KR102074038B1 - Preparation Method of Customized Bone Graft for 3D Printing - Google Patents

Abstract

The present invention provides a method for producing a 3D printing customized bone support and the 3D printing customized bone support produced thereby. The 3D printing customized bone support produced by the method of the present invention is excellent in biocompatibility and cell adhesion, and when implanted into the bone damage, shows excellent bone formation ability.

Description

Preparation method of customized bone support 3D printing {Preparation Method of Customized Bone Graft for 3D Printing}

The present invention relates to a method for producing a 3D printing customized bone support.

As of 2012, the global industrial 3D printer industry is expected to reach US $ 2.24 billion and reach US $ 13.3 billion by 2021 with rapid growth of more than 19.3% per year. In particular, the application of 3D printing technology by field can be used in various fields such as oral and maxillofacial surgery, plastic surgery, and orthopedic surgery, and the weight of the medical field is expected to increase gradually with the advantage of enabling patient-specific technology. If the successful development of artificial support for tissue regeneration using PCL material in the previous previous research, the present invention establishes the technology of 3D printing process using biodegradable organic / inorganic composite material and biocompatible collagen with specific inducing ability for bone regeneration. And an optimization thereof. The 3D printed artificial support made of PCL material has been made for the patient's customized, but many studies have been conducted, but it is analyzed that the defect site is limited to regeneration into bone tissue.

The present invention has been intensive research efforts to produce a 3D printing customized bone support using xenografts. As a result, the distal bone was pulverized to a nano level to prepare HA (hydroxyapatite) nanoparticles, and then mixed with the molten PCL to prepare a PCL / HA support, and then reacted with an EDC / NHS crosslinked collagen solution for biocompatibility and The present invention was completed by elucidating that 3D printing customized bone support having excellent cell adhesion ability can be prepared.

Accordingly, it is an object of the present invention to provide a method for producing a 3D printing customized bone support.

Another object of the present invention is to provide a 3D printing customized bone support produced by the method of the present invention.

Other objects and advantages of the present invention will become apparent from the following detailed description and claims.

According to one aspect of the present invention, the present invention provides a method for producing a 3D printing customized bone support comprising the following steps:

(a) pulverizing a heterologous bone to produce HA (hydroxyapatitde) nanoparticles;

(b) reacting collagen with an EDC (1-ethyl-3- (3-dimethyl aminopropyl) carbodiimide) / NHS ( N- hydroxysuccinimide) solution, neutralizing and washing the cellulose dialysis membrane to perform crosslinking reaction;

(c) mixing HA nanoparticles with molten PCL (polycaprolactone) and fabricating a bone support using a 3D printer; And

(d) reacting the bone support with the EDC / NHS crosslinked collagen solution.

The present invention has been intensive research efforts to produce a 3D printing customized bone support using xenografts. As a result, crushed xenografts to nanoscale to produce HA nanoparticles, mixed with molten PCL to produce a PCL / HA support, and then reacted with EDC / NHS crosslinked collagen solution to increase biocompatibility and cell adhesion. It has been found that excellent 3D printing custom bone supports can be produced.

The present invention is to prepare a 3D printing customized bone support using nano-level pig bone-derived xenograft, mixed with PCL to produce a customized 3D printing PCL / HA support, and then reacted with EDC / NHS crosslinked collagen solution Overcoming the limitations of bone regeneration, which is a disadvantage of 3D printing bone support using PCL material in previous studies, by identifying that 3D printing customized bone support with excellent biocompatibility and cell adhesion ability can be prepared.

The manufacturing method of the 3D printing customized bone support of the present invention will be described in detail for each step as follows:

Step (a): Preparation of HA Nanoparticles

According to the present invention, first, the HA bone is pulverized to produce HA nanoparticles.

The heterologous bone used in the present invention is selected from the group consisting of bovine bone, horse bone and pork bone. Preferably, swine spongy bone is used as the xenograft.

According to one embodiment of the present invention, in order to manufacture HA nanoparticles using a mortar and pestle, the bone is divided, and then dispersed in water, and then the ultra-fast nanoparticle disperser and milling of 1 mm zirconium ball to the nano level. Crush. The prepared HA nanoparticles have a size of 100-300 nm, more preferably 200-300 nm.

Step (b): Collagen Crosslinking Reaction

Collagen extracted from porcine skin is reacted with EDC (1-ethyl-3- (3-dimethyl aminopropyl) carbodiimide) / NHS ( N- hydroxysuccinimide) solution, and neutralized and washed on a cellulose dialysis membrane to perform crosslinking reaction.

Extracting collagen from pig skin may use a variety of methods known in the art. Preferably it is extracted according to the contents described in Example 1-2 below.

According to one embodiment of the present invention, the collagen solution extracted from porcine skin is reacted with EDC / NHS solution at pH 5.5 and neutralized and washed for 1-2 days in a cellulose dialysis membrane to perform crosslinking reaction.

In the present invention, since the crosslinking reaction of collagen is performed in the liquid state, the reaction proceeds uniformly.

Step (c): Golgi Support Preparation

The molten PCL of the HA nanoparticles prepared in step (a) is preferably mixed at a ratio of 1: 9 to 4: 6 (w / w). More preferably, the mixing is carried out at a ratio of 1: 9 to 3: 7 (w / w), most preferably 2: 8 (w / w).

According to one embodiment of the present invention, HA nanoparticles and molten PCL are mixed and then placed in a spray syringe to maintain a temperature of 120-150. Preferably 130-140, more preferably 135 temperature is maintained. Then, PCL / HA support is produced using an extruded 3D printer. The size and shape of the PCL / HA support can be varied depending on the location of the implant.

Step (d): Golgi Support and Collagen Solution Reaction

After reacting the EDC / NHS cross-linked collagen solution to the PCL / HA support produced through the 3D printer and lyophilized to finally produce a customized 3D printing bone support.

According to another aspect of the present invention, the present invention provides a 3D printing customized bone support produced by the manufacturing method.

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides a method for producing a 3D printing customized bone support and a 3D printing customized bone support produced thereby.

(b) 3D printing customized bone support produced by the method of the present invention is excellent in biocompatibility and cell adhesion, and when implanted into bone defects, it shows excellent bone formation ability can be used industrially as a bone graft material have.

Figure 1a is an electron micrograph of the HA nanoparticles prepared by using a pig spongy bone removed organic matter.
Figure 1b is a combination of PCL / PLGA / TCP, PCL / PLGA / Bio-OSS and PCL / made of a mixture of TCP powder, Bio-OSS powder, pig spongy bone bone powder (PBP) and PCL / PLGA removed organic matter Morphological characteristics of PLGA / PBP 3D bone support were observed by scanning electron microscopy (SEM).
Figure 1c is the result of the CCK-8 analysis to evaluate the cell affinity of the PCL / PLGA / TCP, PCL / PLGA / Bio-OSS and PCL / PLGA / PBP 3D bone support of FIG.
Figure 2 is a process diagram for the development process of PCL / TCP, PCL / HA and Col-PCL / HA 3D printing customized bone support.
3 is a result of evaluating the compressive strength of the 3D printing bone support.
Figure 4a is a result of evaluating the cell proliferation of PCL / TCP, PCL / HA and Col-PCL / HA 3D printing customized bone support.
Figure 4b is a photograph of the staining cells 7 days cell culture for PCL / TCP and observed under a fluorescence microscope and confocal microscopy.
Figure 4c is a photograph of the staining cells 7 days cell culture for PCL / HA and observed by fluorescence microscope and confocal microscopy.
Figure 4d is a photograph of the stained cells 7 days of cell culture for Col-PCL / HA 3D printing customized bone support and observed by fluorescence microscope and confocal microscopy.
5 is a performance evaluation results of PCL / TCP, PCL / HA and Col-PCL / HA 3D printing custom bone support in the rabbit bone defect model.
Figure 6a is a micro-CT analysis (BV) of the PCL / TCP, PCL / HA and Col-PCL / HA 3D printing custom bone support in the rabbit bone defect model.
FIG. 6B shows the results of micro-CT analysis (Tb.Th) of PCL / TCP, PCL / HA and Col-PCL / HA 3D printing custom bone support in rabbit bone defect model.
FIG. 6C shows the results of analysis of Micro-CT (Tb.N) of PCL / TCP, PCL / HA and Col-PCL / HA 3D printing custom bone support in rabbit bone defect model.
FIG. 6D shows the results of analysis of Micro-CT (Tb.Sp) of PCL / TCP, PCL / HA and Col-PCL / HA 3D printing custom bone support in rabbit bone defect model.
Figure 7a is a photograph observed by optical microscopy after non-limeous tissue sampling for the control group (Critical defect group) in the rabbit bone defect model.
Figure 7b is a photograph observed by optical microscopy after non-limbized tissue specimens for the performance evaluation of PCL / TCP in the rabbit bone defect model.
Figure 7c is a photograph observed by optical microscopy after the non-limeous tissue specimens for the performance evaluation of PCL / HA in the rabbit bone defect model.
Figure 7d is a photograph observed by optical microscopy after non-limbized tissue specimens for the performance evaluation of Col-PCL / HA 3D printing custom bone support in the rabbit bone defect model.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Example 1: Development of 3D printing customized bone support

1-1. Manufacture of HA (Hydroxyapatite) nanoparticles

Porcine spongy bone from which organic matter was removed was ground using a mortar and pestle to disperse bone powder in 500 ml water, and then ultra-high nano-disperser (Laboratory Agitator Mill MiniCer, NETZSCH) and 1 mm zirconium ball were used. 3 hours milling (2,500 rpm, feed level 4, 1.2 bar) was used, and this was repeated three times to prepare HA nanoparticles (200-300 nm).

1-2. Produced using 3D printing based on particle size Corrugated  Cell affinity assessment

Porcine spongy bone (PBP) from which Bio-OSS bone material and organic material is removed is made into particles of 1 μm to 100 μm and uniformly mixed with PCL / PLGA at a weight ratio of 2: 8, and the prepared material is placed in a spray syringe and 3D printed PCL. / PLGA / Bio-OSS and PCL / PLGA / PBP bone support were produced. TCP (b-tricalcium phosphate nanopowder, Berkeley Advanced Biomaterials Inc. USA) was uniformly mixed with PCL / PLGA and weight ratio 2: 8 as a powder to produce 3D printed PCL / PLGA / TCP corrugated support in the same manner (FIG. 1b). .

Scanning electron microscopy (SEM) photographs of Bio-OSS particles made of 100 μm or less and PBP particles showed that the Bio-OSS particles and PBP particles were larger than TCP particles (100 nm) and had irregular shapes. Was observed.

3D printing PCL / PLGA / TCP corrugated supports made of homogeneous TCP particles have a scanning electron microscope compared to PCL / PLGA / Bio-OSS and PCL / PLGA / PBP corrugated supports made of particles of irregular shape and size. In the SEM) picture, the shape was homogeneous and the surface was rough (FIG.

In the CCK-8 kit assay analysis using human-derived mesoderm stem cells, the cell growth rate gradually increased as the surface of the particles became more homogeneous and smaller in size (FIG. 1C).

Therefore, it has been proved that the shape and size of the inorganic particles used for the bone support for 3D printing have an effect on the cell proliferation rate. Therefore, 3D printing bone support made of HA (Hydroxyapatite) nanoparticles can have a positive effect on bone formation. I could confirm it.

1-2. Collagen Extraction and Crosslinking

Acetic acid and pepsin were treated to porcine skin to extract collagen, and then crosslinked with 1-ethyl-3- (3-dimethyl aminopropyl) carbodiimide (EDC) / N- hydroxysuccinimide (NHS).

Biomedical collagen extraction was performed according to the following method.

1) Collecting Donpi

Samples were used to receive pig skin at the slaughterhouse.

A pig's dorsal skin is taken with a surgical knife. Peeled skin is fresh skin, ie, peeled within 1 day prior to use, preferably used within 5 hours.

2) swamp blood swelling

The collected skin tissue is cut into 5 cm wide and 15 cm long and then immersed in 1 M (pH 2.18) acetic acid solution, followed by swelling for 4 to 24 hours to remove the epidermal layer and subcutaneous fat.

3) washing and storage

The separated dermis is washed with sterile distilled water, filtered, and drained, and then packed in 50 g each and stored at -80.

4) Collagen Extract

After thawing the cryopreserved dermis, soak in 99% ethanol and sterilize and degrease for 4 to 24 hours. Prepare dermis (50 g) and 2.5 L of acetic acid (0.5 M, pH 2.31) and grind the mixture into a grinder with 0.5 M acetic acid. The solution is then treated with pepsin (5 g,> 400 units / mg) and stirred for 4 to 24 hours.

5) Vacuum filtration

Filtration is carried out in 30 water ore (7 ply) to remove impurities (hair, etc.).

6) collagen precipitation

To 100 ml of the vacuum filtered solution is added dropwise NaCl (5 M, 17.5 ml) solution and after 4 to 12 hours stirring collagen precipitate is obtained.

7) Centrifugation

The precipitate is centrifuged at 7,000 rpm at 4 for 10 minutes to obtain pellets.

8) cellulose dialysis

50 ml of pellet was diluted in 250 ml of sodium phosphate buffer (mixing & dilution solution with 0.2 M NaH ₂ PO ₄ and 0.2 M Na ₂ HPO _{4 ,} pH 7.4) and 50 ml of 99% ethanol and tubed on cellulose dialysis membrane to obtain 7 L of distilled water. Dialysis for 24 hours. Distilled water should be exchanged three times in total.

9) Lyophilization

The dialysis result obtained by the above method is centrifuged at 12,500 rpm for 4 hours for 1 hour, and then the supernatant is lyophilized for 2-3 days.

In the case of products made using pure collagen, the physical properties (tensile strength, elasticity, degradability, etc.) are weak, so it is difficult to perform a procedure requiring physical properties. Therefore, cross-linking between molecules is required in order to facilitate manipulation in surgical operation and to prevent rapid degradation in vivo.

As a method, there are largely a method using a chemical crosslinking agent (reacts with glutaraldehyde, hexamethylene diisocyanate, carbodiimide, etc.) and a physical method (dehydration heat treatment, irradiation with ultraviolet rays or microwaves). These cross-linking reactions can cause calcification and inflammatory reactions in tissues. Thus, in vitro cross-linking improved mechanical and physical properties. However, studies on maintaining biocompatibility without side effects are still ongoing.

In particular, chemical crosslinking methods are widely used in collagen-based biomaterial research and development because they can easily induce crosslinking reactions without special equipment. As a side effect, cell and tissue toxicity may be caused by residual material after crosslinking reaction. One commonly used crosslinking agent is 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC), which induces cross-linking between collagen molecules and is then removed in the washing process. This is because it is relatively small. The efficiency of crosslinking reaction by EDC can be increased by the use of N-hydroxysuccinimide (NHS).

In general, the cross-linking process of collagen-based biomaterials using EDC / NHS is as follows. 1) Preparation of the desired concentration of collagen solution (neutralization reaction is optional), 2) Preparation and drying of collagen-based biomaterials, 3) Reaction of 1 with EDC and NHS solution (pH 5.5), 4) Neutralization (Optimal EDC crosslinking reaction) To neutralize acidity for bioapplication) and washing to remove product after reaction with unreacted material, and 5) drying.

Biomedical collagen crosslinking reaction in the present invention was carried out according to the following method.

1) Prepare collagen solution of desired concentration.

2) React 1) with EDC and NHS solution (pH 5.5).

3) Neutralize and wash in cellulose dialysis membrane for 1-2 days at room temperature (20).

4) Collagen-based biomaterials are manufactured and dried.

The difference from the conventional method is that EDC / NHS crosslinking reaction proceeds in the solid state, so if the product thickness is thick, the crosslinking reaction may occur relatively weakly inside, and neutralization and washing may be incomplete. This may result in degradation of biomaterials and cell and tissue toxicity.

However, the method developed by the present inventors can induce a uniform reaction compared to the conventional method by proceeding the EDC / NHS crosslinking reaction in a liquid state (collagen solution), not a solid, and neutralized and washed through dialysis Minimize possible side effects.

1-3. Material Preparation for 3D Printing

HA or β-TCP (average diameter: 100 nm, Berkeley Advanced Biomaterials Inc., Berkeley, Calif., USA) particles were melted with molten PCL (19561-500G, 43,000-50,000 Mw; Polysciences Inc., Warrington, PA, USA). The mixture was uniformly mixed at a weight ratio of 2: 8, and the prepared material was placed in a spray syringe to maintain a temperature of 135.

1-4. Support fabrication using 3D printing

PCL / TCP or PCL / HA supports were fabricated using an extrusion-based 3D printing system. In order to implant the rabbit radial defects, a cylindrical support having a diameter of 4 mm and a length of 20 mm was prepared, the thickness of the sprayed strand was 300 μm, and the pore size was 400 μm. Collagen-coated PCL / HA support was prepared by lyophilization of the 3D printed PCL / HA support with an EDC / NHS crosslinked collagen solution for 24 hours (FIG. 2).

Example 2: 3D printing bone support performance evaluation

2-1. Evaluation of Compressive Strength of 3D Printing Corrugated Support

PCL / TCP or PCL / HA supports were fabricated to a size of 5 × 5 × 5 mm, and some PCL / HA supports were lyophilized after 24 hours reaction with EDC / NHS cross-linked collagen solution. The compressive strength of each sample was measured using a universal testing machine (Instron 3304, Instron, USA) with the crosshead moving speed of 1 mm / min.

As a result of the evaluation of the compressive strength, as shown in FIG. Although the PCL / TCP support has a higher compressive strength than the PCL / HA support, it is believed that the collagen coating significantly increases the compressive strength when the Col-PCL / HA has a higher compressive strength than the PCL / TCP support.

2-2. Cell proliferation assessment on 3D scaffold

MG63 cells were cultured in a 5% CO ₂ incubator using DMEM medium containing 10% FBS and 1% penicillin-streptomycin. Cells were cultured after aliquoting with 1 × 10 ⁵ cells / scaffold to confirm cell adhesion to the support. After 1, 3, and 7 days of incubation, the CCK reagent was treated with 30 μl, and then reacted in the incubator for 2 hours, and then measured at 450 nm using a multiplate analyzer.

As can be seen in Figure 4a, as the cell culture period increases the amount of cells was confirmed that the support is not cytotoxic, through the results of the day 7 cell culture collagen-coated PCL / HA support to the other group It was confirmed that the cell affinity was significantly higher than. As can be seen in Figures 4b to 4d, the cells were stained on day 7 of the cell culture and the same results were obtained in fluorescence microscopy and confocal micrographs.

2-3. Evaluation of Bone Formation Ability of 3D Support in Rabbit Bone Defect Model

After forming a 20 mm long bone defect between the rabbit radial bone, PCL / TCP, PCL / HA or collagen coated PCL / HA (Col-PCL / HA) scaffolds were implanted, respectively. The implant was fixed with wires to minimize movement of the support at the implant. After 8 weeks of transplantation, rabbits were sacrificed to perform a simple radiograph of the transplanted area, tissues were collected, micro-CT analysis and non-regressive tissue specimens were prepared, and histological analysis was performed.

5 is a normal radiograph and micro-CT photograph 8 weeks after the formation of the 20 mm bone defect in the rabbit radius and after fixing the 3D support.

As a result, as can be seen in Figures 6a to 6d, it was confirmed that the bone formation ability increased in the order of PCL / TCP, PCL / HA, Col-PCL / HA. In addition, as can be seen in Figures 7a to 7d, after the production of non-mineral tissue specimens, it was confirmed that the bone formation ability increased in the order of PCL / TCP, PCL / HA, Col-PCL / HA in the results of histological analysis.

Claims (8)

A method of manufacturing a patient-specific 3D printing bone support comprising the following steps:
(a) pulverizing pork bone to produce HA (hydroxyapatitde) nanoparticles;
(b) crosslinking the collagen solution with EDC (1-ethyl-3- (3-dimethyl aminopropyl) carbodiimide) / NHS (N-hydroxysuccinimide) solution, neutralizing and washing in a cellulose dialysis membrane;
(c) mixing the HA nanoparticles with molten PCL (polycaprolactone) and fabricating a bone support using a 3D printer; And
(d) coating the bone support with the EDC / NHS crosslinked collagen solution and not including washing after the coating.
delete The method of claim 1, wherein the collagen is extracted from pig skin.
The method according to claim 1, wherein the HA nanoparticles of 100-300 nm obtained by milling the pork bone and dispersed in water, and then milling with ultra-high nano-disperser and zirconium ball It is HA nanoparticles, a method for producing a patient-specific 3D printing bone support.
The method of claim 1, wherein the HA nanoparticles and the molten PCL in step (c) is mixed at a ratio of 1: 9 to 2: 8 (w / w).
The patient-specific 3D printing Golgi of claim 1, further comprising mixing HA nanoparticles and the molten PCL in step (c), and then placing the molten PCL in a spray syringe. Method of making a delay.
The method of claim 1, wherein the manufacturing method further comprises lyophilizing after step (d).
A patient-specific 3D printing bone support manufactured by the method of any one of claims 1 and 3 to 7.

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