KR101999098B1 - Scaffold using material from marine organisms for bone tissue generation and method for preparing the same - Google Patents

Abstract

The present invention relates to a support for bone tissue regeneration comprising a marine organism-derived substance, specifically fish-derived collagen or alginic acid, and an abalone hydrolyzate, and a method for producing the same.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a scaffold for regenerating bone tissue containing a marine organism derived material and a method for preparing the same,

The present invention relates to a support for bone tissue regeneration comprising a marine biologically-derived material and a preparation method thereof, and more particularly to a support for bone tissue regeneration comprising fish-derived collagen or alginic acid and an abalone hydrolyzate, .

The development of science and the resulting increase in human longevity have increased the problems of various diseases, aging and long term damage due to various accidents. Therefore, interest in tissue engineering that regenerates tissues is getting hotter day by day. Tissue engineering is a discipline that combines life sciences and engineering to restore, regenerate, and replace the structure and function of living tissues to function properly. Among the various tissues, bone is an important tissue that also plays a role of a reservoir of calcium to regulate the calcium ion concentration in the body in addition to the mechanical function of supporting the human body and performing an action, and also possessing an important physiological function of producing red blood cells and white blood cells for human body in bone marrow . Bones can be damaged by aging and other physiological reasons, or can be damaged by various accidents. Currently, bone damage is treated primarily by mechanical and physical methods. Bone regeneration and treatment for bone loss is an important goal in oral surgery or orthopedic surgery caused by accidents, illnesses, infections, and the like.

Supports for tissue engineering should satisfy key conditions such as cell affinity, proper porosity for nutrients and oxygen permeability, and surface activity to promote cell attachment and differentiation for optimized tissue formation. In particular, in the case of the supporter for bone tissue regeneration, osteoinduction, migration and binding of surrounding cells, decomposition in the body, support of mechanical load, and similarity of bone environment must be satisfied. However, currently used supports for bone tissue regeneration each have a certain limit. Specifically, the PLGA-based biodegradable synthetic polymer has excellent biocompatibility and natural decomposition properties in the body, and thus is widely used as a support material in the field of tissue engineering. However, PLGA-based biodegradable synthetic polymers have low bioactivity, high hydrophobicity, Affinity, it still needs to be optimized to be suitable for tissue engineering. In addition, although most of the biocompatible materials are used for tissue regeneration, there are safety problems such as inflammation reaction and immune response when inserted into the body. In addition, when inserted into a damaged area, there are problems such as a problem of regulating a recovery speed and a wound remaining after completion of tissue regeneration.

Accordingly, the present inventors have found that a supporter for bone tissue regeneration, in which a marine-derived physiologically active substance and a natural polymer substance are fused, can provide an optimal environment for cell differentiation and homeostasis for bone tissue regeneration, Immune response, and the like, thereby completing the present invention.
<Prior Art Literature>
Registered Patent No. 10-0824352 (Apr. 13, 2008)

It is an object of the present invention to provide a three-dimensional bone tissue regeneration support having safety in the body and optimized for cell production for bone tissue regeneration.

Another object of the present invention is to provide a method for producing a supporter for regenerating bone tissue.

One embodiment is a biodegradable polymeric scaffold; A coating layer containing an overturned hydrolyzate; And a coating layer containing fish-derived collagen and / or alginic acid.

As used herein, the term "bone regeneration" refers to any phenomenon that treats, alleviates, or alleviates bone damage through such processes as proliferation of damaged bone tissue by osteoblasts or differentiation of osteoblasts into bone cells And, although not limited thereto, the bone regeneration may be particularly due to promotion of bone differentiation.

The term "bone regeneration support " refers to a structure that replaces a portion of a damaged bone tissue in vivo and can complement or replace the function of the bone. The supporter for regenerating the bone tissue may serve as a support to allow related cells to adhere and to proliferate or differentiate for restoration of bones, for example. Regardless of the shape of the bone regeneration support, the bone regeneration support need not necessarily correspond to the shape of the bone defect site to be implanted, and the bone regeneration support body may be in a state of being processed to a similar shape. However, if necessary, the shape of the bone damage site to be transplanted can be grasped in advance, and a shape suitable for the shape may be prepared, or it may be previously shaped into a specific structure so as to be generally applicable to various situations.

The term "biodegradable polymer scaffold" means a structure made of a biodegradable polymer. The biodegradable polymer scaffold may be the base of the scaffold for regenerating bone tissue of the present invention, and the shape of the scaffold of the present invention may be determined by the shape of the biodegradable polymer scaffold.

Wherein the biodegradable polymer is selected from the group consisting of poly (epsilon -caprolactone), polylactic acid, polyglycolic acid, polylactic acid-glycolic acid copolymer, polydioxanone, polyhydroxybutyrate, polyhydroxybutyric acid- A poly (? -Ethyl glutamate), a polyanhydride copolymer, and a mixture thereof. By using the biodegradable polymer material, the support of the present invention can be completely decomposed and eliminated in vivo after the support is maintained until its function and role are sufficiently performed. The weight average molecular weight of the biodegradable polymer used in the support of the present invention may be 1,000 to 1,000,000 g / mol. Specifically, the polymer has a weight average molecular weight of 1,000 to 900,000 g / mol, 1,000 to 800,000 g / mol, 1,000 to 700,000 g / mol, 1,000 to 600,000 g / mol, 1,000 to 500,000 g / mol, 1,000 to 300,000 g / mol, or 1,000 to 200,000 g / mol. Outside the range of the weight average molecular weight, the physical properties of the support for tissue regeneration are not suitable. Particularly, when the weight average molecular weight is within the above-mentioned range, it can be decomposed before the function of tissue regeneration is sufficiently performed. The biodegradable polymer may preferably be poly (epsilon -caprolactone), polylactic acid or polyglycolic acid, and more preferably poly (epsilon -caprolactone). The poly (epsilon -caprolactone) has biocompatibility and biodegradability, and has a higher tensile strength than other polymers and is more suitable for use in a support for bone tissue regeneration.

The biodegradable polymer scaffold may preferably have a three-dimensional lattice form. The three-dimensional lattice form may be formed by arranging thread-like strands made of biodegradable polymers in parallel in each layer, and arranging polymer strands in each layer in parallel at right angles with adjacent layers ( 1). The biodegradable polymeric strands may preferably have a diameter of 300 to 500 mu m, 350 to 500 mu m, 300 to 450 mu m, or 350 to 450 mu m. As used herein, the term "diameter" means the length of the minor axis through the center of the fiber. The diameter of the polymeric strand used in the present invention is within the above range so as to be suitable for three dimensional (three dimensional) binding with cells for bone tissue regeneration while securing the minimum mechanical properties required for the tissue regeneration support. . The biodegradable polymer scaffold may have a pore size of preferably 400 to 500 mu m, or 450 to 500 mu m.

The term "rollover internal hydrolyzate" refers to a substance obtained by decomposing abalone organisms into enzymes. Wherein the overturned hydrolyzate is obtained by treating the overturned protein with at least one protein hydrolyzing enzyme selected from the group consisting of pepsin, trypsin, chymotrypsin, pancreatin, alcalase, flavosome, papain and mixtures thereof . The protein hydrolase may preferably be pepsin, trypsin, and / or chymotrypsin. The treatment ratio of the enzyme to the overturned viscous protein may be about 1: 100. The overturned protein can be present in the form of a short peptide by the above enzyme treatment. The overturned hydrolyzate may be a fraction obtained by treating the inverted embryo with a protein hydrolyzing enzyme and then fractionating it by an ultrafiltration membrane. The overturned hydrolyzate may more preferably have a molecular weight of 1 to 10 KDa, more preferably a peptide having a molecular weight of more than 1 KDa and less than 10 KDa.

The term "fish-derived collagen" refers to collagen obtained from fish. The fish-derived collagen can be preferably obtained from the skin of the fish. The fish may be, for example, at least one selected from the group consisting of salmon neck salmon, sea bass tuna, sea bream mackerel, sea mackerel, seagrass sechiride, cod fish, and sole, and preferably the fish is a fish such as a flounder, It can be a fluke.

The alginic acid (or alginate) may preferably be one obtained from algae. The alginic acid may be preferably in the form of a salt, more preferably sodium alginate.

The bone tissue regenerating support of the present invention is characterized in that a coating layer containing overturned hydrolysates and a coating layer containing fish-derived collagen and / or alginic acid are formed on the surface of the biodegradable polymer scaffold. Therefore, the support of the present invention binds a natural bioactive substance derived from a marine organism capable of providing a more cell-friendly environment on the surface of the biodegradable polymer scaffold, and as a result, promotes cell adhesion, proliferation or differentiation for bone regeneration effectively do.

The bone tissue regenerating support of the present invention may more preferably comprise a coating layer containing the overturned hydrolyzate and a coating layer containing fish-derived collagen. In one embodiment, the support comprising the coating layer containing the overturned hydrolyzate and the coating layer containing the collagen-derived collagen is higher than the support comprising the coating layer containing the overturned hydrolyzate and the coating layer containing sodium alginate, It was confirmed that osteoblast differentiation effect was present.

The bone tissue regeneration support may further include a physiological activity factor for bone regeneration. The term "physiologically active factor" means a substance that enhances or inhibits the function of a living body, and can refer to vitamins, hormones, enzymes, neurotransmitters, and the like. The physiologically active factors for bone regeneration include, for example, bone morphogenetic protein (BMP), transforming growth factor (TGF), fibroblast growth factor (FGF) And may be at least one selected from the group consisting of vascular endothelial growth factor (VEGF) and epidermal growth factor (EGF), and may be preferably an osteogenic protein, and / or a transforming growth factor.

The bone tissue regeneration support of the present invention may be specifically used for bone regeneration or regeneration of cartilage, and more specifically, it may be used as a bone conduction support or a support for regenerating cartilage. The term "osteoconduction" occurs when osteoblasts or osteoclasts migrate from the surrounding bone tissue to the graft site and bone is formed by mineral deposition, resulting in bone tissue or differentiated mesenchymal cells in the periphery. The term "osteoconductive support" refers to an environment that is stimulated to become an osteogenic cell line on the surface of the support, or in its pores, channels or other internal pores, such as primitive cells, undifferentiated cells and omnipotent cells, &Lt; / RTI &gt; In one embodiment, it was confirmed that the support of the present invention provides a fine environment for the differentiation of mesenchymal stem cells into bone cells.

Another aspect is a method for producing a biodegradable polymer scaffold comprising: preparing a biodegradable polymer scaffold; Forming a coating layer containing the overturned hydrolyzate on the surface of the support; And a step of forming a coating layer containing fish-derived collagen and / or alginic acid.

In the above method, the step of preparing the biodegradable polymer scaffold may be such that the strand of the biodegradable polymer exhibits a three-dimensional lattice pattern. The three-dimensional lattice form may be formed by arranging thread-like strands made of biodegradable polymers in parallel in each layer, and arranging polymer strands in each layer in parallel at right angles with adjacent layers ( 1). The biodegradable polymer scaffold can be preferably produced by a 3D printing technique. The term "3D printing" is used in the same sense as Rapid Prototyping (RP). By layering materials in a systematic manner by two-dimensional sectioning of three-dimensional image data, Refers to technology that can be produced in time. The biodegradable polymeric strands may preferably have a diameter of 300 to 500 mu m, 350 to 500 mu m, 300 to 450 mu m, or 350 to 450 mu m. The biodegradable polymer scaffold may have a pore size of preferably 400 to 500 mu m, or 450 to 500 mu m.

Wherein the biodegradable polymer is selected from the group consisting of poly (epsilon -caprolactone), polylactic acid, polyglycolic acid, polylactic acid-glycolic acid copolymer, polydioxanone, polyhydroxybutyrate, polyhydroxybutyric acid- (? -Ethylglutamate), a polyanhydride copolymer, and a mixture thereof. It is also preferably poly (epsilon -caprolactone), polylactic acid or polyglycolic acid, and more preferably poly (epsilon -caprolactone).

In the above method, the step of forming the coating layer containing the overturned hydrolyzate may be performed by immersing the prepared biodegradable polymer scaffold in a solution containing the overturned hydrolyzate.

Wherein the overturned hydrolyzate is obtained by treating the overturned protein with at least one protein hydrolyzing enzyme selected from the group consisting of pepsin, trypsin, chymotrypsin, pancreatin, alcalase, flavosome, papain and mixtures thereof And may be preferably obtained by treatment with pepsin, trypsin, and / or chymotrypsin. The above-mentioned overturned hydrolyzate is as described for the support.

In the above method, the step of forming the coating layer containing the fish-derived collagen and / or the alginic acid may include forming a coating layer containing the overturned hydrolyzate in a solution containing the fish-derived collagen, alginic acid, or a combination of collagen and alginic acid Can be carried out by immersing the biodegradable polymer scaffold. The fish-derived collagen and the alginic acid are as described for the support.

The support for bone tissue regeneration according to the present invention has excellent biocompatibility and appropriate mechanical strength by introducing a coating layer containing a natural bioactive substance derived from marine organism into a biodegradable polymer scaffold, It is advantageous for attachment, proliferation and differentiation. Therefore, it can be utilized as a material for implant and bone regeneration for bone tissue.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic diagram illustrating the fabrication of a PCL support fabricated using 3D printing.
FIG. 2 (A) is a graph showing the ratio of collagen extracted from (A) SDS-PAGE pattern (? -Chain;? 1 -chain;? 2 -chain). FIG. 2 (B) is a graph showing the results of MSC cell viability according to IGDP concentration measured by MTT assay. 2 (C) is a graph showing the effect of alkaline phosphatase (ALP) activity on the IGDP concentration in MSC cells.
FIG. 3 shows FTIR spectral results of PCL, PCL / IGDP, PCL / Col, PCL / Sa, PCL / IGDP / Col and PCL / IGDP / Sa supports.
4 shows SEM images (A) and porosity (B) for PCL, PCL / IGDP, PCL / Col, PCL / Sa, PCL / IGDP / Col and PCL / IGDP / Sa supports.
Figure 5 (A) shows the form of water droplets on the surface of each support over time (1 second, 2 seconds, 3 seconds, and 20 minutes). 5 (B) shows the contact angle of water droplets of each support. Figure 5 (C) shows the degree of protein adsorption of each support. 5 (D) shows the stress-strain curves.
Figure 6 (A) is a graph showing the results of MSC proliferation (n = 4, * P < 0.05 (compared with MSC cells cultured on PCL scaffolds) cultured on different supports for different incubation periods (1, 2 and 7 days) Figure 6 (B) shows the results of staining with MSC for 4 days and 7 days, followed by fluorescein diacetate (FDA), on each support.
Figure 7 (A) shows the results of ALP activity of MSC cells cultured on each support. Figure 7 (B) shows the calcium deposition results of MSC cells cultured on each support identified through alizarin red S staining. FIG. 7 (C) shows the result of confirming the mineralization of MSC cells cultured on each support analyzed by absorbance measured using a microplate reader (n = 4, * p <0.05).
FIG. 8 shows the result of RT-PCR analysis of osteogenic differentiation-related genes of MSCs distributed in each support. GADPH was used as a control.
Figure 9 (A) is a μ-CT image showing the formation of new tissue of tibial defects containing control (PCL), PCL / IGDP, PCL / IGDP / Col, and PCL / IGDP / Sa scaffolds. Fig. 9 (B) shows regenerated soft tissue and hard tissue at a ratio of 0.48 mm ² area at 2 weeks and 6 weeks after scaffold implantation. (* p < 0.05).

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited by these examples.

Manufacturing example  1. Preparation of supporter for bone tissue regeneration

1.1. Abalone digestion Hydrolyzate Peptides , Fish-derived collagen solution and Algin Prepare sodium acid solution

Abalone ( Haliotis discus hannai ) was washed and lyophilized after isolation. abalone The products of the intestine gastro-intestinal digests peptides (IGDP) were adjusted to pH 2.2 using 1M HCl and 10M NaOH. Pepsin (EC 3.4.23.1) was added at an enzyme and substrate ratio of 1/100 (w / w) and then stirred at 37 ° C for 2 hours. The pH was set to 6.5 to obtain the conditions of the viscous hydrolyzate. Trypsin (EC 3.4.21.4) and a-chymotrypsin (EC 3.4.21.1) were supplemented with a ratio of enzyme and substrate of 1/100 (w / w) and then stirred at 37 ° C for 2 hours. IGDP was centrifuged at 4 캜 for 15 minutes, and the supernatant was lyophilized to obtain a powder (5.65 g). The resulting IGDP was separated into two different ultrafiltration (UF) membranes with a range of molecular weight cutoffs of 1 kDa < IGDP < 10 kDa and lyophilized.

For the production of fish-derived collagen solution, flounder ( Paralichthys olivaceus ) (bought in Korea, Jeju, Dongmun Fisheries Market) Collagen (Col) was extracted from the skin. The extracted collagen was diluted in 0.5 M acetic acid at 4 DEG C for 24 hours (0.5%).

Sodium alginate (Sa) was obtained from brown algae-derived sodium alginate (A0682, Sigma Aldrich, USA). Sodium alginate (2% w / v) was dissolved in distilled water at 50 ° C for 6 hours and then transferred to room temperature.

1.2. PCL  Production of three-dimensional support

A support was made using a 3D printer IEI system (Iwashita Engineering, Inc., Tokyo, Japan) with hot melt, pressure pump TCD-200, nozzle, and x-y-z stage. PCL (polycaprolactone) was placed in an iron cylinder, the hot-melt was treated at 110 占 폚, and the printer was operated at a linear speed of 0.6 mm / sec. PCL was used in the form of pellets and printed with a length of 13.5 mm. PCL was hot-melted and air pressure of 500 kPa was applied and extruded into 3D strands and plotted layer by layer until it was 8 layers (see Fig. 1). The nozzle size was 400 ㎛ and the pore size was 400 - 500 ㎛.

The surface of the prepared PCL support was treated with NaOH (3M, 37 ° C) for 24 hours, then immersed in 1M HCl, and then washed with distilled water. The PCL / IGDP, PCL / Col and PCL / IGDP / Col scaffolds were crosslinked in 50 mM EDC / NHS solution for 24 hours, washed three times with 98 wt% ethanol and distilled water and then lyophilized at -80 ° C. The PCL / Sa and PCL / IGDP / Sa supports were incubated at 4 ° C in 0.5 M CaCl ₂ Dipped for 24 hours, then washed with ethanol and lyophilized at -80 &lt; 0 &gt; C.

1.3. PCL support coating

To improve the surface of the PCL support, carboxyl group was formed by reacting with 3M NaOH at 37 ° C for 24 hours. Then, remaining NaOH was removed using 1M HCl and distilled water. PCL / IGDP or PCL / Col scaffolds were prepared by cross-linking IGDP or collagen with surface-modified PCL scaffolds for 24 hours using 50 mM EDC / NHS solution. In addition, PCL / IGDP / Col scaffolds were prepared by cross-linking the PCL / IGDP scaffold with 50 mM EDC / NHS solution for 24 hours in the presence of collagen. The PCL / IGDP / Sa support was prepared by crosslinking alginate with 0.5M calcium chloride using PCL / IGDP support, and the PCL / Sa support was prepared by crosslinking the alginate with 0.5M calcium chloride to the surface- Respectively. The supernatant was washed three times with 95 wt% ethanol and distilled water to remove remaining substances and then lyophilized.

Example  1. Characterization of fish collagen and cell Survival  And ALP IGDP  Check the effect

Collagen extracted from the flounder skin with acid solution was analyzed by SDS-PAGE. As can be seen in Fig. 2A, the collagen contained? 1 and? 2 -chain with a molecular weight of about 130 kDa (MW) and also contained a? -Chain with a molecular weight of about 270 kDa. The protein pattern of the extracted flounder collagen was similar to that described for Ovis aries- derived collagen (Fauzi MB, Lokanathan Y et al. 2016). The extract of collagen derived from flounder skin was similar to the molecular weight of Ty-I collagen from pig skin (Sigma, St. Louis, Mo., USA).

The overturned intrinsic hydrolyzate peptide (IGDP) was prepared according to the method described in Nguyen VT, Qian ZJ et al. (2012) (slightly modified). Cytotoxicity and ALP activity of the prepared IGDP were confirmed using MSC cells. ALP is a typical enzyme expressed early in osteoblast differentiation and plays an important role in bone formation. The results of cytotoxicity of MTT assay showed that IGDP did not show any toxicity in MSC cells and MSC cell proliferation was induced at 5 days in IGDP treatment. In addition, it was confirmed that ALP activity was induced by IGDP (Fig. 2B and C). As shown in Figures 2B and C, IGDP treatment significantly increased ALP activity in a concentration dependent manner. The maximal effect was observed at 500 ㎍ / ml of IGDP, after 5 days of incubation, ALP activity was increased by 273.21% compared to the control. These observations suggest that IGDP stimulates osteoblast differentiation. Thus, a support was prepared using IGDP at a concentration of 0.5 ± 0.038 mg / ml in a single support (length 13.5 × width 13.5 mm thickness 2.3 mm).

Example 2. Fourier transform infrared spectroscopy (FTIR) analysis of a support

To confirm the presence of specific chemical functional groups on each support, FTIR spectral analysis was performed using FTIR-Perkin-Elmer Spectrum GX (Washington DC, USA) at 4000-650 cm ^-1 spectra range. Infrared results of the support by KBr method are as follows.

FTIR spectral results of PCL, PCL / IGDP, PCL / Col, PCL / Sa, PCL / IGDP / Col, and PCL / IGDP / Sa supports are shown in FIG. PCL spectrum 2926 - 2949 cm ^-1 and 2852 - showed a major peak at 2865 cm ^-1, respectively, which is due to the asymmetric and symmetric stretching CH _2. Strong bands at 1719 - 1721 cm ^-1 indicate carbonyl (C = O) stretching signals and peaks at 1295 cm ^-1 and 1239 cm ^-1 relate to CO and CC stretching and asymmetric COC stretching vibrations in the crystalline phase. Another significant peak at 1164 cm ^-1 corresponds to the stretching of the symmetric COC. The amide A band position is 3386 cm ^-1 , and stretching vibration of NH and OH groups appears in the region. The amide B band was found at 2949 cm ^-1 due to the characteristic of CH ₂ asymmetric stretching. 1635 cm ^-1 (C = O stretch / COO ^- with Hydrogen bond), 1539 cm ^-1 (CN stretch and bending that couples NH ring), and 1239 cm ^-1 (CN stretching vibration and the absorption peak of amide NH in the a-plane bending vibration) is IGDP and amide I in the collagen, amide II, and amide. The PCL / Sa and PCL / IGDP / Sa supports exhibited a characteristic band of 1239 cm ^-1 corresponding to the (S = O) group. The peak at 3340 cm ^-1 corresponds to the -OH functional group of PCL, IGDP and sodium alginate. The FTIR spectra of sodium alginate (Sa) showed characteristic peaks at 3260-3380 cm ^-1 and 2926-2949 cm ^-1 due to OH and CH groups. The peaks at 1541 and 1419 cm ^-1 are due to CO and COOH functional groups, respectively. The spectra of PCL, PCL / Sa and PCL / Sa supports were very similar. This is probably due to the reaction of the PCL / IGDP support with sodium alginate. 1400-1669 cm ^-1 peak of PCL / IGDP, PCL / Col, PCL / Sa, PCL / IGDP / Col and PCL / IGDP / Sa, containing IGDP, sodium alginate (Sa) and collagen Was higher than the characteristic peak. On the other hand, the spectra of PCL / IGDP, PCL / Col, PCL / Sa, PCL / IGDP / Col, and PCL / IGDP / Sa are similar to those of pure material and due to the chemical interaction of PCL and IGDP, Sa and Col A typical absorption band for one PCL was shown.

Example  3. Support of  Scanning Electron Microscope (SEM) images and porosity analysis

SEM analysis was performed to confirm the surface and porosity of PCL, PCL / IGDP, PCL / Col, PCL / Sa, PCL / IGDP / Col, and PCL / IGDP / The average interlayer size of each support was similar in the range of 19 ± 15 μm to 250 ± 21 μm. Also, the pore size and the strut diameter of each support were confirmed to be about 421 ± 29 to 463 ± 24 μm and 355 ± 12 to 394 ± 21 μm, respectively (Table 1 and FIG. 4A).

The strand diameter, interlayer spacing, pore size of each support PCL PCL / IGDP PCL / Col PCL / IGDP / Col PCL / Sa PCL / IGDP / Sa Strut diameter (탆) 353 ± 16 394 ± 21 388 ± 13 365 ± 17 355 ± 12 385 ± 21 Interlayer spacing (탆) 236 ± 18 237 ± 14 221 ± 20 250 ± 19 237 ± 11 219 ± 15 Pore size (탆) 454 ± 21 421 ± 29 425 ± 10 449 ± 19 463 ± 24 437 ± 25

The porosity of PCL, PCL / IGDP, PCL / Col, PCL / Sa, PCL / IGDP / Col and PCL / IGDP / Sa supports with the same size (length 13.5 x width 13.5 mm thickness 2.3 mm) Respectively. Each support was placed in a cylinder with a known volume (T ₁ ) of ethanol for 10 minutes, and the liquid was allowed to penetrate into the pores of the support through a continuous vacuum-press cycle. The volume of the ethanol before and after removal of the liquid-immersed support (T ₂ and T ₃ , respectively) was measured and the porosity was calculated using the following formula.

As a result, it was confirmed that each support had a high porosity and a large surface area. 4B, it was confirmed that addition of IGDP, Col, and Sa improved porosity (PCL: 66.67 + 0.88%; PCL / IGDP: 71.03 + 0.92%; PCL / Col: 74.74 + 1.93%; PCL / Sa: 73.81 ± 1.21%; PCL / IGDP / Col: 80.61 ± 1.14%; and PCL / IGDP / Sa: 78.52 ± 1.37%). Especially, PCL / IGDP / Col and PCL / IGDP / Sa supports had high porosity. Thus, it was observed that the addition of components such as sodium alginate and collagen in the PCL / IGDP support positively affected the porosity.

Example 4. Confirmation of Water Absorption Rate and Protein Attachment of Support

The contact angle with water was measured to examine the degree of hydrophobicity of the support. To measure the water absorption rate of the support, a drop of DMEM was dropped onto a 1 ml tip onto PCL, PCL / IGDP, PCL / Col, PCL / Sa, PCL / IGDP / Col and PCL / IGDP / Sa scaffolds. The average contact angle of each support was obtained from the water absorption image using image analysis software 3D (ImageJ 1.49v, National Institutes of Health, USA). Specifically, the average contact angle (θ _C ) of PCL, PCL / IGDP, PCL / Col, PCL / Sa, PCL / IGDP / , 2 seconds, 3 seconds, and after 20 minutes are shown in Figs. 5A and 5B. After 1 second and 20 minutes, the water contact angles of the PCL were? _C = 82.69 占 1.21 占 and 48.89 占 1.25 占 respectively. Indicating that the PCL support is hydrophobic. However, the average contact angles of the PCL / IGDP, PCL / Col, PCL / Sa, PCL / IGDP / Col and PCL / IGDP / Sa supports are respectively _C = 32.25 ± 1.02 °, 66.80 ± 1.11 °, 34.18 ± 1.32 °, ± 0.98 ° and 15.72 ± 0.85 °, respectively. PCL / Sa and PCL / IGDP / Sa were taken directly after placing the DMEM droplets on each support, and the contact angle dropped to near zero after 2 seconds (FIG. 5B). On the other hand, PCL / IGDP, PCL / Col and PCL / IGDP / Col contact angles fell to near zero after three seconds. IGDP is hydrophilic in its chemical structure by amine and carboxyl groups, and can form hydrogen bonds with water in DMEM. The alginate is an anionic polysaccharide containing a carboxyl group. These results indicate that θ _C is significantly higher in PCL / IGDP / Col and PCL / IGDP / Sa supporters than in PCL, PCL / IGDP, PCL / Col and PCL / Sa because of the increased -COOH and -OH groups of IGDP, collagen or sodium alginate Respectively.

The amount of total protein adsorbed on PCL, PCL / IGDP, PCL / Col, PCL / Sa, PCL / IGDP / Col and PCL / IGDP / Sa supports was evaluated using BCA assays. Protein adsorption is an important characteristic of biomaterial scaffolds, and proteins adsorbed on the surface of the scaffold can regulate cell attachment and survival (Jaiswal AK, Chhabra H et al. 2013). Secreted by serum or cells, it is believed to mediate cell adhesion, differentiation and migration to form neo-tissue. Therefore, protein adsorption is an important factor in evaluating and studying scaffolds for tissue engineering. Specifically, PCL, PCL / IGDP, PCL / Col, PCL / Sa, PCL / IGDP / Col and PCL / IGDP / Sa scaffolds with the same size (length 13.5 x width 13.5 mm thickness 2.3 mm) Immersed in 0.1 M PBS (pH = 7.4) and measured using the BCA method. Each substrate was immersed in 70% ethanol for 30 minutes, then washed three times with PBS to remove ethanol, and then subjected to UV treatment for 30 minutes before immersing in a medium containing protein. The support was placed into a 24-well culture plate and 1.5 ml PBS solution with 2.5% FBS and 1% penicillin-streptomycin (containing one support per well) was added to each well. The solution added to each well was collected according to immersion time, and the protein content was measured using BCA measurement method. The amount of adsorbed protein was determined by subtracting the amount of protein remaining in the FBS / PBS solution after adsorption from the amount of protein in the control FBS / PBS solution (without support) under the same incubation conditions. The results of measuring the protein adsorption of PCL, PCL / IGDP, PCL / Col, PCL / Sa, PCL / IGDP / Col and PCL / IGDP / Sa supports are shown in FIG. As a result, it was confirmed that the protein adsorption of the supporter exhibited the maximum adsorption value at 25 hours (PCL / Col, PCL / IGDP / Col, PCL / IGDP / Sa) and 30 hours (PCL, PCL / IGDP and PCL / Sa) I could. It was also found that the addition of collagen and sodium alginate to PCL / IGDP increased protein adsorption, and the addition of collagen, IGDP and sodium alginate to the PCL supernatant significantly increased protein adsorption compared to PCL (1.97 (PCL / IGDP / Sa), 2.45 times (PCL / IGDP), 2.57 times (PCL / Col), 4.48 times (PCL / IGDP / Col) and 3.16 times (PCL / IGDP / Sa)).

Example  5. Support of  Cell viability and cell adhesion Confirm

PCL, PCL / IGDP, PCL / Col, PCL / Sa, PCL / IGDP / Col and PCL / IGDP / Sa supports were stirred in a 48-well plate with 70% ethanol overnight. The plates were washed with PBS to remove ethanol, treated with UV for 30 min, and then immersed in DMEM (no FBS) medium at 37 째 C before cell seeding. MSC cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin. The cell culture 37 ℃, in 5% CO ₂ Lt; / RTI &gt; The supporter was placed in a well plate, and 5x10 ⁴ cells in a DMEM medium were dispensed on a supporter. Living cells were counted using MTT method (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) on days 1, 2, 4 and 7. After culturing the cells on each supporter, 200 μl of 1 mg / ml MTT reaction reagent in 100 μl DMEM was added to each well, and the plate was further cultured for 4 hours. The mitochondrial succinate dehydrogenase converts MTT to visible formazan crystals in living cells during incubation at 37 ° C. After removing the solution from each well, dimethylsulfoxide (DMSO) was added to dissolve the formazan product, and the absorbance at 540 nm was measured using a microplate reader and compared with the absorbance of the control cells.

Cell growth and proliferation characteristics on PCL / IGDP, PCL / Col, PCL / Sa, PCL / IGDP / Col and PCL / IGDP / Sa scaffolds after 1, 2, 4, The cell viability of PCL, PCL / IGDP, PCL / Col, PCL / Sa, PCL / IGDP / Col and PCL / IGDP / Sa scaffolds showed no significant difference on the first day of culture 6A). However, two days after cell culture, PCL / IGDP / Col and PCL / IGDP / Sa showed higher cell proliferation than PCL, PCL / IGDP, PCL / Col and PCL / Sa. PCL / IGDP / Col showed the highest cell proliferation after 4 and 7 days of culture, and PCL, PCL / IGDP, and PCL / Col in both PCL / IGDP / Col and PCL / IGDP / , And PCL / Sa supporters. This indicates that the combination of collagen and / or sodium alginate and other substances has a high cell adhesion and proliferative effect. Thus, the addition of IGDP, collagen and sodium alginate to PCL can enhance cell viability and PCL / IGDP / Col and PCL / IGDP / Sa supports provide a suitable surface for cell adhesion and proliferation.

In addition, FDA staining confirmed cell attachment and proliferation on the supporter. The growth of cells cultured for 4 days and 7 days in PCL, PCL / IGDP, PCL / Col, PCL / Sa, PCL / IGDP / Col and PCL / IGDP / Sa scaffolds (13.5 × 13.5 mm in thickness 2.3 mm) FDA (Sigma-Aldrich, USA) fluorescence reagent was used for evaluation. 1 ml of FDA solution was added directly to the support of each well, and each support was stained in the dark room for 5 minutes. Then, the cells were rinsed with PBS and the morphology of the cells was observed through a fluorescence microscope. After 4 days and 7 days of incubation, the structures of the support were kept almost the same, and the different cell attachment and propagation results were shown for each support. These results support the quantitative results of the MTT assay. Figure 6B shows a stained micrograph of MSC cells cultured for 4 days and 7 days in PCL, PCL / IGDP, PCL / Col, PCL / Sa, PCL / IGDP / Col and PCL / IGDP / Sa scaffolds. MSC cells were identified as green fluorescence by FDA. Live cells attached to PCL / IGDP / Col scaffolds had higher cell density than PCL, PCL / IGDP, PCL / Col, PCL / Sa and PCL / IGDP / Sa scaffolds, It was confirmed that the cells proliferated better.

Example  6. Cells Alkaline Phosphatase  Activity and Calcium Content Analysis

ALP activity is well known as an important factor in the measurement of osteoblast differentiation and mineralization by biochemical markers for the measurement of osteoblastic phenotype (Han P, Cheng P et al., 2015; Mohammadi Y, Soleimani M et al. Pan L, Pei X et al. 2012). Therefore, we investigated whether the scaffold enhances the differentiation of MSC cells through ALP activity analysis of cells. Prior to seeding the cells, the support was incubated overnight in 70% ethanol, treated with UV for 30 minutes, and then incubated in DMEM medium (no FBS) for 2 hours at 37 ° C. MSC cells (5x 10 ⁵ cells / well) of the same size having a 12-well plate PCL (length × width 13.5 mm thickness 2.3 mm 13.5), PCL / IGDP, PCL / Col, PCL / Sa, PCL / IGDP / Col And PCL / IGDP / Sa scaffolds and then rinsed three times with PBS. Cells were cultured for 2 and 7 days on each support and then lysed with lysis buffer [containing 25 mM (Na ₂ CO ₃ , NaHCO ₃ ), 0.1% Triton 100X] to give 25 mM (Na ₂ CO ₃ , NaHCO ₃ ) (containing 1.5 mM MgCl ₂ and 1 X p-nitrophenylphosphate disodium (pNPP)) at 37 ° C. for 1 hour and 30 minutes, then reacted with 1 N NaOH And the absorbance at 405 nm was measured.

As a result, it was confirmed that ALP activity of cells cultured in PCL / IGDP / Col and PCL / IGDP / Sa scaffold for 7 days of incubation was higher than that of PCL, PCL / IGDP, PCL / Col, and PCL / Sa supports. In addition, the activity of ALP in cells cultured on PCL / IGDP / Col and PCL / IGDP / Sa supernatants increased with increasing cell adhesion and proliferation after 7 days. Therefore, it was confirmed that the introduction of collagen and sodium alginate into PCL / IGDP significantly increased the level of ALP activity.

In addition, calcium content of MSC cells was examined through calcification assays. Calcium content is an important indicator of osteoblast differentiation and mineralization (Karadeniz F, Ahn BN et al. 2015). The amount of calcium deposited on MSC cells cultured for 28 days in PCL, PCL / IGDP, PCL / Col, PCL / Sa, PCL / IGDP / Respectively. Specifically, the support was incubated with 70% ethanol overnight, treated with UV for 30 minutes, and then incubated with DMEM medium (without FBS) for 2 hours at 37 ° C. MSC cells (1x10 ⁵ cells / well) cultured on each support having the same size (13.5 x 13.5 mm wide and 2.3 mm thick) in a 12-well plate were rinsed three times with PBS. The cells were then cultured in ODM medium containing 5% serum for 28 days on each different support, fixed with cold 70% ethanol, and stained with alizarin red to determine calcification. After quantification, cells stained with alizarin red were decolored with ethylpyridinium chloride (n > 3), the dye was transferred to a 96-well plate, and absorbance was measured at 562 nm using a microplate reader.

As a result, after 28 days of culture, MSC cells cultured on PCL / IGDP / Col and PCL / IGDP / Sa supporter surface showed higher calcium content than PCL, PCL / IGDP, PCL / (Fig. 7B). In addition, the PCL / IGDP / Col scaffold showed the highest amount of calcium deposition (430.40% as compared to the PCL scaffold control) (Figure 7C). These results of ALP and calcium deposition showed that PCL / IGDP / Col and PCL / IGDP / Sa supporters promote cell viability, osteoblast differentiation and mineralization.

Example 7. Confirmation of differentiation effect for bone formation of supporter

Strengthening of bone formation is an important procedure for bone strength by gene expression of bone formation markers, which is regulated by the activity of Runx2 transcription factor and is accompanied by increased expression of bone matrix proteins such as OPN and OSC , And then stimulate mineralization and lead to bone formation (Beederman M, Lamplot JD et al 2013, Do AV, Khorsand B et al 2015, Jeong YT and Baek SH et al 2016). Relative mRNA expression levels of bone markers commonly used by RT-PCR were measured to analyze osteogenic differentiation characteristics of scaffolds. Five samples per group (length 13.5 x width 13.5 mm thickness 2.3 mm) were used. The support was placed in a 12-well plate, incubated overnight with 70% ethanol, treated with UV for 30 minutes and then incubated in DMEM medium (without FBS) for 2 hours at 37 ° C. 5 × 10 ⁵ cells / well of MSC were cultured on a supernatant in DMEM culture medium for 5 days at 37 ° C. in 5% CO ₂ for 1 day. On the second day of culture, the culture medium of each well was replaced with ODM medium, and the culture medium was changed every 2 days. After 4, 7, and 14 days of culture, total RNA of osteoblasts was isolated using TRIzol ^® reagent according to the manufacturer's instructions, and RT-PCR was performed to check the specific mRNA expression of the differentiated cells. One μg of total RNA was mixed with diethyl pyrocarbonate (DEPC) -treated water to reach a total volume of 19 μl in a Maxime PreMix kit, followed by reverse transcription at 45 ° C. for 30 minutes, Lt; 0 &gt; C for 5 minutes. The target cDNA was amplified using the following primers:

ALP: 5'-ACGTGGCTAAGAATGTCATC-3 '(forward) / 5'-CTGGTAGGCGATGTCCTTA-3' (reverse direction);

OSC: 5'-ATGAGAGCCCTCACACTCCTC-3 '(forward) / 5'-GCCGTAGAAGCGCCGATAGGC-3' (reverse direction);

OPN: 5'-GACCACATGGACCGACGATG-3 '(forward) / 5'-TGGAACTTGCTTGACTATCGA-3' (reverse direction);

Runx2: 5'-TGGCGTCAAACAGCCTCTTC-3 '(forward) / 5'-TGGTGCTCGGATCCCAAA-3' (reverse direction); And

GAPDH: 5'-GCCACCCAGAAGACTGTGGAT-3 '(forward) / 5'-TGGTCCAGGGTTTCTTACTCC-3' (backward).

 Finally, the relative mRNA expression levels of each gene were averaged with GAPDH and the PCR products were electrophoresed on 1.2% agarose gels.

Figure 8 shows mRNA expression levels of ALP, OSC, Runx2 and OPN of MSCs cultured in PCL and PCL / IGDP, PCL / Col, PCL / Sa, PCL / IGDP / Col, and PCL / IGDP / Sa scaffolds. PCL / IGDP, PCL / Col, and PCL / Sa in both PCL / IGDP / Col and PCL / IGDP / Sa scaffolds at day 7 (ALP) and day 14 (OPN and OSC) ALP, OSC, and OPN expression were found. In addition, PCL / IGDP / Col and PCL / IGDP / Sa supernatant showed significantly higher Runx2 expression on day 7 of culture than PCL alone. Indicating that the PCL / IGDP / Col and PCL / IGDP / Sa scaffolds are more amenable to bone differentiation.

Example  8. Support of  Confirming the effect of rabbit bone regeneration

PCL / IGDP / PCL / IGDP / Col and PCL / IGDP / Sa scaffolds were inserted into the rabbit tibia. A 10 week old male rabbit was used in the experiment to confirm bone regeneration and did not show any inflammation or immune response after the transplantation. Animals were adapted to a controlled environment with a 12 h person / cancer cycle, relative humidity (40 - 70%) and temperature (20 - 24 ° C) before the experiment. Four groups of scaffolds (PCL, PCL / IGDP, PCL / IGDP / Col, and PCL / IGDP / Sa scaffolds (length 6.0 mm x width 2.0 mm thickness 2.0 mm) were pre-sterilized. The skin of the tibia was removed and the site was aseptically treated with povidone and 70% ethanol for surgery. Anesthesia was induced by intramuscular injection of a mixture of ketamine (80 mg / kg) and xylazine (10 mg / kg). Animals were maintained under general anesthesia during the entire surgical procedure and 2.1 mm diameter bone damage was made in the middle of each rabbit leg tibia bone. A total of four samples were inserted into the defect sites of each group. After insertion of the supporter, the surgical site was closed and treated with povidone iodine solution. To evaluate the osteogenesis of the rabbit tibial defect site with each support, evaluation was performed using X-ray micro-CT scanning (NFR Polaris-G90, NanoFocusRay, Iksan, Korea). Μ-CT images were used to measure the degree of new tissue and the distribution of newly formed tissues suggests that they are found in soft tissue (Min) and hard tissue (Max). The ratio of new bone volume (RT) was calculated by the following formula. The newly formed tissue area and the existing tissue area are on the surface of 0.48 mm ² , and the mean value is a median factor corresponding to the soft tissue and hard tissue area values.

New soft and hard tissues were clearly observed at the center two weeks and six weeks after transplantation in the control (PCL support), PCL / IGDP, and PCL / IGDP / Sa. PCL / IGDP / Col and PCL / IGDP / Sa scaffolds formed larger tissue than the other implants. The hard tissue of the PCL / IGDP / Col scaffold was the largest in the total defect. A new bone rate (0.606 per new bone maximum at 1.0 (* p <0.05)) after PCL / IGDP / Col support transplantation at 6 weeks was 0.244 per PCL (1.0 (* p <0.05) , PCL / IGDP (0.426 per new bone maximum at 1.0 (* p <0.05)) and PCL / IGDP / Sa (0.441 per new bone maximum at 1.0 .

Claims (14)

Biodegradable polymer scaffolds;
A coating layer formed on the surface of the supporter and containing an abalone built-in hydrolyzate obtained by treating the abalone protein with a protein hydrolyzing enzyme; And
And a coating layer formed on the surface of the coating layer containing the hydrolyzate and containing fish-derived collagen or alginic acid. The biodegradable polymer of claim 1, wherein the biodegradable polymer is selected from the group consisting of poly (epsilon -caprolactone), polylactic acid, polyglycolic acid, polylactic acid-glycolic acid copolymer, polydioxanone, polyhydroxybutyrate, Wherein the support is one or more selected from the group consisting of polyoxyethylene stearate, polyoxyethylene stearate, polyoxyethylene stearate, polyoxyethylene stearate, polyoxyethylene stearate, polyoxyethylene stearate, polyoxyethylene stearate, polyoxyethylene stearate, 2. The bone tissue regeneration support according to claim 1, wherein the protein hydrolyzing enzyme is at least one selected from the group consisting of pepsin, trypsin, chymotrypsin, pancreatin, alcalase, flavosome, papain, . The scaffold of claim 1, wherein the overturned hydrolyzate is a protein having a molecular weight of 1 to 10 KDa. The support for bone tissue regeneration according to claim 1, wherein the fish is at least one member selected from the group consisting of a salmon neck salmon, a perch fish, a perch fish mackerel, a perch fish mackerel, a perch fish mackerel, a perch sechiride fish, a cod fish and a flounder. The support for regeneration of bone tissue according to claim 1, wherein the alginic acid is derived from algae. The support for bone tissue regeneration according to claim 1, wherein the alginic acid is sodium alginate. The support for regeneration of bone tissue according to claim 1, wherein the biodegradable polymer scaffold has a pore size of 400 to 500 탆 and is in the form of a three-dimensional lattice. Preparing a biodegradable polymer scaffold;
Forming a coating layer on the surface of the supporter, wherein the abrasion-resistant protein is treated with a protein hydrolyzing enzyme; And
And forming a coating layer containing fish-derived collagen or alginic acid on the surface of the coating layer containing the hydrolyzate.  [Claim 11] The method of claim 9, wherein the biodegradable polymer scaffold is fabricated such that the strand of the biodegradable polymer exhibits a three-dimensional lattice pattern.   10. The biodegradable polymer of claim 9, wherein the biodegradable polymer is selected from the group consisting of poly (epsilon -caprolactone), polylactic acid, polyglycolic acid, polylactic acid-glycolic acid copolymer, polydioxanone, polyhydroxybutyrate, Wherein the support is at least one member selected from the group consisting of hydroxypropylcellulose, hydroxypropylcellulose, hydroxypropylcellulose, hydroxypropylcellulose, hydroxypropylcellulose, hydroxypropylcellulose, hydroxypropylcellulose, hydroxypropylcellulose, hydroxypropylcellulose, 10. The method according to claim 9, wherein the step of forming the coating layer containing the overturned hydrolyzate is carried out by immersing the biodegradable polymer scaffold in the solution containing the overturned hydrolyzate, / RTI &gt; 11. The bone tissue regeneration support according to claim 9, wherein the protein hydrolyzing enzyme is at least one selected from the group consisting of pepsin, trypsin, chymotrypsin, pancreatin, alcalase, flavosome, papain, &Lt; / RTI &gt; The method according to claim 9, wherein the step of forming the coating layer containing fish-derived collagen or alginic acid comprises: immersing a biodegradable polymer scaffold in which a coating layer containing the overturned hydrolyzate is formed in a solution containing fish-derived collagen or alginic acid &Lt; / RTI &gt; wherein the method is performed by a method comprising the steps of:

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