step such as cell transplantation is not necessary, faster recovery can be expected by implanting a support into a bone injury patient in a short time.

Figure 1 shows the process of manufacturing bdECM.

Figure 2 schematically shows the overall experimental process.

3 is a SEM photograph of a support prepared by 3D printing technology according to an embodiment of the present invention. (a) PCL / PLGA / β-TCP support, and (b) collagen or (c) PCL / PLGA / β-TCP support.

Figures 4a and 4b confirms the attachment, form and proliferation of osteoblasts on a support according to an embodiment of the present invention. (a) SEM photograph of day 1 or 14 of culture of cells attached to each support, and (b) survival and proliferation trend of cells in each support.

Figures 5a and 5b shows the ALP activity of osteoblasts in the support according to an embodiment of the present invention. (a) ALP expression of osteoblasts on support at day 14 of culture and (b) quantitative analysis of ALP activity.

Figures 6a to 6c shows bone mineralization of osteoblasts in the support according to an embodiment of the present invention. (a) Alizarin Red S staining, a calcium accumulation marker on the support on day 21, (b) quantitative analysis of Alizarin Red S solution accumulated on day 21, and (c) calcium content on days 7, 14 and 21. 6D is quantification of bone differentiation mRNA expression (right: OC, left: COL1A1) by qRT-PCR.

7A and 7B show in vivo bone regeneration in a support according to one embodiment of the invention. The mouse cranial damage model was treated with each support alone or with osteoblasts with or without osteoblasts. (a) Evaluation of bone regeneration by micro-CT. Representative micro-CT picture of mouse skull. Damaged sites were treated with PCL / PLGA / β-TCP, PCL / PLGA / β-TCP / Col, or PCL / PLGA / β-TCP / bdECM. The photo above shows only the support without osteoblasts, and the photo below shows the treatment of osteoblasts with the support. Scale bar = 4 mm. (b) The volume of injury site filled with new bone was measured by micro-CT analysis program (n = 10, number of damages per group). * P <0.05 compared to PCL / PLGA / TCP group and #p <0.05 compared to PCL / PLGA / TCP / Col group.

FIG. 8A shows the bone regeneration by histological analysis of Goldner's trichrome staining on the mouse skull injury site. Arrows indicate damage site boundaries. Scale bar = 2 mm. All photos are 40x magnification. 8b and 8c confirm the new bone area and bone density by histomorphologic analysis (n = 10, number of injuries). * P <0.05 compared to PCL / PLGA / β-TCP group and #p <0.05 compared to PCL / PLGA / β-TCP / Col group.

The present invention provides, in one embodiment, a first layer comprising polycaprolactone (PCL), poly (D, L-lactic-co-glycolic acid) (PLGA), and tricalcium phosphate (β-TCP); And a second layer comprising a demineralized and decellularized bone extracellular matrix (bdECM), the second layer surrounding the first layer to provide a support for bone regeneration.

In another aspect, the present invention provides a method for preparing a support by mixing (a) polycaprolactone (PCL), poly (D, L-lactic-co-glycolic acid) (PLGA), and tricalcium phosphate (β-TCP). ; And (b) coating the support with a demineralized and decellularized bone extracellular matrix (bdECM).

As used herein, the term “scaffold” may refer to a substance that replaces a part of an organ or tissue damaged in vivo and may complement or replace a function thereof. In particular, the polymer support may include a biodegradable polymer material, which may be maintained until the support sufficiently performs its function and role, and then completely decomposed in vivo. In the present invention, as the polymer material of the support, PCL (polycaprolactone) and PLGA (poly (D, L-lactic-co-glycolic acid)) together with the most extensive bone reconstitution ability of bio-ceramic and forms a solid bond with the target bone tissue Β-TCP (tricalcium phosphate), which is known to cause bone formation, was mixed and used.

Specific manufacturing method of the support for bone regeneration of the present invention is as follows.

First step (a) is a step of preparing a support (PCL / PLGA / β-TCP) by mixing PCL, PLGA and β-TCP, the mixing and manufacturing process is not limited to this, in particular using 3D printing technology It may be performed by.

The term used in the present invention, "3D printing" is a term commonly used in the same sense as the rapid prototyping technology (RP, Rapid Prototype). It is a technology that can produce 3D models in a short time.

In particular, the PCL / PLGA / β-TCP support is not limited thereto, but may include PCL: PLGA: β-TCP in a weight ratio of 1.5 to 2.5: 1.5 to 2.5: 0.5 to 1.5.

In a specific embodiment of the present invention, PCL, PLGA and β-TCP were mixed in a weight ratio of 2: 2: 1 and transferred to a syringe of a 3D printer and sprayed through a precision nozzle to prepare a PCL / PLGA / β-TCP support ( 3a).

Next step (b) is a step of coating the prepared PCL / PLGA / β-TCP support with bdECM. Step (b) is not limited thereto, but specifically, the first step of dipping and centrifuging a PCL / PLGA / β-TCP support in bdECM; A second step of keeping the support submerged in bdECM; And a third step of drying the support. The second step may be to maintain the support to be immersed in bdECM for 5 to 30 minutes.

As used herein, the term "bdECM (demineralized and decellularized bone extracellular matrix)" refers to decellularization of a demineralized bone matrix (DBM) to reduce an immune response upon transplantation into an individual, and the decellularization process is performed using trypsin-EDTA. It may be, but is not limited thereto.

The DBM refers to demineralized bone matrix, and was developed to reproduce a microenvironment including growth factors, collagen and non-collagenous protein (NCP). It is a semi-transparent, flexible rubber-like substance that contains bone morphogenetic protein (BMP) that promotes bone growth. Since the protein is not modified by the acid, such as hydrochloric acid and citric acid, DBM left after demineralization can effectively promote bone regeneration, including BMP.

In the present invention, the term "demineralization" refers to extracting minerals from a biological tissue containing minerals such as calcium using a chelating agent, and the like and remaining in the solution remaining after separating DBM from bone tissue. It contains a large amount of minerals such as calcium phosphate that make up the human bone. Several methods can be used to demineralize bone tissue: hydrochloric acid, ethylenediaminetetraacetic acid (EDTA), formic acid, citric acid, acetic acid, nitric acid, nitrous acid ( It is possible by using a variety of acid solutions such as nitrous acid, and may be made by stirring the bone fragments in hydrochloric acid, in particular, but is not limited thereto.

The coating is a process in which the PCL / PLGA / β-TCP support prepared in step (a) is dipped in the prepared bdECM and dried, thereby forming a first layer including the PCL / PLGA / β-TCP support. The second layer containing bdECM is enclosed. Specifically, the support may be immersed in bdECM and centrifuged to subside the material between the pores, followed by incubation and drying.

In particular, the present invention is not limited thereto, but the process of submerging the material between the pores by centrifugation may be performed by centrifugation for 30 seconds to 5 minutes at 1000 to 2000 rpm at 1 to 10 ° C., and the process may be performed once to 5 times. Can be performed repeatedly. In addition, the incubation may be in particular incubated for 5 to 30 minutes at 30 to 40 ℃, the drying may be performed for 5 to 30 minutes, but is not limited thereto. Step (b) may be performed repeatedly once to five times.

In addition, the solution containing bdECM of step (b) may be a solution of bdECM dissolved in distilled water at 5 to 20 mg / ㎖.

In a specific embodiment of the present invention, bdECM was dissolved in distilled water at 10 mg / ml to prepare a coating solution, and the PCL / PLGA / β-TCP support prepared by 3D printing was dipped in the coating solution for 20 minutes. After drying, a PCL / PLGA / β-TCP support (PCL / PLGA / β-TCP / bdECM) coated with bdECM was prepared.

As used herein, the term "bone regeneration" may mean any phenomenon of treating, alleviating, or ameliorating bone damage through a process in which bone cells multiply damaged or osteoblasts differentiate into bone cells. Although not limited to this, in particular, the bone regeneration may be by promoting bone differentiation.

In the present invention, the term "support for bone regeneration" does not necessarily correspond to the shape of the bone injury site to be transplanted, regardless of its shape, and it is sufficient if the shape is processed to a similar shape. However, if necessary, the shape of the bone injury site of the transplant target may be grasped in advance, and a shape suitable for the graft may be prepared, and the shape of the membrane or the strip may be preformed so as to be generally applicable to various situations.

To this end, various materials for increasing the moldability of the support for bone regeneration may be further added. That is, a linear material, a tubular material, a particulate material, an amorphous material, and the like, which are excellent in biocompatibility, may be further added to improve physical properties of the support for bone regeneration. This type of material may be selected from materials such as collagen, carboxymethylcellulose (CMC) or animal-derived gelatin, and there is no particular limitation as long as it does not cause an immune response to the transplant target.

In a specific embodiment of the present invention, the PCL / PLGA / β-TCP / bdECM support was observed with a scanning electron microscope (SEM) to confirm that pores and interconnections were present (FIG. 3), not coated. Or it was confirmed that the cell adhesion rate is superior to the support with the collagen coating (Fig. 4).

In another specific embodiment of the present invention, when inoculating osteoblasts to the PCL / PLGA / β-TCP / bdECM scaffold and cultured in bone differentiation medium, ALP expression, calcium accumulation, and bone such as OCN and COL1A1 It was confirmed that the expression of differentiation-related mRNA was increased, it was confirmed that the bone differentiation induction function is excellent (Figs. 5 and 6).

Furthermore, in another specific embodiment of the present invention, when the PCL / PLGA / β-TCP / bdECM scaffold is implanted together with osteoblasts or the scaffold alone in a bone damaged mouse model, the new bone volume and bone density increase. By confirming that the damaged area is recovered (FIGS. 7 and 8), it can be seen that the PCL / PLGA / β-TCP / bdECM support of the present invention has an excellent effect as a composition for bone regeneration. This effect is also significantly superior to the PCL / PLGA / β-TCP support or the PCL / PLGA / β-TCP / Col support.

In conclusion, the PCL / PLGA / β-TCP / bdECM scaffold of the present invention is coated with bdECM prepared by decellularizing DBM and has bone regeneration effect without transplantation with osteoblasts. It can be used to improve the adhesion of bone cells and promote bone differentiation, which is excellent as a support for bone regeneration.

Hereinafter, the present invention will be described in more detail with reference to the following examples. These examples are only intended to illustrate the invention and are not to be construed as limiting the scope of the invention by these examples.

Example  One : bdECM (demineralized and decellularized  bone extracellular  manufacturing of matrix

In order to prepare a support having excellent bone regeneration effect, bdECM was used to coat the support. The specific manufacturing process is as follows, the overall process is shown in FIG.

(1) bone processing

Tibia were isolated from calves 12 to 24 months old. The bone was divided into pieces and divided into cancellous and cortical groups, and then the spongy group was used. Phosphate-buffered saline (PBS) containing 0.1% (w / v) gentamycin (Gentamicin, Invitrogen, Carlsbad, Calif., USA) was used to remove and wash the residual tissue of the spongy flakes. The pieces were then frozen in liquid nitrogen and cut into sections up to 4 x 4 x 4 mm. The sections were washed with distilled water, immersed in liquid nitrogen, and ground in a coffee mill (Kordia Co., Daegu, Gyeongbuk, Korea) and ground.

(2) Demineralization and Decellularization

The bone was demineralized by stirring (300 rpm) in 0.5 N HCl (25 mL / g g) at room temperature for 24 hours. After demineralization, the result (hereinafter referred to as 'bDBM') was filtered under vacuum and rinsed with distilled water. The lipids in the demineralized powder were then extracted for 1 hour in chloroform (fisher Scientific, Loughborough, UK) and methanol (Fisher Scientific) 1: 1 mixtures and rinsed first with methanol and then with distilled water. The bDBM was snap frozen, lyophilized overnight under reduced pressure and stored at -20 ° C.

The bDBM was then rinsed with distilled water and evacuated with continuous stirring for 24 hours at 37 ° C. and 5% CO _{2 in} 0.05% trypsin (Sigma-Aldrich) and 0.02% ethylenediamine tetraacetic acid (Sigma-Aldrich) mixed solution. Saturated. The result (hereinafter referred to as 'bdECM') was rinsed with PBS containing 1% (w / v) penicillin / streptomycin for 24 hours at 4 ° C. continuously to remove residual cellular material. It was then frozen immediately, lyophilized overnight under reduced pressure and stored at -20 ° C.

Example 2 Preparation and Characterization of Supported Coatings with bdECM

A support (PCL / PLGA / β-TCP) comprising PCL, PLGA, and β-TCP was prepared using 3D printing technology, and coated with the prepared bdECM to support bone regeneration (PCL / PLGA / β-). TCP / bdECM). In order to confirm the bone regeneration effect of the finished support, the support alone (PCL / PLGA / β-TCP) or the support was collagen (PCL / PLGA / β-TCP / Col) or bdECM (PCL / PLGA / β-TCP / In vivo and ex vivo bone regeneration effect of the support coated with bdECM) was confirmed (Fig. 2). The specific manufacturing process is as follows.

(1) Preparation of PCL / PLGA / β-TCP support using 3D printing technology

0.4 g of PCL (MW 43,000-50,000; Polysciences Inc., Warrington, PA, USA) and 0.4 g of PLGA (MW 50,000-75,000; Sigma-Aldrich, St. Louis, MO, USA) were placed in a glass container at 130 ° C. for 10 minutes. Put it. The melted polymers were mixed with 0.2 g of β-TCP (Sigma-Aldrich) powder to a final concentration of 20% by weight. The mixture was transferred to a 10 ml syringe of MHDS and sprayed through a precision nozzle at 650 kPa, 140 ° C. The printed scaffolds were sterilized for 30 minutes in 70% ethanol and exposed to UV overnight prior to the coating step.

(2) coating on PCL / PLGA / β-TCP support

The coating solution was prepared by dissolving bdECM at distilled water at 10 mg / ml, and as a control, atelocollagen solution (Koken, Shizuoka, Japan) was prepared at a concentration of 10 mg / ml. The PCL / PLGA / TCP support was coated with the prepared coating solution. To fill the pores in the pores of the support, the support was immersed in a bdECM solution and centrifuged at 1,500 rpm for 1 minute at 4 ° C. to fill the pores of the pores. The support filled with pores was incubated at 37 ° C. for 20 minutes and then dried under sterile conditions for 20 minutes. This process was repeated three times.

(3) Characterization of the prepared support

In order to analyze the characteristics of the prepared support, the shape of the support was observed at 10 kV with a scanning electron microscope (JSM-5300, JEOL, Tokyo, Japan). The support was coated with platinum for 120 seconds with a sputter-coater and the surface morphology of the PCL / PLGA / β-TCP support was compared with the PCL / PLGA support. Uncoated PCL / PLGA / β-TCP support was used as a control in all experiments. The degree of bdECM or collagen coating on the support was confirmed by spectra of X-ray photoelectron spectroscopy (XPS) for nitrogen (N), oxygen (O), and carbon (ESCALAB 220iXL; VG Scientific, East Grinstead, West Sussex , UK).

Observation of the support by SEM revealed that the support had a diameter of 4 mm and a height of 1 mm, a string thickness of about 200 μm and a pore size of about 300 μm. PCL / PLGA / β-TCP supports have a rough surface because they contain β-TCP powders (FIG. 3A), and collagen or bdECM coated supports have been found to have smooth surfaces without blocking pores (FIGS. 3B and 3C). ). The smooth surface of the support means that the collagen and bdECM have been successfully coated. In addition, the supports all maintained well interconnected, which is important for bone regeneration because it allows the movement of host cells from the bone marrow and the movement of surrounding osteoblasts.

On the other hand, the degree of the collagen or bdECM coated on the support was quantified by XPS analysis, shown in Table 1 below.

element	PCL / PLGA / β-TCP	PCL / PLGA / β-TCP / Col	PCL / PLGA / β-TCP / bdECM
C	74.7%	63.6%	64.7%
N	0%	11.8%	10.6%
O	25.3%	24.6%	24.7%

Example  3: confirm the cell proliferation effect according to the support

(1) cell culture

To analyze in vitro cellular behaviors, isolated primary osteoblasts were seeded at 1 x 10 ⁴ cells in each support. Calvarial osteoblasts were isolated from the cranial canal of Sprague-Dawley rat embryos (SLC, Tokyo, Japan). The scaffolds inoculated with cells for one day for cell adhesion were immersed in growth medium, and then the culture medium was incubated with 50 mM ascorbic acid-2-phosphate (Sigma-Aldrich), 10 mM to induce osteogenic function of osteoblasts. It was replaced with complete αMEM containing β-glycerophosphate (Sigma-Aldrich), and 100 nM dexamethasone (Sigma-Aldrich). The medium was changed daily.

(2) Osteoblast Morphology

SEM analysis was performed to observe the cell morphology of the scaffolds 1 or 14 days after inoculation with osteoblasts. The support was fixed for 4 hours at 4 ° C. with a modified Karnovsky's fixative containing 2% paraformaldehyde and 2% glutaraldehyde in 0.05M sodium cacodylate buffer (SC buffer, pH 7.2; Sigma-Aldrich). The primary immobilized support was washed three times at 4 ° C. with 0.05 M SC buffer and soaked in 0.05 M SC buffer containing 1% osmium tetroxid (Sigma-Aldrich) at 4 ° C. for 2 hours. Then dehydrated sequentially and dried for 15 minutes with hexamethyldisilazane (Sigma-Aldrich). The support and cell morphology on the support were identified at 15 kV using FE-SEM (JEOL). All samples were coated on carbon tape and coated with platinum for 60 seconds using a sputter coater.

(3) cell proliferation assay

Osteoblast proliferation assay was performed on the support using Cell Count Kit-8 (CCK-8, Dojindo, Kumamoto, Japan). The support was immersed in CCK-8 solution (1:10 ratio) diluted with growth medium and incubated at 37 ° C. for 2 hours. The solution was then extracted and absorbance was measured at 450 nm using a microplate reader (Asys UVM 340; Biochrom, Cambridge, UK). The support was washed with PBS and incubated again with fresh culture medium.

As a result of inoculating the cells and confirming the shape of the support by SEM, it was confirmed that the cells were attached on the support on day 1, and the attached cells grew through the pores of the support over time (14 days) (FIG. 4a). In particular, the cells of the PCL / PLGA / β-TCP / bdECM scaffold showed more ordered morphology closer to the anisotropic shape of the collagen matrix. Such morphology is known to affect the mechanical properties of bone tissue.

Furthermore, as a result of measuring the absorbance in the cell proliferation assay, it was confirmed that osteoblasts adhered better on the support coated with collagen or bdECM than the uncoated support, from which the support coated with protein showed the cell adhesion rate. It was found to improve. The osteoblast proliferation rate of the three groups showed a similar tendency to each other (Fig. 4b).

The bdECM coating reduced the pore size of the support by about 30% (FIG. 3), but further increased osteoblast adhesion. This result means that the pores of the support are not filled with bdECM, so osteoblasts can migrate inside the support. On the other hand, the collagen coating showed a lower cell adhesion rate compared to the bdECM coating, and the bdECM can provide a better environment than the collagen coating.

In conclusion, even when the support for bone regeneration coated with the bdECM of the present invention is actually implanted in the human body, the cells of surrounding tissues can effectively grow along the pores of the support.

Example 4 Confirmation of the Bone Differentiation Enhancement Effect of Rat-derived Osteoblasts

(1) ALP expression confirmation

In order to confirm whether the support of the present invention has an effect of improving bone differentiation, bone differentiation was observed while culturing rat-derived osteoblasts on the support. First, the expression of ALP, most widely known as a biochemical marker of osteoblast activity in cells inoculated to each support, was confirmed by immunofluorescence staining. The support incubating osteoblasts in bone production medium was washed with PBS and fixed with 4% paraformaldehyde solution on day 14. Then treated with PBS containing 2% BSA to prevent nonspecific binding, immunostained with anti-ALP antibody (1: 200; Santa Cruz Biotechnology, Santa Cruz, CA, USA) followed by Alex Fluor 488 goat anti-rabbit Incubated with antibody (1: 100; Invitrogen). All samples were counterstained with DAPI (1: 100; Sigma-Aldrich) and observed with a laser scanning confocal microscope (Olympus FluoView ^™ FV1000, Tokyo, Japan).

In addition, ALP activity was quantified by p-NPP (p-nitrophenyl phosphate, Sigma-Aldrich) at 14 days. Osteoblasts inoculated on the support were lysed with RIPA Lysis Buffer (Millipore, billerica, Mass., USA) and the lysates were incubated at 37 ° C. for 30 minutes with ALP substrate buffer containing 5 mM pNPP. The enzyme activity of ALP was measured at 405 nm using a microplate reader.

As a result, ALP marker expression expressed by osteoblasts throughout the support was confirmed, which means that osteoblast activity is very high on the support (FIG. 5A). Among them, the cells on the support coated with bdECM showed an aligned form as in the SEM analysis on day 1. Quantitatively measured ALP activity shows the highest activity in the lysate isolated from the bdECM coated support (FIG. 5B).

(2) check calcium accumulation

When osteoblasts were incubated with bone differentiation medium on a support, the degree of bone mineralization was confirmed through calcium accumulation. Ca ^{2 +} concentrations in the lysate was measured with a spectrophotometer according to the manufacturer's instructions using the (Abcam, Cambridge, MA, UK ) Calcium Detection Kit. On day 21, the inoculated scaffolds were fixed with 4% paraformaldehyde to stain calcium accumulation. Cells were then stained with 2% Alizarin Red S (Sigma-Aldrich) solution (pH 4.3) at room temperature for 20 minutes. The calcium accumulation region was observed by digital microscope (Dino lite, New Taipei, Taiwan), and calcium accumulation was quantified by extracting Alizarin Red from cells using DMSO (dimethyl sulfoxide, Sigma-Aldrich). The extract was measured at 570 nm using a microplate reader.

As a result, it was confirmed that osteoblasts on the support coated with collagen or bdECM contained calcium minerals that were visible to the naked eye after 21 days. However, more calcium mineral amount was observed in the bdECM coated support compared to the collagen coated support (FIG. 6A). Colorimetric analysis results were also in agreement with the staining results (FIG. 6B). The calcium content of the bdECM-coated PCL / PLGA / TCP support showed a significantly higher calcium content than the other groups, in particular, it was confirmed that the continual increase over time (Fig. 6c).

(3) Confirmation of mRNA expression related to bone differentiation

Bone differentiation-related mRNA expression of cultured osteoblasts was analyzed by qRT-PCR. RNA was isolated from cells with RNAiso reagent (Takara, Japan) according to the manufacturer's instructions on day 21 of culture in differentiation medium. RNA concentration was measured using Nanodrop (ThermoScientific, USA) and reverse transcription was performed with an amfiRivert cDNA synthesis platinum master mix kit (GenDEPOT, USA). Gene expression was analyzed by LightCycler SYBR Green I Master mix using LightCycler 480 Real-Time PCR equipment (Roche Biochemicals, IN, USA). Primer sequences were prepared using NCBI and PubMed databases and the sequence numbers are shown in Table 2 below.

gene	Primer sequence
GAPDH	Sense Antisense	5'- GGC ACA GTC AAG GCT GAG AAT G -3 '(SEQ ID NO: 1) 5'- ATG GTG GTG AAG ACG CCA GTA -3' (SEQ ID NO: 2)
OCN	Sense Antisense	5'- GGT GCA GAC CTA GCA GAC ACC A -3 '(SEQ ID NO: 3) 5'- AGG TAG CGC CGG AGT CTA TTC A -3' (SEQ ID NO: 4)
COL1A1	Sense Antisense	5'- ACG TCC TGG TGA AGT TGG TC -3 '(SEQ ID NO: 5) 5'- CAG GGA AGC CTC TTT CTC CT -3' (SEQ ID NO: 6)

As a result, mRNA expression associated with bone production, including osteocalcin (OCN) and type I collagen (COL1A1), was also higher in the bdECM-coated PCL / PLGA / TCP support group. These results also suggest that bdECM-coated scaffolds are superior in bone tissue regeneration in mineralization as well as tissue maturation (FIG. 6D).

Taken together, it can be seen that osteoblasts cultured on bdECM-coated PCL / PLGA / TCP scaffolds exhibit significantly enhanced in vitro osteogenic activity compared to other groups, which may be due to the biomolecules present in bdECM. have. bdECM contains osteogenic biomolecules such as bone morphogenetic protein-2 (BMP-2), BMP-7 and other unknown factors. These bone-induced biomolecules affect bone differentiation and mineralization of osteoblasts. In addition, osteoblasts cultured in PCL / PLGA / TCP / bdECM showed the highest cell adhesion rates. This means that various cell attachment sites have been provided by the bdECM bioactive factor.

In conclusion, ALP expression, Alizarin Red S staining and qRT-PCR results of OCN and COL1A1 showed that the support for bone regeneration coated with bdECM of the present invention promoted bone differentiation.

Example 5: Confirmation of the bone regeneration effect in vivo of the support

A mouse cranial canal injury model was used to confirm the in vivo bone regeneration effect of PCL / PLGA / β-TCP / bdECM scaffolds. In vivo bone formation was achieved by implanting each scaffold with or without osteoblasts. The efficiency was confirmed. Specific experimental methods and contents are as follows.

(1) cranial canal damage model to assess bone regeneration

Six-week-old mice obtained from the Institute of Cancer Research (Koatech, Sungnam, Kyunggi-do, South Korea) were anesthetized with xylazine (20 mg / kg) and ketamine (100 mg / kg). The hair of the mouse head was pushed, the center of the skull was inverted vertically from the nasal bone to the back of the nape, and the periosteum was lifted to expose the parietal bone surface. Surgical trephine burrs (Ace Surgical Supply Co., Brockton, Mass., USA) and low-speed micromoters were used to create two circular bone injuries (4 mm in diameter) in the skull. The damage size was lethal to the mouse skull injury model. The drill site was washed with saline and the bleeding site was electrically built. Five mice (10 injuries) were used for each group.

(2) micro-CT analysis

Eight weeks after transplantation, mice were euthanized with CO ₂ and the skulls were harvested for analysis. Bone formation was assessed by micro-CT scan (n = 7 per group). micro-CT images were obtained with a micro-CT scanner (SkyScan-1172, Skyscan, Kontich, Belgium). New bone volume was confirmed using a CT analysis program (CT-An, Skyscan).

(3) histological analysis

After micro-CT imaging, samples were prepared for histological and histomorphometric analysis. Samples were immersed in 10% (v / v) buffered formalin solution, dehydrated in increased concentration of alcohol solution, cleared with xylene, and embedded with paraffin. One thalamus and one anterior section were obtained from two samples per mouse using microcutting and grinding techniques. The sections were stained with Goldner's trichrome stain. The bone formation area was determined by calculating the percentage of newly formed mineralized bone using Adobe Photoshop software (Adobe Systems, Inc., San Jose, Calif., USA). The percentage of bone formation area at the site of injury was calculated as (new bone area / bone injury area) × 100. Bone density was calculated as [new bone area / (new bone area + fibrous tissue area + remaining biomass area)] × 100.

As a result, microcomputed tophography (micro-CT) photographs showed that PCL / PLGA / TCP / bdECM scaffold transplantation further improved bone regeneration compared to other scaffold implants (FIG. 7A). In addition, PCL / PLGA / TCP / bdECM supports significantly increased regenerated bone volume compared to other supports (FIG. 7B). In particular, PCL / PLGA / TCP / bdECM scaffolds were found to improve bone regeneration and increase regenerated bone volume to levels similar to those with osteoblasts even without osteoblasts. This means that the PCL / PLGA / TCP / bdECM scaffold of the present invention can bring about a bone regeneration effect only by implanting the scaffold alone without mixing with other cells such as osteoblasts.

Furthermore, histological analysis using Goldner's trichrome staining confirmed that PCL / PLGA / TCP / bdECM transplantation improved bone regeneration efficiency (FIG. 8A). PCL / PLGA / TCP / bdECM successfully filled the regenerated bone, including the newly formed bone marrow at the site of injury. In contrast, implantation of the PCL / PLGA / TCP and PCL / PLGA / TCP / Col scaffolds showed only fibrous tissue at the site of injury.

In addition, the implantation of PCL / PLGA / TCP / Col support with osteoblasts significantly improved bone formation area and bone density compared to the absence of osteoblasts (FIGS. 8B and 8C). In addition, the implantation of PCL / PLGA / TCP / bdECM with osteoblasts improved the bone formation area and bone density more than the implantation of PCL / PLGA / TCP / Col scaffold. However, in the case of transplanting PCL / PLGA / TCP with osteoblasts, it was confirmed that only fibrous tissues were formed, similar to the case without transplantation of osteoblasts.

The support prepared by the 3D printing technology of the present invention can be used in other kinds of bone tissue engineering because it can control the microstructure and interconnected pore structure that can improve the growth of patient tissue into the support implanted at the bone injury site. have. The PCL / PLGA / TCP / bdECM of the present invention can allow cells of a host such as MSC and osteoblasts to grow inside, and the cells migrated into the support can be differentiated into bone in contact with the biomolecules contained in the bdECM. .

Supports prepared by uncoated 3D printing made of synthetic artificial materials (PCL / PLGA / TCP) lack bone-specific bioactive signals that induce bone regeneration. In addition, the collagen-coated PCL / PLGA / TCP / Col scaffold did not effectively treat bone damage alone without osteoblasts. This is due to the lack of bone-generating biomolecules compared to PCL / PLGA / TCP / bdECM. Poor collagen inferior cell adhesion rate and osteogenic activity may have influenced these results. On the other hand, PCL / PLGA / TCP / bdECM showed no difference in the condition with or without osteoblasts. This may be because a significant amount of host cells migrated compared to PCL / PLGA / TCP / Col. Therefore, PCL / PLGA / TCP / bdECM does not require osteoblasts cultured in vitro for the treatment of bone damage, bdECM was prepared by decellularizing DBM, and the scaffold of the present invention is an immune response caused by cell transplantation. Can reduce side effects and reduce the cost and time for cell culture.

In conclusion, the support for bone regeneration coated with bdECM of the present invention promotes bone cell proliferation, improves bone differentiation, and thus has an excellent bone regeneration effect, and thus can be used as a support for bone regeneration.

From the above description, those skilled in the art will appreciate that the present invention can be implemented in other specific forms without changing the technical spirit or essential features. In this regard, it should be understood that the embodiments described above are exemplary in all respects and not limiting. The scope of the present invention should be construed that all changes or modifications derived from the meaning and scope of the following claims and equivalent concepts rather than the detailed description are included in the scope of the present invention.

Claims (11)

1. A first layer comprising polycaprolactone (PCL), poly (D, L-lactic-co-glycolic acid) (PLGA), and tricalcium phosphate (β-TCP); And

   and a second layer comprising a demineralized and decellularized bone extracellular matrix (bdECM), the second layer surrounding the first layer.
2. The support for bone regeneration according to claim 1, wherein the first layer comprises PCL: PLGA: β-TCP in a weight ratio of 1.5 to 2.5: 1.5 to 2.5: 0.5 to 1.5.
3. The support for bone regeneration of claim 1, wherein the second layer is formed by coating the first layer with a coating solution in which bdECM is dissolved at 5 to 20 mg / ml in distilled water.
4. The support for bone regeneration according to claim 1, wherein the bone regeneration is by promoting bone differentiation.
5. (a) preparing a support by mixing polycaprolactone (PCL), poly (D, L-lactic-co-glycolic acid) (PLGA), and tricalcium phosphate (β-TCP); And

   (b) coating the support with a solution containing a demineralized and decellularized bone extracellular matrix (bdECM).
6. The method of claim 5, wherein step (a) comprises mixing PCL: PLGA: β-TCP in a weight ratio of 1.5 to 2.5: 1.5 to 2.5: 0.5 to 1.5.
7. The method of claim 5, wherein step (b)

   A first step of dipping and centrifuging the PCL / PLGA / β-TCP support in bdECM;

   A second step of keeping the support submerged in bdECM; And

   Comprising a third step of drying the support, bone support support production method.
8. The method of claim 7, wherein the second step is to maintain the support to be immersed in bdECM for 5 to 30 minutes.
9. The method of claim 5, wherein the solution containing bdECM of step (b) is bdECM dissolved in distilled water at 5 to 20 mg / ㎖, support for bone regeneration support.
10. The method of claim 5, wherein step (a) is performed using a 3D printer.
11. The method of claim 5, wherein the bone regeneration is by promoting bone differentiation.

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