KR20180076478A - PLGA/Hydroxyapatite hybrid bone graft containing duck's feet-derived collagen and a fabrication method of it - Google Patents

Abstract

The present invention relates to a hybrid bone graft material containing duck′s feet-derived collagen and to a manufacturing method thereof, and specifically, to a bone graft material, which improves mechanical properties and greatly reduces inflammatory reactions by mixing a duck′s feet-derived collagen, a natural polymer, with poly (lactic-coglycolic acid) (PLGA), a bio-synthetic polymer, and hydroxyapatite (HAp), a bioceramic-based bone inducer, and to a manufacturing method thereof.

Description

TECHNICAL FIELD [0001] The present invention relates to a hybrid bone graft material containing collagen derived from flippers and a method for manufacturing the hybrid bone graft material,

The present invention relates to a hybrid bone graft containing a collagen derived from flakes and a method for producing the same, and more particularly, to a biodegradable polymeric material such as poly (lactic-coglycolic acid) (PLGA) (HAp), which is a ceramic-based bone inducing material, to improve mechanical properties and significantly reduce inflammation, and a method for producing the same.

Bone defects are often caused by injuries or fractures as well as diseases such as arthritis and osteoporosis, and there is a demand for replaceable bone graft materials to maintain their function. Bone defects can cause functional, social, and mental problems. [S. Bhumiratana, JC Bernhard, DM Alfi, K. Yeager, RE Eton, J. Bova, F. Shah, JM Gimble, MJ Lopez, SBEisig, G. Vunjak-Novakovic, et al. , Tissue-engineered autologous grafts for facial bone reconstruction , Science translational medicine , 8 (343), 343 r83, 2016]

A variety of bone grafts are used to treat bone defects. In the case of autologous bone graft, bone conduction and bone induction are good, but there is a limitation in harvesting. In allografts, a large amount of bone can be obtained, but the risk of infection is high.

Therefore, there is a need for transplantation using biocompatible biopolymers as a tissue engineering treatment method. As mentioned above, the biomaterial should not be toxic to the human body after the use period and after use, and should be made of a material that minimizes adverse effects in the body due to the biodejection reaction at the time of transplantation.

Collagen, which accounts for 80% of the bone organic matter, is the main protein that forms the connective tissue. It has low antigenicity, excellent cell attachment ability, and biodegradability. Collagen is extracted mainly from the bones and skin of cattle and pigs. It causes problems in the stability of acquired infectious diseases due to diseases such as foot-and-mouth disease and mad cow disease.

Therefore, in order to solve the problem of stability of such infectious diseases, Korean Patent No. 10-1489916 (Kang, Gil-sun) has proposed for the first time a technology to use collagen derived from flippers with a relatively low infectious disease as a bone graft material. In this patented invention, since collagen extracted from flippers contains a type 1 collagen which maintains the elasticity and flexibility of the bone and has low inflammation, it is proposed to manufacture a support having an optimized condition using bone graft have.

Korean Patent No. 10-1629204 also proposes a technique relating to silk fibroin / collagen composite implants using a collagen derived from flippers.

However, despite the excellent effect of collagen derived from flippers, the patented inventions have a limited problem of being used as a graft material due to their low mechanical properties, and development of improved bone graft materials is urgently required.

Therefore, in the research and development field of bone graft materials, there is a great need to develop a new bone graft material having superior mechanical properties as well as solving the aforementioned problem of infectious diseases.

Korean Patent No. 10-1489916 Korean Patent No. 10-1629204

In order to solve the problems of the prior art as described above, the present invention provides a collagen-derived collagen, which has advantages such as low antigenicity, biodegradability, and biocompatibility, To improve the physical properties of the bone graft material by complementing the disadvantage that the mechanical strength is lowered when the support is manufactured.

 Therefore, it is an object of the present invention to provide a collagen-based collagen composition that uses Duck's feet-derived collagen and utilizes PLGA (poly (lactic-coglycolic acid)) and HAp (hydroxyapatite) And to provide a bone graft material with a new structure applicable as a bone substitute having mechanical strength.

Another object of the present invention is to provide a hybrid osteoclast, which is excellent in bone regeneration effect without any concern for infection diseases, further increases the activity of alkaline phosphatase after bone grafting, cell proliferation, and bone mineralization, Bone graft material.

Still another object of the present invention is to provide a hybrid bone graft material composition containing bone marrow-derived mesenchymal stem cells in a new bone graft material as described above so as to have better human applicability.

It is still another object of the present invention to provide a method of manufacturing the hybrid bone graft material of the new composition.

In order to solve the above-mentioned problems, the present invention provides a hybrid bone graft material characterized in that PLGA (poly (lactic-coglycolic acid)) and HAp (hydroxyapatite) are contained in collagen derived from flippers.

The present invention also provides a hybrid bone graft material comprising bone marrow-derived mesenchymal stem cells in addition to the hybrid bone graft material containing PLGA and HAp as the above-described collagen derived from the flippers.

The present invention also provides a method for preparing a PLGA solution, comprising: preparing a PLGA solution;

Mixing powdery HAp with collagen derived from powdery flakes into a PLGA solution; And

Preparing a bone graft material by solvent casting or salt extraction;

Wherein the collagen-derived collagen-containing collagen-containing collagen-containing collagen-containing collagen-containing collagen-containing collagen-containing collagen-containing collagen-containing collagen-containing collagen-

By constructing the new composition as described above, the present invention can provide a bone regeneration effect in comparison with a bone graft made only of PLGA / HAp or a bone graft containing only DC and PLGA, which does not contain existing bone graft materials or flippers collagen (DC) There is an excellent effect.

Particularly, in the case of a bone graft material obtained by seeding a bone graft material prepared from a bone graft material according to the present invention, the activity of the alkaline phosphatase, the proliferation of the cell, In addition, it was confirmed that the bone mineralization was remarkably increased, and since the inflammation reaction is greatly reduced while having the advantage of the collagen derived from the flippers, the problem caused by the infectious diseases hardly occurs.

1 is a photograph showing a naked eye image of a bone graft material of 10, 20, 40, 60 and 80 wt% DC / PLGA / HAp including PLGA / HAp bone graft materials manufactured according to the present invention.
FIG. 2 is a graph showing FT-IR for PLGA / HAp bone graft materials, DC / PLGA / HAp bone graft materials, collagen derived from pigs, PLGA, and HAp prepared for comparison of characteristics in the present invention.
FIG. 3 shows the compressive strengths of DC / PLGA / HAp bone graft materials prepared according to the present invention.
Figure 4 shows the porosity of DC / PLGA / HAp bone grafts prepared according to the present invention.
5 is an SEM photograph showing the size and shape of the pores of the DC / PLGA / HAp bone graft materials prepared according to the embodiment of the present invention.
FIG. 6 shows the results of evaluation of cell proliferation capacity of DC / PLGA / HAp bone grafts prepared according to the present invention.
FIG. 7 shows results of evaluation of alkaline phosphatase activity of bone marrow-derived mesenchymal stem cells in DC / PLGA / HAp bone grafts prepared according to the present invention.
FIG. 8 is a graph showing the bone-specific gene expression of (a) bone-specific gene and (b) gene expression in DC / PLGA / HAp bone grafts prepared according to an embodiment of the present invention Respectively.
FIG. 9 is a SEM photograph showing attachment and migration of cells in DC / PLGA / HAp bone grafts prepared according to an embodiment of the present invention. FIG.
FIG. 10 shows the degree of mineralization of each bone graft material after implantation of the DC / PLGA / HAp bone graft materials prepared according to the embodiment of the present invention into each bone defect model, using (a) two-dimensional photographs and (b) three- Respectively.
FIG. 11 is a graph showing the bone mineral density, bone mineral density (B), and bone density (B) of each bone graft material after implanting the DC / PLGA / HAp bone graft materials prepared according to the embodiment of the present invention, volume.
FIG. 12 is a graph showing the results of transplantation of DC / PLGA / HAp bone grafts prepared according to an embodiment of the present invention into each bone defect model, and H & E staining, Masson's trichrome staining, and ED- Formation and inflammatory reaction.

Hereinafter, the present invention will be described in more detail as an embodiment.

In the present invention, DC means 'floret collagen', PLGA means 'poly (lactic-coglycolic acid)' and HAp means 'hydroxyapatite'. Also, for example, the expressions such as DC / PLGA / HAp or PLGA / HAp are expressed as meaning that each component is mixed or applied in various conditions and amounts depending on the purpose.

The term " collagen derived from flounder (DC) " in the present invention means not only collagen which has not been sterilized or sterilized by refining derived from flippers, but also derived from collagen components having the same conditions such as ducks and other animal wastes And collagen having substantially the same or similar properties as flake-derived collagen.

In the present invention, the term "bone graft material" refers to a raw material used for bone grafting, means a raw material for constituting or substituting bone in the human body through bone grafting, A composition, a combination, a mixture, a kneaded product, a molded article, a foamed structure, a stem cell-containing implant, and the like, or any one or more of them.

The bone graft material using the flap-based collagen of the present invention can be applied more commercially and advantageously, and various advantages of the bone graft material containing the collagen derived from the flap are retained, but the infectious disease does not occur after the bone graft And to provide a new technology to solve a very important problem.

Preferably, the present invention is characterized in that PLGA (lactic-coglycolic acid) and HAp (hydroxyapatite) are essentially contained in collagen derived from flippers. This form is a special form of bone graft containing collagen derived from flippers, and this form is called a hybrid bone graft.

Herein, what is called hybrid bone graft material includes two or more biomaterials.

The collagen derived from flippers used in the present invention is prepared, for example, by modifying the method proposed in Korean Patent No. 10-1489916 developed by the present inventor. More specifically, for example, it is possible to selectively clean flippers not contaminated with diseases and to cut them as they are to prepare cutting tissues; A fat removal step in which the cut tissue is removed from the 0.5 M NaOH solution and the organic solvent (washed with a 3: 1 mixture of methanol and chloroform, and further washed with alcohol and acetone); A collagen extraction step in which an organic acid solution (adding 8 times (w / v) of 5% citric acid in a tissue volume) is added to the cut tissue from which the fat has been removed and the resultant is stirred to extract collagen; Passing the collagen solution obtained in the collagen extraction step through a filter (2.0 탆 filter paper, 0.22 탆 membrane), and then removing the impurities by centrifugation; The collagen (DC) derived from flippers can be prepared by the same method as the extraction method of high purity collagen, which comprises washing the collagen solution from which the impurities have been removed and freeze-drying to obtain collagen powder.

According to a preferred embodiment of the present invention, such a DC can be applied in the form of powder.

According to a preferred embodiment of the present invention, DC in the DC / PLGA / HAp bone graft may contain 5-45% by weight of the total composition. More preferably, DC may contain 25-42 wt%, most preferably 35-42 wt%.

According to a preferred embodiment of the present invention, in the DC / PLGA / HAp bone graft material, PLGA may be contained in an amount of 50-90% by weight in the entire structure. More preferably 50-70% by weight, most preferably 50-60% by weight, especially 55% by weight.

According to a preferred embodiment of the present invention, the preferred composition of the DC / PLGA / HAp bone graft material is DC / PLGA / HAp = 5-45 weight / 50-90 weight / -10% by weight.

According to a preferred embodiment of the invention, the DC in the overall composition can be contained in an amount of 70-85% by weight of the PLGA content.

According to a preferred embodiment of the present invention, the hybrid bone graft material containing PLGA and HAp in the above-described collagen derived from the flippers according to the present invention may be preferably applied to the human body by containing bone marrow-derived mesenchymal stem cells.

According to a preferred embodiment of the present invention, bone marrow-derived mesenchymal stem cells may be contained in an amount of 0.1-20 parts by weight, more preferably 1-10 parts by weight, per 100 parts by weight of the DC / PLGA / HAp bone graft material.

The method for manufacturing the hybrid bone graft material of the DC / PLGA / HAp configuration according to the present invention may be performed as follows.

First, a step of preparing a PLGA solution is performed. The preparation of such a PLGA solution may be performed by, for example, dissolving PLGA in methyl chloride.

In the present invention, the powdery HAp is mixed with the powdered collagen (DC) from the flake-formulated PLGA solution. The DC used herein may be one obtained by the conventional method described above. According to the present invention, in the process of extracting collagen from flippers, the methanol and chloroform mixed solutions used for cleaning the flippers may have a volume ratio of 3: 0.5-2, most preferably 3: 0.8-1.2.

The thus mixed mixture can be used to produce a bone graft material using a solvent casting method or a salt extraction method.

According to the present invention, the bone graft material as described above may further contain pore inducing material NaCl particles for the purpose of forming pores. The content can be used in a proportion of 70 to 85% by weight with respect to the whole composition.

The pore-generating material NaCl used in the present invention preferably has a particle size (maximum diameter) of 250 to 355 μm. By using such a pore inducing substance, a bone graft material having pores similar in shape to the bone due to the pore inducing material can be manufactured after manufacturing the bone graft material.

According to the present invention, the DC / PLGA / HAp bone graft material is a composition in which each component is mixed, or a composition made by applying such a composition at a pressure of 40-80 kgf / cm ² .

According to a preferred embodiment of the present invention, the DC / PLGA / HAp bone graft material prepared as described above is sterilized using ethanol, more preferably 70% ethanol, washed with PBS, It is preferable to use stem cells for sowing.

The bone graft material according to the present invention exhibits excellent mechanical properties due to mixing of three components such as DC / PLGA / HAp unlike the conventional bone graft material.

Despite advantages such as low antigenicity, biodegradability, and biocompatibility of flippers collagen derived from flippers, in order to compensate for the disadvantage that the mechanical strength is lowered when the support is manufactured alone, the PLGA and HAp It is possible to provide a goal frame structure having high mechanical strength.

In addition, it greatly reduces the risk of infectious diseases that may occur after bone grafting and is therefore very useful as a bone graft material.

Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention is not limited to the Examples.

Production Example 1: Production method of collagen derived from flippers

After washing the flippers with distilled water, pressure is applied to soften the tissue. To remove fat in tissues, the mixture was stirred at 250 rpm for 24 hours in 0.5 M NaOH and washed with distilled water. The washed tissue was washed twice with 8% (w / v) 5% citric acid (w / v) for 48 hours at 250C at 4 ° C, and then washed with a mixture of methanol and chloroform rpm, and the tissue was pulverized with a blender.

The supernatant is collected and passed through a 2.0 μm filter paper and a 0.22 μm membrane in order to remove any remaining impurities. The pH is adjusted by adding acid and centrifuged (12,000 rpm, 15 min, 4 ° C). The collected collagen was washed twice with 100% alcohol, centrifuged (3,500 rpm, 5 minutes, 4 ° C), and lyophilized and pulverized to obtain powder form.

Examples 1-5, Comparative Example: Preparation of DC / PLGA / HAp bone graft material

Bone grafts were prepared as a support for the preparation and performance of bone graft materials. The prepared support was prepared by solvent casting / salt extraction method using PLGA / HAp as control (Comparative Example 1) and PLGA / HAp support (Example 1-5) containing DC as experimental group. 1 g of PLGA was dissolved in 4 mL of Methyl Chloride and then the high purity collagen extracted from the flippers was added to the PLGA in an amount of 0 (Comparative Example), 10, 20, 40, 60, 80 wt% and a pressure of 60 kgf / cm ² for 24 hours and then placed in a silicon mold. soaked in distilled water to lay over the support prepared in order to remove the NaCl after 24 hours 48 hours distilled water is changed every 6 hours. supporter remove NaCl Was cooled and then lyophilized for 24 hours) as shown in Table 1 below.

DC PLGA HAp Total weight (g) PLGA / HAp 0 One 0.1 1.1 10 wt% DC / PLGA / HAp 0.1 One 0.1 1.2 20 wt% DC / PLGA / HAp 0.2 One 0.1 1.3 40 wt% DC / PLGA / HAp 0.4 One 0.1 1.5 60 wt% DC / PLGA / HAp 0.6 One 0.1 1.7 80 wt% DC / PLGA / HAp 0.8 One 0.1 1.9

The bone graft materials of each support type thus manufactured are shown in comparison with FIG.

1 is a photograph showing a naked eye image of a bone graft material of 10, 20, 40, 60 and 80 wt% DC / PLGA / HAp including PLGA / HAp bone graft material, respectively.

Production Example 2: Cell culture

Bone marrow stem cells (BMSCs) used in this study were isolated from the thighs of four - week - old white New Zealand rabbits.

The bones of the rabbit legs were cut and then cut and harvested using 18 gauge syringes. The collected bone marrow was cultured in a culture vessel at a concentration of 1 × 10 ⁵ cells / mL in a 5% CO ₂ incubator at 37 ° C. using 20% FBS and 100 unit / mL of 1% penicillin / streptomycin (Lonza, USA) culture medium containing α-MEM. When the cells were attached to the culture vessel, the culture medium was exchanged, and the culture medium was exchanged every 2 days. When the bottom of the culture vessel was filled with 80 ~ 90% of the cells, cells were harvested using 0.25% trypsin / 1 mM EDTA.

Experimental Example 1: Characterization of implant materials

1-1. FT-IR analysis

In order to confirm the chemical changes of the graft material, it was analyzed at 4,000 ~ 500 cm ^-1 wavelength using an infrared spectrophotometer (FT-IR, GX, Perkin Elmer, USA). Through this evaluation, it was found that the inherent peaks of PLGA, DC, and HAp appeared in each bone graft material. Most DC / PLGA / HAp supports have an NH peak at DC of approximately 3750-3500 cm ^-1 And the peaks of amide I and amide II were observed between 2000 and 1500 cm ^-1 and that the peak related to the PO bending mode of phosphate group (PO ₄ ^3- ) in HAp appeared between 1250 and 1000 cm ^-1 I could. In the IR spectra of PLGA, the intrinsic peaks at 1750 ~ 1500 cm ^-1 and 1250 ~ 1000 cm ^-1 were also observed in DC / PLGA / HAp scaffolds. From the results of FT-IR measurements, it was found that the IR spectra of DC, PLGA and HAp show distinct IR peaks of DC, PLGA and HAp, and DC, PLGA and HAp It was confirmed that they were chemically mixed well.

FIG. 2 is a graph showing FT-IR for PLGA / HAp bone graft materials, DC / PLGA / HAp bone graft materials for each DC content, collagen derived from flippers, PLGA, and HAp prepared for characterization.

1-2. Evaluation of compressive strength

(TMS-pro, Food Technoogy Corporation, Sterling, Virginia, USA) was used to measure the compressive strength of DC / PLGA / HAp grafts by content. The measurement distance was 1.5 mm and the measurement speed was 10 mm / min , And the measurement force was 1N. The results of this study showed that the compressive strength increased with increasing content of collagen derived from flounder, and showed the highest compressive strength in 80 wt% DC / PLGA / HAp bone graft.

FIG. 3 shows the compressive strengths of the DC / PLGA / HAp bone graft materials prepared through the present experiment.

1-3 Measurement of porosity

To measure the voids in the support made of bone graft material, voids were measured as a percentage using the following equation (1).

[Equation 1]

(Where V ₁ is the volume of distilled water for the first time, V ₂ is the volume of the distilled water after the support is added, and V ₃ is the volume of the distilled water after removing the support)

The results showed that porosity of bone graft material decreased with increasing content of collagen derived from flippers.

Figure 4 shows the porosity of DC / PLGA / HAp bone graft materials.

1-4. Internal morphology observation (scanning electron microscope observation)

(SEM, Hitachi, S-2250N, Japan) to confirm the morphological characteristics of the supporter made of bone graft material. Each of the prepared samples was fixed to a metal plate using a carbon tape and platinum coated with a thickness of 200 in a plasma sputter (Model SC 500K, Emscope, UK) under an argon gas for 2 minutes. The shape and size of the pores of the DC / PLGA / HAp graft were observed using a scanning electron microscope (Hitachi Co, Model S-2250N, Japan).

Through this evaluation, all the bone graft materials produced were in the form of rectangular shaped pores, and most of the pores were about 250 ~ 355μm in size.

FIG. 5 is a SEM photograph showing the size and shape of the pores of the DC / PLGA / HAp bone grafts measured through the present experiment.

Experimental Example 2: Evaluation of in vitro behavior

2-1. Evaluation of cell proliferation rate

Cell growth rate was observed using MTT assay. The cultured cells were seeded at a concentration of 1 × 10 ⁵ cells / scaffold per supernatant, and samples were collected on days 1, 7, 14, 21 and 28, and MTT (3- [4,5- Dimethythiazol-2-yl] -2,5-diphenyltetrazolium bromide, Sigma Co.) was added and incubated for 4 hours in a 5% CO ₂ incubator at 37 ° C. When purple crystals were formed, 1 mL of dimethylsulfoxide solution (Sigma Co.) was added and pipetting was carried out until the crystals were dissolved. The dissolved solutions were dispensed into 96-well plates in an amount of 100 μL, and the absorbance was measured at 570 nm using an ELISA plate reader (E-max, Molecular Device, USA).

Through this evaluation, the cell proliferated gradually in all bone graft materials over the period of culture, and showed the highest cell proliferation among 80wt% DC / PLGA / HAp bone graft materials.

FIG. 6 shows the results of evaluating the cell proliferation capacity of DC / PLGA / HAp bone grafts prepared according to the example.

2-2. Assessment of alkaline phosphatase activity

The bone marrow-derived mesenchymal stem cells were inoculated on the supporter prepared in the above example, and then the alkaline phosphatase (ALP) was measured to confirm bone differentiation. ALP analysis was performed at 1, 7, 14, 21 and 28 days after sowing. The ALP activity kit (Takara) was used for analysis. After removing the supernatant from the supernatant, the supernatant was washed once with 1x PBS and cells were extracted from supernatant with 5% Extraction solution. After adding nNPP solution to the extract solution, the reaction was carried out at 37 ° C for 1 hour, 50 μL of 0.9 N NaOH aqueous solution was injected, and the activity of alkaline phosphatase was analyzed using absorbance at 405 nm.

The activity of alkaline phosphatase increased gradually during the incubation period, and the activity of alkaline phosphatase was highest in 80 wt% DC / PLGA / HAp bone graft. The bone morphogenesis of bone marrow-derived mesenchymal stem cells was most prominent in 80wt% DC / PLGA / HAp bone graft material.

FIG. 7 shows results of evaluation of alkaline phosphatase activity of bone marrow-derived mesenchymal stem cells in DC / PLGA / HAp bone graft materials as described above.

2-3. Gene expression analysis

The expression of bone-specific genes was examined by reverse transcription polymerase chain reaction (RT-PCR). The mRNA was extracted from the cells proliferating in the ball graft material and the cDNA was amplified through the bone-specific cytokines Collagen type I (Col1), osteocalcin (OCN) and Runt-related transcription factor 2 (Runx2) The resulting DNA was electrophoresed on a 1% agarose gel containing EtBr solution in 0.5% TAE buffer and set at 100 V for 30 minutes. The gene was observed at 360 nm ultraviolet wavelength, Expression was quantified by Image J program.

Through this evaluation, it was found that bone-specific gene expression was observed in all the bone graft cells. The quantification of these expression values revealed that bone-specific gene expression was highest in the 80 wt% DC / PLGA / HAp bone graft material I could.

FIG. 8 shows the result of quantitative analysis of (a) bone-specific gene expression and (b) cell gene expression in DC / PLGA / HAp bone graft materials in each bone graft material.

2-4. Observation of attachment and migration of cells inside the graft material (scanning electron microscope observation)

(SEM, Hitachi, S-2250N, Japan) in order to confirm the attachment pattern of bone marrow-derived mesenchymal stem cells in bone graft material. BMSCs were seeded at a density of 1 × 10 ⁵ cells / supporter on the supporter, cultured for 1 and 28 days, and then the culture solution was removed and washed with PBS. This was fixed with 2.5% glutaraldehyde (Sigma) for 24 hours and the dehydration process was performed for 20 minutes using an alcohol gradient solution (50, 60, 70, 80, 90, 100%). The surface of the support was cut and the cross section was observed. Each sample was fixed to a metal plate using carbon dope and platinum coated to 200 thickness with a plasma sputter (Model SC 500K, Emscope, UK) under argon gas for 2 minutes. Cell attachment and morphology of DC / PLGA / HAp grafts were observed using a scanning electron microscope (Hitachi Co, Model S-2250N, Japan).

In this study, cell attachment and migration were observed in DC / PLGA / HAp bone graft materials, and cell attachment and migration were observed to be actively observed in 80wt% DC / PLGA / HAp bone graft material there was.

FIG. 9 is a SEM photograph showing cell adhesion and migration in DC / PLGA / HAp bone graft materials.

Experimental Example 3: Evaluation of in vivo behavior

Sprague Dawley rats (female, 6 weeks old) were purchased from Korean-Japanese experimental animals. After general anesthesia, head hair removal was performed and disinfected with povidone iodine. The upper surface of the skull was exposed by incising the scalp along the midline from the anterior to the posterior part of the frontal bone of the white paper. A 4 mm diameter circular defect was formed on the upper surface of the exposed skull using a 4 mm trephine bur.

In the control group, the control group was the control group, and the experimental group was the PLGA / HAp graft material and the 80 wt% PLGA / DC / HAp graft material. The rBMSCs were sown on the scaffolds, Respectively.

3-1. Micro-CT evaluation

Bone mineral density (BMD) and bone volume (BV) were measured using a CTAn program using Micro-CT (Skyscan 1076, Optoscan, Belgium) , And Percent bone volume (BV / TV) were analyzed. Three - dimensional images of the skull defect model were obtained using the CTvox program.

The results of this study showed that bone mineralization was the most abundant in the 80wt% DC / PLGA / HAp bone graft when the two- and three-dimensional cranial defect models were examined. When quantitative evaluation was made, PLGA / HAp bone graft Bone mineral density, bone volume, and percentage bone volume were found to be highest in 80% by weight of DC / PLGA / HAp bone graft materials.

FIG. 10 shows results of (a) two-dimensional photographs and (b) three-dimensional photographs showing the degree of mineralization in each bone graft material after transplanting DC / PLGA / HAp bone graft materials to bone defect models in the experiment.

Fig. 11 shows the bone mineral density, (b) bone volume and (c) peritoneal bone volume of each bone graft after DC / PLGA / HAp bone grafts were transplanted into the bone defect model. The results are evaluated.

3-2. Histological analysis

After 2, 4, and 8 weeks after transplantation, the tissues were excised and fixed with 4% neutral formalin (Sigma, USA) solution for 24 hours. The cells were demineralized for one day with deionized water, dehydrated with alcohol and embedded in paraffin. 7 μm in thickness and fixed on PLL coated slides. The tissue sections were subjected to deparaffinization and then subjected to H & E, Masson's trichrome staining, and ED-1 staining for histological evaluation. Microscopic photographs (Nikon TE-2000, Japan) were taken at 40 and 100 times.

Through this evaluation, tissue and collagen formation was highest in 80wt% DC / PLGA / HAp bone graft material than PLGA / HAp bone graft material. In addition, the inflammatory response was significantly reduced in 80wt% DC / PLGA / HAp bone graft material compared to PLGA / HAp bone graft material.

FIG. 12 shows the result of the above-mentioned experiment. After DC / PLGA / HAp bone grafts were transplanted into bone defect models, each bone graft material was examined for H & E staining, Masson's trichrome staining and ED- It is a photograph.

The DC / PLGA / HAp bone graft material for bone regeneration according to the present invention significantly reduces infection diseases, unlike conventional bone graft materials, and is particularly useful for tissue regeneration of injured bone due to its excellent mechanical properties. , Tissue engineering and regenerative medicine.

Claims (13)

A hybrid bone graft material containing collagen derived from flippers, wherein PLGA (poly (lactic-coglycolic acid)) and HAp (hydroxyapatite) are contained in collagen derived from flippers. The hybrid bone graft material according to claim 1, wherein DC is contained in an amount of 5-45% by weight of the whole composition. The hybrid bone graft material according to claim 2, wherein the DC is contained in an amount of 35-42% by weight of the whole composition. The hybrid bone graft material according to claim 1, wherein the PLGA is contained in an amount of 50-90 wt% in the entire structure. The hybrid bone graft material according to claim 1, wherein DC / PLGA / HAp is contained in an amount of 5-45% by weight / 50-90% by weight / 1-10% by weight. The flip-based collagen-containing hybrid bone graft material according to claim 1, wherein DC is contained in an amount of 70-85% by weight of the PLGA content in the whole composition. The hybrid bone graft material according to claim 1, wherein PLGA is contained in an amount of 50 to 60% by weight in the whole composition. The hybrid bone graft material according to claim 1, further comprising bone marrow-derived mesenchymal stem cells. Preparing a solution of PLGA (poly (lactic-coglycolic acid));
Mixing HAp (hydroxyapatite) in the form of powder and collagen (DC) in powder form in the PLGA solution; And
Preparing a bone graft material by solvent casting or salt extraction;
Wherein the collagen-derived collagen-containing collagen-containing collagen-containing collagen-containing collagen-containing collagen-containing collagen-derived collagen-containing collagen-derived collagen- [Claim 12] The method according to claim 9, wherein preparing the PLGA solution comprises dissolving PLGA in methyl chloride and dissolving the PLGA solution in methyl chloride. The hybrid bone graft material according to claim 9, wherein the mixture is produced by using NaCl having a particle size (maximum diameter) of 250 to 355 μm as a pore inducing material for forming pores in addition to the mixture . The hybrid bone graft material according to claim 9, wherein the mixture is formed into a compact by applying a pressure of 40-80 kgf / cm ² . [12] The method of claim 9, wherein the dentifrice collagen (DC) is washed using a mixed solution of methanol: chloroform having a volume ratio of 3: 0.5 - A method for manufacturing a hybrid bone graft material containing collagen derived from flippers.

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