KR20160121727A - A scaffold with bone derived extracellular matrix for bone regeneration - Google Patents

Abstract

The present invention provides a biodegradable polymer comprising a first layer comprising a biodegradable polymer; And a second layer comprising a demineralized and decellularized bone extracellular matrix (bdECM), the second layer surrounding the first layer, and a method of making the same.
The support for bone regeneration coated with the extracellular matrix of the present invention can be made into a support having a shape exactly matching the lesion affected by bone damage through a 3D printer. It can bind to the lesion faster than the existing support, Proliferation and bone differentiation can be improved and a faster bone regeneration effect can be expected. In addition, since the step such as cell transplantation is not required, a faster recovery can be expected by transplanting the scaffold into the bone injured patient in a short time.

Description

[0001] The present invention relates to a scaffold with bone-derived extracellular matrix for bone regeneration,

The present invention provides a biodegradable polymer comprising a first layer comprising a biodegradable polymer; And a second layer comprising a demineralized and decellularized bone extracellular matrix (bdECM), the second layer surrounding the first layer, and a method of making the same.

BACKGROUND ART Bone tissue is an important tissue for maintaining the skeleton of human body, and various materials and forms of bone tissue replacement and regenerative bone graft materials have been researched and developed for bone tissue regeneration. Bone graft materials can be classified into osteogenic materials, osteoconductive materials, and bone-inducing materials according to the healing mechanism. According to the graft materials used for transplating or implantation, autografts, allografts, And transplantation.

As a method of minimizing the dual immune response, autologous bone grafting using autogenous bone minimizes the immune response when transplanted to the site of bone injury, thereby enabling stable bone tissue regeneration. However, it has disadvantages such as secondary bone loss and recovery period of other parts due to extraction of autogenous bone, and has a disadvantage that its amount is very limited. To compensate for this, there are other transplantations that use other people 's bone. However, unlike autogenous bone, it causes a lot of immune reactions and is very expensive. Therefore, studies on bone tissue engineering to synthesize and transplant synthetic bone to be used by many people while minimizing the immune response are under way.

The scaffold for application to bone tissue engineering must meet several key requirements for optimized tissue formation of the host tissue. These conditions include cell affinity, proper porosity for nutrients and oxygen permeation, and surface activity to promote cell attachment and differentiation. In addition, ideally, the support must be degraded and replaced by the host cell in succession. Synthetic degradable plastics such as PCL (polycarprolactone), PLGA (poly (D, L-lactic-co-glycolic acid) and copolymers thereof are more economical and clinically widespread .

In particular, as 3D printing technology has been developed, it is possible to produce an implant that exactly matches the lesion of the patient. In addition, such 3D printing techniques have repeatable reproducibility. However, the biodegradable polymer used in the preparation of implants to date has a disadvantage that it is not well fused with surrounding bone tissue-derived cells, has low cell adhesion, and can not induce bone differentiation of stem cells. This is due to the surface characteristics of the polymer, and if the fusion with the bone tissue around the lesion does not occur immediately, the biodegradable polymer may be degraded and the original intended shape of the bone may not be regenerated.

Meanwhile, the inventors of the present invention have found that the PCL / PLGA / β-TCP scaffold is somewhat effective in bone regeneration by mixing β-TCP with PCL / PLGA scaffolds (PCL / PLGA / (Tissue Eng. Part A 2012, 19, 317). However, the cell adhesion rate was decreased and the new bone formation and bone density were not satisfactory.

The present inventors have made intensive efforts to develop a supporter having excellent bone regeneration effect. As a result, they have found that a porous support containing a biodegradable polymer is produced using 3D printing technology, and an ECM derived from a bone tissue is extracted, (BdECM), a demineralized and decellularized bone extracellular matrix, was coated on the prepared support, and the bdECM-coated bdECM-coated bone matrix The present inventors confirmed that the support enhances the adhesion and differentiation of osteocytes and promotes bone regeneration, thereby completing the present invention.

One object of the present invention is to provide a biodegradable polymer comprising a first layer comprising a biodegradable polymer; And a second layer comprising a demineralized and decellularized bone extracellular matrix (bdECM), the second layer surrounding the first layer.

It is another object of the present invention to provide a method of manufacturing a bone regeneration support comprising coating a support containing a biodegradable polymer with bdECM (demineralized and decellularized bone extracellular matrix).

In order to accomplish the above object, the present invention provides, as one embodiment, a first layer comprising a biodegradable polymer; And a second layer comprising a demineralized and decellularized bone extracellular matrix (bdECM), said second layer surrounding said first layer.

According to another aspect of the present invention, there is provided a method for producing a bone regeneration support comprising coating a support containing a biodegradable polymer with a solution containing bdECM (demineralized and decellularized bone extracellular matrix).

As used herein, the term "scaffold" may refer to a substance that replaces or partially replaces damaged organs or parts of tissues in vivo. In particular, the polymer scaffold may include a biodegradable polymer material, and the scaffold may be completely decomposed in vivo after the scaffold is maintained until its function and role are sufficiently performed.

The biodegradable polymer includes poly-L-lactic acid, poly-glycolic acid, poly-D-lactic acid-co-glycolic acid, poly- But are not limited to, at least one selected from the group consisting of acid, poly-caprolactone, poly-valerolactone, poly-hydroxybutyrate and poly-hydroxyvalerate.

In addition, the support may further include a ceramic material in the biodegradable polymer, and the ceramic material may exhibit a bone remodeling capability and may form a firm bond with the bone tissue of the subject to cause bone formation. The ceramic material may in particular be composed of alumina, zircon, silica, magnesia, zinc oxide, titanium oxide, mixed oxides such as PZT, barium titanate, silicate, hydroxyapatite, tricalcium phosphate and mixtures thereof But is not limited to.

A specific method for producing the bone regeneration support of the present invention is as follows.

First, the support including the biodegradable polymer may be produced by a 3D printing technique, but not limited thereto.

The term "3D printing " used in the present invention is a term used in the same sense as Rapid Prototyping (RP). It is a term in which materials are systematically layered one by one through two- It refers to a technique that can produce a 3D model in a short time.

In a specific embodiment of the present invention, a PCL / PLGA / β-TCP support was prepared by mixing PCL, PLGA and β-TCP at a weight ratio of 2: 2: 1, transferring the mixture to a 3D printer syringe and injecting through a precise nozzle 3a).

Next, the support prepared above may be coated with a solution containing bdECM to prepare the support for bone regeneration of the present invention. The step of coating with the solution containing bdECM is not limited thereto. Specifically, the first step of immersing the prepared support in bdECM and centrifuging; A second step of keeping the support immersed in bdECM; And a third step of drying the support. The second step may be to keep the support immersed in the bdECM for 5 to 30 minutes.

The term " bdECM (demineralized and decellularized bone extracellular matrix) " in the present invention means depletion of a demineralized bone matrix (DBM) to reduce immune response upon transplantation to a subject, and degreasing is performed using trypsin- But is not limited thereto.

The DBM means a demineralized bone matrix, which has been developed to reproduce a microenvironment including growth factors, collagen and NCP (non-collagenous protein). It is a semitransparent and flexible rubbery substance, which contains bone morphogenetic protein (BMP) that promotes bone growth. Since the original structure of the protein is not modified by acid such as hydrochloric acid or citric acid, DBM remaining after the demineralization treatment can promote bone regeneration effectively including BMP.

The term "demineralization" in the present invention means extraction of minerals with a chelating agent or the like in a biotissue containing an inorganic substance such as calcium, and the remaining solution after separating DBM from bone tissue And contains a large amount of inorganic components such as calcium phosphate which constitute the human bone. Several methods can be used to demineralize bone tissue, such as hydrochloric acid, ethylenediaminetetraacetic acid (EDTA), formic acid, citric acid, acetic acid, nitric acid, nitrous acid, and the like. In particular, it may be formed by stirring the bone fragments in hydrochloric acid, but the present invention is not limited thereto.

The coating is a process of immersing a support containing a biodegradable polymer in the bdECM and then drying the biodegradable polymer. Through this process, the first layer containing the biodegradable polymer is surrounded by the second layer containing the bdECM. Specifically, the support may be immersed in bdECM and centrifuged to allow the material between the pores to settle, followed by incubation and drying.

Particularly, though not limited thereto, the process of centrifugally separating the material between the pores may be centrifugation at 1 to 10 ° C at 1000 to 2000 rpm for 30 seconds to 5 minutes, Can be repeatedly performed. In addition, the incubation may particularly be performed at 30 to 40 캜 for 5 to 30 minutes, and the drying may be performed for 5 to 30 minutes, but is not limited thereto. The step (b) may be repeated one to five times.

In addition, the coating may be coating the support with bdECM dissolved in distilled water at 5 to 20 mg / ml.

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

In the present invention, the term "bone regeneration" may mean any phenomenon that treats, alleviates, or alleviates bone damage through proliferation of damaged bone tissue or differentiation of osteoblast into osteocyte, In particular, but not limited to, the bone regeneration may be by promoting bone differentiation.

The term "support for bone regeneration" in the present invention does not necessarily correspond to the shape of the bone damage site to be implanted, regardless of its shape, and it is sufficient that the support is in a state of being processed to a similar shape. However, if necessary, the shape of the bone damage site to be implanted can be grasped in advance, and a shape suitable for the shape may be prepared, or it may be previously shaped by a structure of a membrane or a structure of a band so as to be generally applicable to various situations.

To this end, various materials for increasing the formability of the bone regeneration support may be further added. That is, a linear material, a tubular material, a granular material, and an indefinite material excellent in biocompatibility can be further added to improve physical properties of the bone regeneration support. Materials of this type may be selected from materials such as collagen, carboxymethylcellulose (CMC) or animal-derived gelatin, and there is no particular limitation as long as they do not cause an immune response to the recipient.

In a specific example of the present invention, it was confirmed that the PCL / PLGA / β-TCP / bdECM support was observed by SEM (scanning electron microscope) to show that pores and interconnection existed (FIG. 3) (Fig. 4), compared to a collagen-coated support (Fig. 4).

In another specific embodiment of the present invention, when osteoblast cells are inoculated into the PCL / PLGA / β-TCP / bdECM support and cultured in a 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 and it was confirmed that the function of inducing bone differentiation was excellent (Figs. 5 to 6).

Further, in another specific embodiment of the present invention, when the PCL / PLGA / β-TCP / bdECM scaffold is implanted in a bone-damaged mouse model with or without osteoblasts alone, (FIG. 7 to FIG. 8), it was found that the PCL / PLGA / β-TCP / bdECM support of the present invention had an excellent effect as a composition for bone regeneration. In addition, this effect is significantly superior to PCL / PLGA / β-TCP supports or PCL / PLGA / β-TCP / Col supports.

In conclusion, the supporter of the present invention is coated with bdECM prepared by depleting DBM, and there is no fear of side effects such as immune reaction during bone marrow transplantation, It is possible to promote bone differentiation, and there is an excellent effect as a support for bone regeneration.

The support for bone regeneration coated with the extracellular matrix of the present invention can be made into a support having a shape exactly matching the lesion affected by bone damage through a 3D printer. It can bind to the lesion faster than the existing support, Proliferation and bone differentiation can be improved and a faster bone regeneration effect can be expected. In addition, since the step such as cell transplantation is not required, a faster recovery can be expected by transplanting the scaffold into the bone injured patient in a short time.

Fig. 1 shows a process for producing bdECM.
2 schematically shows an overall experimental procedure.
3 is a SEM photograph of a support prepared by 3D printing technology. PCL / PLGA /? -TCP support coated with (a) PCL / PLGA /? -TCP support and (b) collagen or (b) bdECM
Figures 4a and 4b show the adhesion, morphology and proliferation of osteoblasts on a support. (a) SEM photographs at 1 or 14 days of incubation of cells attached to each support, and (b) cell viability and proliferation tendency in each support.
Figures 5A and 5B show the ALP activity of osteoblasts in the support. (a) Quantitative analysis of ALP expression of osteoblastic cells and (b) ALP activity in the supernatant after 14 days of culture.
Figures 6a to 6c show bone mineralization of osteoblasts in a support. (a) Alizarin Red S staining as a calcium accumulation marker in the supporter 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. FIG. 6D shows the expression of bone morphogenetic mRNA (right: OC, left: COL1A1) by qRT-PCR.
Figures 7a and 7b show in vivo bone regeneration in a support. In the mouse skull injury model, each scaffold and osteoblast were used together or each scaffold alone without osteoclasts. (a) Evaluation of bone regeneration with micro-CT. It is a typical micro-CT photograph of mouse skull. The damaged areas were treated with PCL / PLGA / β-TCP, PCL / PLGA / β-TCP / Col, or PCL / PLGA / β-TCP / bdECM. The top photo shows only the support without the osteocyte, and the bottom photo shows the osteocyte treated 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 injuries per group). * P <0.05 compared with PCL / PLGA / β-TCP group and #p <0.05 compared with PCL / PLGA / β-TCP / Col group.
FIG. 8A is a histological evaluation of bone regeneration after Goldner's trichrome staining on the injured area of the mouse skull. The arrow indicates the damage site boundary. Scale bar = 2 mm. All photos are 40x magnification. Figures 8b and 8c are histometric morphometric analysis of new bone area and bone density (n = 10, number of injuries). * P <0.05 compared with PCL / PLGA / β-TCP group and #p <0.05 compared with PCL / PLGA / β-TCP / Col group.

Hereinafter, the present invention will be described in more detail with reference to examples. These examples are for illustrative purposes only and are not to be construed as limiting the scope of the present invention.

Example  1: bdECM ( demineralized and decellularized bone extracellular  matrix

In order to prepare a support having excellent bone regeneration effect, bdECM to be used for coating a support was prepared. The detailed manufacturing process is as follows, and the overall process is shown in FIG.

(1) Corrugating

Tibiae were isolated from calves 12 to 24 months old. The bone was fragmented and divided into cancellous and cortical groups, and the spongy group was used. The remaining tissue of the caul explants was removed with PBS (phosphate-buffered saline) containing 0.1% (w / v) gentamicin (Invitrogen, Carlsbad, Calif., USA) and washed. The slices were then frozen in liquid nitrogen and cut into sections 4 x 4 x 4 mm or less. The sections were washed with distilled water, immersed in liquid nitrogen and ground in a coffee mill (Kordia Co., Daegu, Gyeongbuk, Korea).

(2) demineralization and de-saturation

The bone was demineralized by stirring (300 rpm) in 0.5 N HCl (25 mL / well g) at room temperature for 24 hours. After demineralization, the resultant (hereinafter referred to as &quot; bDBM &quot;) was filtered under vacuum and rinsed with distilled water. The lipid was then extracted in a 1: 1 mixture of chloroform (Fisher Scientific, Loughborough, UK) and methanol (Fisher Scientific) for 1 hour and then rinsed with methanol and then with distilled water. The bDBM was snap frozen, lyophilized overnight under reduced pressure, and stored at -20 [deg.] C.

And then rinsing the bDBM with distilled water, 0.05% trypsin (Sigma-Aldrich) and 0.02% EDTA (ethylenediamine tetraacetic acid, Sigma-Aldrich) was stirred for 24 hours in 37 ℃, 5% CO ₂ condition in the mixed solution continued evasion Saturated. The resultant (hereinafter referred to as "bdECM") was rinsed with 4% (w / v) penicillin / streptomycin-containing PBS at 24 ° C for 24 hours to remove residual cellular material. It was then frozen for an instant, lyophilized overnight under reduced pressure and stored at -20 ° C.

Example  2 : bdECM Preparation and Characterization of Coated Supports

(PCL / PLGA /? -TCP) containing PCL, PLGA, and? -TCP was prepared using 3D printing technology and coated with bdECM to prepare a bone regeneration support (PCL / PLGA / TCP / bdECM). (PCL / PLGA /? -TCP / Col) or bdECM (PCL / PLGA /? -TCP) in order to confirm the bone regeneration effect of the completed support. bdECM) -coated support in vitro and in vivo (Fig. 2). A specific manufacturing process is as follows.

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

PCL (MW 43,000-50,000; Polysciences Inc., Warrington, PA, USA) and PLGA (MW 50,000-75,000; Sigma-Aldrich, St. Louis, Mo., USA) were placed in glass containers at 130 ° C for 10 minutes. The melted polymers were mixed with 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 and 140 ° C. The printed scaffold was sterilized in 70% ethanol for 30 minutes 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 in distilled water at a concentration of 10 mg / ml, and as a control, atelocollagen solution (Koken, Shizuoka, Japan) was prepared at a concentration of 10 mg / ml. PCL / PLGA / β-TCP support was coated with the coating solution prepared above. In order to fill the voids in the pores of the support, the support was immersed in the bdECM solution and centrifuged at 1,500 rpm for 1 minute at 4 ° C. The pores of the pores were filled in two steps. The supporter filled with pores was incubated at 37 DEG C for 20 minutes and then dried for 20 minutes under sterilized conditions. This process was repeated three times.

(3) Characterization of the prepared support

In order to analyze the characteristics of the support, the shape of the support was observed with a scanning electron microscope (JSM-5300, JEOL, Tokyo, Japan) at 10 kV. The support was coated with platinum for 120 seconds in 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 scaffolds were used as controls in all experiments. The degree of bdECM or collagen coating on the support was confirmed by X-ray photoelectron spectroscopy (XPS) spectroscopy of nitrogen (N), oxygen (O), and carbon (C) (ESCALAB 220iXL; VG Scientific, East Grinstead, West Sussex , UK).

As a result of observing the support by SEM, the support had a diameter of 4 mm, a height of 1 mm, a line thickness of about 200 μm, and a pore size of about 300 μm. It was confirmed that the PCL / PLGA / β-TCP support had a rough surface because it contained β-TCP powder (FIG. 3A) and the support coated with collagen or bdECM had a smooth surface without blocking the pores (FIGS. 3B and 3C ). The smooth surface of the support means that collagen and bdECM have been successfully coated. In addition, all of the supports have maintained good interconnection, which is very important for bone regeneration because it allows migration of host cells and surrounding osteoblasts from bone marrow.

Meanwhile, the extent to which the collagen or bdECM was coated on the support was quantified by XPS analysis, and is 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: Confirmation of cell proliferation effect by supporter

(1) Cell culture

To analyze in vitro cell behavior, isolated primary osteoblast cells were inoculated at 1 x 10 ⁴ cells per support. Two calvarial osteoblasts were isolated from the two openings of Sprague-Dawley rat embryos (SLC, Tokyo, Japan). For the cell attachment, the supernatant of cells inoculated for one day was immersed in the growth medium, and then the culture medium was supplemented with 50 mM ascorbic acid-2-phosphate (Sigma-Aldrich), 10 mM beta-glycerophosphate (Sigma-Aldrich), and 100 nM dexamethasone (Sigma-Aldrich). The medium was changed daily.

(2) Observation of osteoblast morphology

SEM analysis was performed to observe the morphology of the scaffolds that were inoculated with osteoblasts for 1 day or 14 days. The supernatant was fixed with modified Karnovsky's fixative containing 2% paraformaldehyde and 2% glutaraldehyde in 0.05M sodium cacodylate buffer (SC buffer, pH 7.2; Sigma-Aldrich) for 4 hours at 4 ° C. The primary fixed support was washed with 0.05 M SC buffer 3 times at 4 ° C and immersed in 0.05 M SC buffer containing 1% osmium tetroxide (Sigma-Aldrich) for 2 hours at 4 ° C. It was then dehydrated sequentially and dried with hexamethyldisilazane (Sigma-Aldrich) for 15 minutes. The cell morphology on the supporter and support was confirmed at 15 kV using FE-SEM (JEOL). All specimens were plated on carbon tape and plated for 60 seconds using a sputter coater.

(3) Cell proliferation assay

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 DEG 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 reincubated with fresh culture medium.

Cells were inoculated and the morphology of the supporter was confirmed by SEM. As a result, it was confirmed that the cells adhered on the supporter on the first day, and that the adherent cells were grown through the pores of the supporter (14 days) 4a). In particular, the cells of the PCL / PLGA / β-TCP / bdECM scaffold showed a more ordered arrangement close to the anisotropic shape of the collagen matrix. Such forms are known to affect the mechanical properties of bone tissue.

Furthermore, the absorbance was measured in the cell proliferation assay. As a result, it was confirmed that the osteoblast was more adhered on the collagen or bdECM-coated supporter than the uncoated supporter. From this result, . The osteoblast proliferation rates of the three groups were similar to each other (Fig. 4B).

The bdECM coating reduced the pore size of the support by about 30% (Fig. 3), but further increased osteoclast attachment. This result means that the pores of the support are not filled with bdECM, and thus the osteoblasts can migrate into the support. On the other hand, the collagen coating showed a lower cell adhesion ratio than the bdECM coating, and the bdECM can provide a better environment than the collagen coating.

As a result, it can be seen that even when the bone regeneration support coated with the bdECM of the present invention is actually implanted into the human body through the above-described experiment, cells of the surrounding tissues can effectively propagate along the pores of the support.

Example  4 : Rat  origin Osteocyte  Confirmation of improvement of bone differentiation

(1) Confirmation of expression of ALP

In order to confirm whether the supporter of the present invention has an effect of improving bone differentiation, the bone differentiation pattern was observed while the rat-derived osteoblasts were cultured on a supporter. First, the expression of ALP, which is most known as a biochemical marker of osteoblast activity, was confirmed by immunofluorescence staining for cells inoculated on each scaffold. The supernatant from which osteoblast cells were cultured in the osteogenic medium was washed with PBS and fixed with 4% paraformaldehyde solution on the 14th day. The cells were then treated with PBS containing 2% BSA and immunoprecipitated with an anti-ALP antibody (1: 200; Santa Cruz Biotechnology, Santa Cruz, Calif., USA) to prevent nonspecific binding, followed by Alex Fluor 488 goat anti-rabbit And incubated with antibody (1: 100; Invitrogen). All specimens were counterstained with DAPI (1: 100; Sigma-Aldrich) and observed with a laser scanning confocal microscope (Olympus FluoView ^™ FV1000, Tokyo, Japan).

ALP activity was quantified on day 14 with p-NPP (p-nitrophenyl phosphate, Sigma-Aldrich). The osteocytes inoculated into the support were lysed with RIPA lysis buffer (Millipore, Billerica, MA, USA) and the lysates were incubated with ALP substrate buffer containing 5 mM pNPP for 30 min at 37 &lt; 0 &gt; C. The enzyme activity of ALP was measured at 405 nm using a microplate reader.

As a result, the expression of ALP marker expressed by osteoblasts was confirmed throughout the support, which means that the osteoblast activity is very high on the support (Fig. 5A). Among these, the cells on the bdECM-coated supporter were aligned on the first day as in the SEM analysis. Quantitatively measured ALP activity has the highest activity in lysates isolated from bdECM coated supports (Figure 5b).

(2) Calcium accumulation confirmation

When osteoblasts were cultured on a bone differentiation medium on supporter, the degree of bone mineralization was ascertained 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 cells inoculated with the cells were fixed with 4% paraformaldehyde to stain calcium accumulation. The cells were then stained with 2% Alizarin Red S (Sigma-Aldrich) solution (pH 4.3) for 20 min at room temperature. The calcium accumulation area was observed with a digital microscope (Dino lite, New Taipei, Taiwan) and the amount of 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 the osteoblasts on the support coated with collagen or bdECM contained a visually recognizable level of calcium mineral after 21 days. However, a greater amount of calcium mineral was observed in the bdECM-coated support than in the collagen-coated support (Fig. 6A). The results of colorimetric analysis were also consistent with the staining results (Fig. 6b). The calcium content in the bdECM-coated PCL / PLGA / β-TCP scaffold was significantly higher than that of the other groups, especially in the course of time (FIG. 6c).

(3) Expression of mRNA expressing bone differentiation

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

Gene Primer sequences 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 osteogenesis including osteocalcin (OCN) and type I collagen (COL1A1) was also higher in bdECM coated PCL / PLGA / β-TCP support groups. These results also suggest that bdECM-coated scaffolds have excellent bone regeneration ability as well as mineralization as well as tissue maturation (Fig. 6d).

As a result of the above experimental results, it can be seen that the osteoblast cultured on the bdECM-coated PCL / PLGA / β-TCP scaffold exhibits significantly improved in vitro bone formation activity as compared with the other groups, . bdECM contains bone morphogenic 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 / β-TCP / bdECM showed the highest cell attachment rate. This means that various cell attachment sites were provided by the bdECM bioactivity factor.

In conclusion, it was found through the results of ALP expression, Alizarin Red S staining, and qRT-PCR of OCN and COL1A1 that the bone regeneration support coated with bdECM of the present invention promotes bone differentiation.

Example  5: Confirmation of in vivo bone regeneration effect of supporter

In order to confirm the in vivo bone regeneration effect of PCL / PLGA / β-TCP / bdECM scaffolds, we used a mouse double open-wound injury model and transplanted each support with or without osteoblasts in the mouse, Efficiency was confirmed. Specific experimental methods and contents are as follows.

(1) Two open-mouth damage models for evaluating bone regeneration

Six-week old mice 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 mouse hair was pushed, the center of the skull was cut longitudinally from the nose to the back of the neck, and the periosteum was lifted to expose the surface of the parietal bone. Two round bone injuries (4 mm in diameter) were made in the skull using a surgical trephine burr (Ace Surgical Supply Co., Brockton, MA, USA) and a low-speed micromoter. The size of the lesion was determined to be catastrophic for the mouse skull injury model. The drill site was cleaned with saline, and the bleeding site was electrically eroded. Five mice (10 lesions) were used per group.

(2) micro-CT analysis

Eight weeks after transplantation, mice were euthanized with CO ₂ and the skull was harvested for analysis. Bone formation was assessed by micro-CT scans (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, specimens were prepared for histomorphometric analysis. Specimens were immersed in 10% (v / v) buffered formalin solution, dehydrated in increasing concentrations of alcohol solution, clarified with xylene, and embedded in paraffin. Microtomy and grinding techniques were used to obtain one sagittal and one frontal section from two specimens per mouse. The sections were stained with Goldner's trichrome stain. The bone formation area was determined by calculating the percentage of newly formed mineralized bones using Adobe Photoshop software (Adobe Systems, Inc., San Jose, Calif., USA). Percentage of bone formation area at injured area was calculated as (new bone area / bone damage area) x 100. Bone density was calculated as [new bone area / (new bone area + fibrous tissue area + remaining biomaterial area)] x 100.

As a result, micro-CT (microcomputed tophography) photographs showed that PCL / PLGA / β-TCP / bdECM scaffold transplantation further improved bone regeneration compared to other scaffold transplantation (FIG. In addition, PCL / PLGA / TCP / bdECM scaffolds significantly increased regenerated bone volume compared to other scaffolds (Fig. 7B). In particular, PCL / PLGA / TCP / bdECM scaffolds showed improved bone regeneration and increased regenerated bone volume in the absence of osteoblasts, similar to that of osteoblasts. This means that the PCL / PLGA / TCP / bdECM scaffold of the present invention can bring about bone regeneration effect only by transplanting the supporter alone without mixing with other cells such as osteoblasts.

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

In addition, transplantation of PCL / PLGA / β-TCP / Col supporters with osteoblasts significantly improved bone formation area and bone density compared to no osteoblasts (FIGS. 8b and 8c). In addition, bone graft area and bone density were improved when PCL / PLGA / β-TCP / bdECM were transplanted with osteoblasts. However, when PCL / PLGA / β-TCP was transplanted with osteoblasts, it was confirmed that only fibrous tissue was formed similarly to the case of osteoblast-free transplantation.

Since the scaffold prepared by the 3D printing technique of the present invention can control the microstructure and the interconnected pore structure that can improve the growth of the patient tissue into the scaffold implanted at the site of bone injury, have. The PCL / PLGA / β-TCP / bdECM of the present invention can allow host cells such as MSC and osteoblast cells to grow inside, and the cells migrated into the support are contacted with biomolecules contained in bdECM to be differentiated into bone .

The support (PCL / PLGA / β-TCP) made from uncoated 3D printing made of synthetic artificial materials lacks bone-specific bioactive signals that lead to bone regeneration. In addition, collagen-coated PCL / PLGA / β-TCP / Col scaffolds did not effectively treat bone damage alone without osteoblasts. This is because osteogenic biomolecules are lacking compared to PCL / PLGA / β-TCP / bdECM. The collagen's in vitro cell adhesion rate and osteogenesis activity may have affected these results. On the other hand, PCL / PLGA / β-TCP / bdECM showed no difference in the presence or absence of osteoblasts. This may be because a significant amount of host cells migrated compared to PCL / PLGA / β-TCP / Col. Therefore, PCL / PLGA / β-TCP / bdECM does not require oocyte cultured in vitro in the treatment of bone damage, bdECM is prepared by depleting DBM. The support of the present invention is immune- It is possible to reduce the side effects such as the reaction and reduce the cost and time for cell culture.

In conclusion, the bdECM-coated support for bone regeneration according to the present invention promotes osteocyte proliferation and improves bone differentiation and has an excellent bone regeneration effect, so that it can be used as a support for bone regeneration.

From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all aspects and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention without departing from the scope of the present invention as defined by the appended claims.

<110> Dankook University Cheonan Campus Industry Academic Cooperation Foundation <120> A scaffold with bone derived extracellular matrix for bone          regeneration <130> KPA150292-KR <160> 6 <170> Kopatentin 2.0 <210> 1 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> GAPDH-F Primer <400> 1 ggcacagtca aggctgagaa tg 22 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> GAPDH-R Primer <400> 2 atggtggtga agacgccagt a 21 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> OCN-F Primer <400> 3 ggtgcagacc tagcagacac ca 22 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> OCN-R Primer <400> 4 aggtagcgcc ggagtctatt ca 22 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> COL1A1-F Primer <400> 5 acgtcctggt gaagttggtc 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> COL1A1-R Primer <400> 6 cagggaagcc tctttctcct 20

Claims (15)

A first layer comprising a biodegradable polymer; And
and a second layer comprising a demineralized and decellularized bone extracellular matrix (bdECM), said second layer surrounding said first layer.
The biodegradable polymer of claim 1, wherein the biodegradable polymer is selected from the group consisting of poly-L-lactic acid, poly-D-lactic acid-co- glycolic acid, poly-L- At least one member selected from the group consisting of polyglycolic acid, poly-caprolactone, poly-valerolactone, poly-hydroxybutyrate and poly-hydroxyvalerate.
The support for bone regeneration according to claim 1, wherein the first layer further comprises a ceramic material.
The method of claim 3, wherein the ceramic material is selected from the group consisting of alumina, zircon, silica, magnesia, zinc oxide, titanium oxide, mixed oxide, barium titanate, silicate, hydroxyapatite, and tricalcium phosphate Wherein the support is at least one selected from the group consisting of:
The support for bone regeneration according to claim 1, wherein the second layer is formed by coating the first layer with a coating solution in which bdECM is dissolved in distilled water at 5 to 20 mg / ml.
The support for bone regeneration according to claim 1, wherein the bone regeneration is by promoting bone differentiation.
A method for producing a scaffold for bone regeneration comprising the step of coating a support comprising a biodegradable polymer with a solution comprising bdECM (demineralized and decellularized bone extracellular matrix).
The biodegradable polymer of claim 7, wherein the biodegradable polymer is selected from the group consisting of poly-L-lactic acid, poly-D-lactic acid-co- glycolic acid, poly- Wherein at least one member selected from the group consisting of glycolic acid, poly-caprolactone, poly-valerolactone, poly-hydroxybutyrate, and poly-hydroxyvalerate is used.
8. The method of claim 7, wherein the support further comprises a ceramic material.
The method of claim 9, wherein the ceramic material is selected from the group consisting of alumina, zircon, silica, magnesia, zinc oxide, titanium oxide, mixed oxide, barium titanate, silicate, hydroxyapatite, and tricalcium phosphate Wherein the support is at least one member selected from the group consisting of polypropylene and polypropylene.
8. The method of claim 7,
A first step of immersing a support containing biodegradable polymer in bdECM and centrifuging;
A second step of keeping the support immersed in bdECM; And
And a third step of drying the support.
12. The method of claim 11, wherein the second step is to keep the support immersed in the bdECM for 5 to 30 minutes.
8. The method of claim 7, wherein the solution containing bdECM is obtained by dissolving bdECM in distilled water at 5 to 20 mg / ml.
8. The method of claim 7, wherein the support is manufactured using a 3D printer.
8. The method of claim 7, wherein the bone regeneration is by promoting bone differentiation.

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