JP2016158680A - Scaffold for osteoanagenesis or for skin regeneration using graphene oxide - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new scaffold excellent in cell proliferation promotion to the inside of the scaffold, which is a scaffold for osteoanagenesis and for skin regeneration in which utilization frequency of the scaffold is high.SOLUTION: In a scaffold for osteoanagenesis or for skin regeneration containing a substrate comprising a porous scaffold, and graphene oxide which is a coating onto at least a part of the surface of the substrate, the coating amount of the graphene oxide is in the range of 0.1-2.5 mass% based on the mass of the substrate.SELECTED DRAWING: None

Description

  The present invention relates to a bone regeneration or skin regeneration scaffold using graphene oxide. More particularly, the present invention relates to a scaffold for bone regeneration or skin regeneration, in which graphene oxide is applied to a collagen biomaterial and tissue regeneration ability is enhanced.

  Regenerative medicine is a treatment method that functionally restores tissues and organs lost due to illness or injury. Regenerative medicine incorporates the principle of tissue engineering consisting of three elements: cells, bioactive substances, and scaffolds (scaffolds). In clinical practice, a method of promoting cell proliferation using scaffolds (scaffold treatment) and a method of directly using cultured cells such as iPS cells are roughly classified.

  The scaffold has an appropriate in vivo strength based on a three-dimensional skeleton, and serves as a scaffold for proliferating cells by securing a regeneration space by transplanting and storing. Cells that proliferate and migrate into the scaffold secrete the extracellular matrix using the scaffold as a scaffold, and at the same time, the vascular network is constructed and self-organizes.

  Further, in the case where accumulation of self cells in the living body is not expected, a method is considered in which cultured cells are seeded in advance in a scaffold and then transplanted into the living body. Similarly, cultured cells transplanted into a living body are proliferated, differentiated and organized using the scaffold as a scaffold.

  For the scaffold, polymers such as collagen, fibrin, chitosan, hyaluronic acid, polylactic acid, and inorganic materials such as hydroxyapatite and calcium triphosphate are applied, and it is necessary to select a substrate according to the regenerated tissue. There are also combinations of physiologically active substances such as fibroblast growth factor (FGF-2), osteoinductive protein (BMP-2), and platelet-derived growth factor (PDGF-BB).

  There are the most orthopedic areas, such as bone and cartilage, for the scaffold treatment, and Medronic Infuse Bone Graft and Olympus Terumo Biomaterials Osferion are commercially available as bone regeneration scaffolds. large. Examples of target diseases include spinal fixation and tumor bone metastasis. Next, there are lots of skin and periodontal tissue, etc., such as LifeCell's allodarm, Olympus Terumo Biomaterials' terdermis, and Koken's bone project. Target diseases include skin burns, breast reconstruction, periodontitis, and implant treatment.

  Scaffolds do not cause inflammation even when placed in vivo, and are required to have excellent biocompatibility that is absorbed and replaced by living tissue. However, for faster self-organization, cells can grow inside the scaffold ( Ingredient) promotion is required. In cases where complete communication holes are provided so that cells can proliferate early in the interior (Non-patent Document 4), a specific structure is imparted to the scaffold surface, inclusion of physiologically active substances, etc. (Non-patent Documents) Reference 5).

Lee WC, Lim CH, Shi H, Tang LA, Wang Y, Lim CT, Loh KP. Origin of enhanced stem cell growth and differentiation on graphene and graphene oxide.ACS Nano, 27, 7334-7341, 2011. E Nishida, H Miyaji, H Takita, I Kanayama, M Tsuji, T Akasaka, T Sugaya, R Sakagami, M Kawanami.Graphene oxide coating facilitates the bioactivity of scaffold material for tissue engineering.Jpn J Appl Phys, 53, 1-7 , 2014. I Kanayama, H Miyaji, H Takita, E Nishida, M Tsuji, B Fugetsu, L Sun, K Inoue, A Ibara, T Akasaka, T Sugaya, M Kawanami. Comparative study of bioactivity of collagen scaffolds coated with graphene oxide and reduced graphene oxide. Int J Nanomedicine, 9, 3363-3373, 2014. H Miyaji, H Yokoyama, Y Kosen, H Nishimura, K Nakane, S Tanaka, K Otani, K Inoue, A Ibara, I Kanayama, T Yoshida, K Ogawa, E Nishida, M Kawanami.Bone augmentation in rat by highly porous β -TCP scaffolds with different open-cell sizes in combination with fibroblast growth factor-2.J Oral Tissue Engin 10, 172-181, 2013. A Ibara, H Miyaji, B Fugetsu, E Nishida, H Takita, S Tanaka, T Sugaya, M Kawanami. Osteoconductivity and biodegradability of collagen scaffold coated with nano-β-TCP and fibroblast growth factor 2.J Nanomater, 2013, 1- 11, 2013.

  As described above, various studies have been made for the purpose of promoting the proliferation of cells into the scaffold (ingulose) for rapid self-assembly (see Non-Patent Documents 4 and 5). However, no scaffold is provided that is capable of promoting a sufficiently satisfactory level of growth. Therefore, an object of the present invention is to provide a new scaffold excellent in promoting the proliferation of cells into the scaffold for rapid self-organization. In particular, an object of the present invention is to provide a new scaffold that is a scaffold for bone regeneration and skin regeneration that frequently uses the scaffold, and that is excellent in promoting the proliferation of cells into the scaffold.

  In recent years, biological applications of nanocarbon have been studied. Nanocarbon includes carbon nanotube, carbon nanohorn, carbon nanofiber, fullerene, graphene and the like. Graphene is a single layer nanosheet of carbon, a nanomaterial about 1 nanometer thick. Graphene oxide (GO) is obtained by imparting a functional group to graphene and improving dispersibility.

  GO has been reported to promote proliferation and differentiation of mesenchymal stem cells in vitro. (Non-patent document 1). It is also known that GO can coat the surface of porous collagen (Non-patent Documents 2 and 3). In addition, the GO-coated collagen scaffold improves strength without changing porosity, imparts GO-derived nanostructures to the collagen surface, and promotes cell ingrowth in subcutaneous connective tissue. It is known (Non-Patent Document 2). However, the promotion of cell growth in subcutaneous connective tissue was obtained only with 0.01 wt% GO coating and could not be observed with 0.1 wt% GO and 0.5 wt% GO coating. Furthermore, cell proliferation when seeded with osteoblast-like cells is 0.01 wt% GO coating, which is equivalent to or slightly inferior to control (no GO coating), 0.1 wt% GO and 0.5 wt% GO coating Was significantly worse than the control.

  The results described in Non-Patent Document 2 show that for cells in subcutaneous connective tissue, 0.01 wt% GO coating has an effect of promoting ingulose, but has no effect on cell proliferation. It was the thing which showed that proliferation was inhibited.

Under these circumstances, the GO coating on the collagen scaffold was considered unsuitable for regenerative therapy, but the present inventors dared to use bone treatment therapy and skin regenerative therapy based on GO treatment. Challenged.
As a result, unexpectedly, in bone and skin, the GO treatment base material has an effect of enhancing the effect of scaffold treatment, and this effect is compared to the cases described in Non-Patent Documents 2 and 3. The present invention was completed by finding that it can be seen in a region where the coating amount is reduced.

The present invention is as follows.
[1]
A base material composed of a porous scaffold, and graphene oxide which is a coating on at least a part of the surface of the base material, and the coating amount of the graphene oxide is 0.1-2. Scaffold for bone regeneration or skin regeneration in the range of 5% by mass.
[2]
The scaffold according to [1], wherein the porous scaffold is made of at least one material selected from the group consisting of collagen, gelatin, fibrin, chitosan, hyaluronic acid, polylactic acid, hydroxyapatite, and calcium triphosphate.
[3]
The scaffold according to [1] or [2], wherein the porous scaffold has a porosity in the range of 50 to 99% and has a continuous pore structure.
[4]
The compressive strength at 50% displacement (measurement condition: crosshead speed 0.5 mm / min) is 1.2 times or more that of the porous scaffold base material, according to any one of [1] to [3]. Scaffold.
[5]
The scaffold according to any one of [1] to [4], wherein the coating amount of graphene oxide is in the range of 1.0 to 2.3 mass% based on the mass of the substrate.
[6]
Any one of [1] to [5], wherein the graphene oxide coating is formed of thin film graphene oxide particles having a thickness of 0.4 to 10 nm and a planar size of 20 nm or more. The described scaffold.
[7]
The scaffold according to any one of [1] to [6], which is used for bone regeneration.
[8]
The scaffold according to [7], wherein the regeneration target is bone and cartilage.
[9]
The scaffold according to any one of [1] to [6], which is for skin regeneration.
[10]
The scaffold according to [9], wherein the regeneration target is epidermis and dermis.
[11]
The scaffold production method according to any one of [1] to [6],
The manufacturing method, comprising injecting a dispersion of graphene oxide particles into a substrate made of a porous scaffold, and coating at least a part of the surface of the substrate with graphene oxide.
[12]
The said dispersion liquid is a manufacturing method as described in [11] which is a thing which disperse | distributed the graphene oxide particle | grains in the mixed solution of water and N-methyl-2-pyrrolidone.
[13]
The production method according to [11] or [12], wherein the concentration of the graphene oxide particles in the dispersion is in the range of 0.0001 to 0.01% by mass.

  According to the present invention, it is possible to provide a scaffold that is particularly effective in bone regeneration therapy and skin regeneration therapy. ADVANTAGE OF THE INVENTION According to this invention, it is a scaffold for bone regeneration and skin regeneration with high use frequency of a scaffold, Comprising: The new scaffold excellent in the proliferation promotion to the inside of a scaffold can be provided.

a: Photograph of collagen scaffold (hereinafter referred to as col), 0.001 GO-coated collagen scaffold (hereinafter referred to as GO scaffold), and 0.01GO scaffold. b: Scanning electron microscope (hereinafter SEM) photograph of col surface. c: SEM photo of the 0.001GO GO scaffold surface. d: SEM photograph of 0.01GO scaffold surface. Compressive strength test results of 0.0003GO, 0.001GO, 0.003GO, 0.01GO scaffold and col. Collagenase dissolution test results for 0.001GO, 0.01GO, 0.1GO scaffold and col. a: Cell proliferation test results of 0.01GO, 0.025GO, 0.05GO, 0.1GO scaffold and col. b: Cell proliferation test results of 0.0003GO, 0.001GO, 0.003GO, 0.01GO scaffold and col. a: Pictures of 0.001GO, 0.01GO coated collagen sponge (hereinafter GO sponge) and collagen sponge (hereinafter cs). b: Cell proliferation test result of 0.001GO, 0.01GO sponge and cs. Tissue image 10 days after rat skull model surgery (hematoxylin eosin (hereinafter referred to as HE) staining). a: After 0.01GO scaffold implantation, b: After col implantation. Tissue image 35 days after rat skull model surgery (HE staining). a: after 0.01 GO scaffold implantation, b: 1 magnified image of a, arrows indicate macrophage-like cells, c: after 0.001 GO scaffold implantation. a: Tissue image of rat skull model col 35 days after implantation (HE staining), b: Rat skull model control (ctrl) 35 days tissue image (HE staining), c: 0.001GO, 0.01GO scaffold, col implantation Results of new bone area measurement after planting and control (ctrl). Tissue image 14 days after rat skull model surgery (peroxidase staining). a: After 0.01GO scaffold implantation, b: After col implantation. Tissue image 14 days after rat skull model surgery. a: Heavy staining with anti-ED1 / anti-Galectin-3 antibody after 0.01GO scaffold and col implantation (staining of macrophages), b: Staining with anti-smooth muscle actin antibody after 0.01GO scaffold and col implantation (blood vessels) Smooth muscle cell staining). Tissue image 14 days after rat skull model surgery. a: Staining with anti-RECA-1 antibody after 0.01GO scaffold and col implantation (vascular endothelial cell staining), b: Staining with anti-Prolyl-4-Hydroxylase beta antibody after 0.01GO scaffold and col implantation ( Staining of fibroblasts). Histological image 14 days after operation in canine extraction socket (HE staining) a: 0.01GO scaffold, b: a 1 enlarged image, c: a 2 enlarged image, d: a 3 enlarged image. a: Histological image (HE staining) 14 days after col implantation in canine tooth extraction socket, b: Measurement result of new bone formation rate after 0.01 GO scaffold and col implantation. Tissue image 10 days after surgery for rat skin defect model (HE staining). a: after 0.01GO scaffold implantation, b: a magnified image of 1 a, c: a magnified image of 2, an arrow indicates the epithelial extension tip, d: a magnified image of 3, an arrow indicates the epithelium The extension tip is shown. Tissue image 10 days after surgery for rat skin defect model (HE staining). After a: col implantation, b: a 1 magnified image, c: a 2 magnified image, arrow shows epithelial extension tip, d: a 3 magnified image, arrow epithelium extension tip Show. Tissue image 10 days after surgery for rat skin defect model (HE staining). a: control (ctrl), b: a magnified image of 1 in c, a magnified image of 2 in c: a, arrow indicates the tip of the epithelium, d: a magnified image of 3, the arrow indicates the tip of the epithelium Show. Measurement results of epithelial invasion rate after 0.01GO scaffold, col implantation and control (ctrl).

<Scarf for bone regeneration or skin regeneration>
The present invention includes a base material composed of a porous scaffold, and graphene oxide which is a coating on at least a part of the surface of the base material, and the coating amount of the graphene oxide is 0. Scaffolds in the range of 1 to 2.5% by weight, which scaffolds are used for bone regeneration or are used for skin regeneration.

  The porous scaffold is not particularly limited as long as it is known as a scaffold used for regenerative therapy or is a scaffold made of a porous biomaterial used for regenerative therapy. For example, collagen, gelatin, fibrin It can be made of a polymer material such as chitosan, hyaluronic acid or polylactic acid, or can be made of an inorganic material such as hydroxyapatite or calcium triphosphate, and can be a composite of these materials. .

  Although there is no restriction | limiting in particular also in the porous of a porous scaffold, From a viewpoint that it can become a favorable scaffold of cell growth, it is preferable that it has a comparatively high porosity, for example, 50-99%. It can have a range of porosity. The porosity here is defined as porosity (%) = 100 × (1−ρ1 / ρ2), ρ1: the density of the scaffold, ρ2: the true density of the material constituting the scaffold. The porous structure is preferably a continuous pore structure. The continuous pore structure is a structure in which at least a part of the pores in the scaffold are in communication and has isolated pores. Preferably not.

  The scaffold of the present invention has a graphene oxide coating on at least a part or the entire surface of a substrate made of a porous scaffold. The surface coating rate varies depending on the coating amount of graphene oxide, the surface area of the porous scaffold, the porosity, and the like. The coating amount of graphene oxide is preferably in the range of 0.1 to 2.5% by mass based on the mass of the base material, and preferably from the viewpoint of providing a particularly effective scaffold in bone regeneration therapy and skin regeneration therapy. It is the range of 1.0-2.3 mass%, More preferably, it is the range of 1.4-2.3 mass%. As shown in the Examples, in SEM observation, collagen scaffolds coated with 1.83% by mass and 2.22% by mass of graphene oxide based on the mass of the substrate (0.001 GO and 0.01 GO, porosity: 97 (.604% and 97.597%), it was confirmed that the entire surface was coated with graphene oxide (FIGS. 1c and d).

  Since the scaffold of the present invention has a graphene oxide coating, the mechanical strength is enhanced, and the compressive strength at 50% displacement (measurement condition: crosshead speed 0.5 mm / min) is the same as that of the porous scaffold base material. There is a significant difference. Specifically, for example, the compressive strength at 50% displacement (measurement condition: crosshead speed 0.5 mm / min) can be 1.2 times or more that of the porous scaffold base material. As shown in Example 1, when the porous scaffold base material is a collagen scaffold base material, the compressive strength at 50% displacement (measurement condition: crosshead speed 0.5 mm / min) has a graphene oxide coating. Compared to no control, it is in the range of about 1.3 times to 1.9 times depending on the amount of graphene oxide coating.

  The coating of graphene oxide can be a layer in which thin-film graphene oxide particles are coated on the surface of a porous scaffold substrate, and the thin-film graphene oxide particles have a thickness of, for example, 0.4 to 10 nm and are planar. The direction size can be more than 20nm. Thus, the scaffold of the present invention having a coating using very fine thin-film graphene oxide particles has an effect of enhancing mechanical strength and promoting ingress in bone regeneration therapy and skin regeneration therapy. Inferred to have.

  The scaffold of the present invention is for bone regeneration, and the regeneration target is bone and cartilage.

  The scaffold of the present invention is for skin regeneration, and the regeneration target is epidermis and dermis.

<Manufacturing method of graphene oxide coating scaffold>
The scaffold manufacturing method of the present invention includes injecting a dispersion of graphene oxide particles into a substrate made of a porous scaffold, and coating at least a part of the surface of the substrate with graphene oxide. . The dispersion of graphene oxide particles, for example, using an injection jig such as a syringe, insert the injection needle into the center of the porous scaffold, and the dispersion from the tip of the injection needle inserted into the center into the interior of the substrate The graphene oxide coating is applied to at least one of the inner surface and the outer surface of the porous scaffold while the injected dispersion travels through the porous scaffold to the outer surface of the substrate. Part or all.

  This graphene oxide coating method is possible because the substrate is porous and the graphene oxide particles are very fine, and the coating is uniformly formed on the inner and outer surfaces of the porous scaffold. There are advantages.

  The dispersion liquid of graphene oxide particles can be a dispersion liquid dispersed in a mixed solution of graphene oxide particles, water, and N-methyl-2-pyrrolidone (NMP). In the mixed solution of water and NMP, the concentration of NMP is, for example, in the range of 0.1 to 99% by mass. A graphene oxide coating can be satisfactorily formed by using a dispersion having a concentration of NMP in this range. However, the higher the ratio of NMP, the better the adhesion of graphene oxide particles to the scaffold substrate surface. Therefore, the concentration of NMP is, for example, in the range of 10 to 99% by mass. The lower limit is preferably 20% by mass or more, more preferably 50% by mass or more.

  The concentration of the graphene oxide particles in the dispersion is suitably in the range of, for example, 0.0001% by mass or more from the viewpoint of forming a coating having good properties as a scaffold for regenerative therapy. More preferably, it is in the range of 0.0001 to 1% by mass, and still more preferably in the range of 0.0001 to 0.05% by mass. The amount of graphene oxide coated on the scaffold for regenerative therapy depends not only on the concentration of the coating solution, but also on the size and internal structure of the scaffold, so depending on the type of scaffold, it contains the desired graphene oxide content. The concentration of the coating solution can be set as appropriate so that the amount is sufficient.

  A dispersion containing water, NMP and graphene oxide particles can be prepared by arbitrarily mixing a commercially available graphene oxide-containing solution and NMP, or water and NMP. When mixing water and NMP, NMP may be added and mixed after mixing the graphene oxide-containing solution and water, or the graphene oxide-containing solution may be added to and mixed with the mixed solution of water and NMP.

  Generally, the graphene oxide particles contained in the graphene oxide-containing solution are graphite intercalation compounds produced by oxidizing graphite by a specific method. Graphite oxidation methods for obtaining graphene oxide include the known Brodie method (using nitric acid and potassium chlorate), the Staudenmeier method (using nitric acid, sulfuric acid and potassium chlorate), and the Hummers-Offeman method (sulfuric acid and sodium nitrate). , Using potassium permanganate). Of these, oxidation proceeds particularly in the Hummers-Offeman method (WS Hummers et al., J. Am. Chem. Soc., 80, 1339 (1958); US Pat. No. 2,798,878 (1957)). This oxidation method is particularly recommended in the present invention. Note that graphene oxide with a large number of layers is usually called graphite oxide, but in the present invention, it is called graphene oxide even when the number of layers is large.

  In order to peel graphene oxide into layers and obtain graphene oxide with a small number of layers, for example, graphene oxide may be sufficiently purified. For the purification operation, known means such as decantation, filtration, centrifugation, dialysis, and ion exchange may be used. At the time of purification, separation of the multilayer structure occurs spontaneously, but in addition to this, addition of a stirring operation such as shaking or a physical force such as a shearing force is desirable because the separation is further promoted. Ultrasonic irradiation can also be used, but the aspect ratio tends to be reduced due to destruction of the basic structure of each layer as the layers are separated. By the above method, a graphene oxide-containing solution can be prepared. Graphene oxide can have a single layer structure or a multilayer structure.

  The oxygen content of graphene oxide in the graphene oxide-containing solution is preferably 5% by mass or more, more preferably 10% by mass or more, and further preferably 30% by mass or more. When the oxygen content is less than 5% by mass, layer separation from graphite oxide becomes difficult. Moreover, if the oxygen content is 30% by mass or more, layer separation from graphite oxide becomes easy. The upper limit of the oxygen content is generally up to about 50% by mass in a sufficiently oxidized state. The oxygen content of graphene oxide in the graphene oxide-containing solution can be evaluated by XPS (X-ray photoelectron spectroscopy) or the like as described above for graphene oxide vacuum-dried at 40 ° C.

  The graphene oxide particles in the graphene oxide-containing solution used in the present invention are preferably thin film particles having a thickness of 0.4 to 10 nm and a planar size of 20 nm or more. The graphene oxide, which is a thin film particle, has less than 20 basic layers of graphene oxide having a layer structure, and can change flexibly even though it has a dense carbon skeleton. For this reason, the substrate surface can be closely adhered along the fine uneven shape of the substrate surface, and as a result, a strong adhesion between the coating and the substrate can be realized. During close contact with the material surface, resistance to external force is relatively small, and resistance to peeling from the substrate surface is also high. In addition, in bone regeneration therapy and skin regeneration therapy, it is presumed that ingrose has an effect of promoting.

  The thinner the graphene oxide particles in the graphene oxide-containing solution are, the more preferable, since a thin graphene oxide layer can be formed. Therefore, the thickness of the graphene oxide particles is preferably 60% or more of graphene oxide particles having a thickness of 5 nm or less, and more preferably 60% or more of graphene oxide particles having a thickness of 1.5 nm or less. preferable. The thickness can be evaluated by the following method using an atomic force microscope. Drop the diluted aqueous dispersion of graphene oxide particles onto the substrate (mica), find isolated particles that do not overlap with the atomic force microscope, and measure the height of the substrate and the isolated particles measured by the atomic force microscope. The difference is the thickness of the particles. In the case where wrinkles are formed in the particles, the wrinkled portion does not reflect the thickness. Therefore, the thickness is evaluated based on the difference in height between the wrinkled portion and the substrate. Because of the influence of adsorbed water, it is considered that the number of graphene oxide layers is one when the thickness is 1.5 nm or less. The content ratio of graphene oxide having a certain thickness or less is calculated by measuring the thickness of 30 particles and calculating the ratio of graphene oxide having a certain thickness or less in 30 particles.

  The graphene oxide used in the present invention has a thickness of 0.4 to 10 nm, a planar size of 20 nm or more, and graphene oxide, which is a thin film-like particle dispersible in a liquid having a relative dielectric constant of 15 or more, For example, it can be manufactured using the manufacturing method described in Japanese Patent No. 4798411. In addition, as the graphene oxide dispersion having the above characteristics, a graphene oxide dispersion (nano GRAX (registered trademark), 1 wt%, Mitsubishi Gas Chemical Co., Ltd.) can also be used.

  Hereinafter, the present invention will be described in more detail based on examples. However, the examples are illustrative of the present invention, and the present invention is not intended to be limited to the examples.

Production Example 1
SEM observation [materials and methods]
GO dispersion (1 wt%, nano GRAX (registered trademark), Mitsubishi Gas Chemical) was diluted with 1-methyl 2-pyrrolidone to prepare 0.0001, 0.001, and 0.01 wt% GO solutions. Col (Terdermis (registered trademark), Olympus Terumo Biomaterial) molded to 6 x 6 x 3 mm (3.7 mg) is immersed in 0.1 mL of GO solution of each concentration, washed with ethanol, dehydrated, dried in air, 0.0001GO, 0.001GO, and 0.01GO scaffolds were used, respectively (FIGS. 1a and 0.0001GO not shown). The GO contents (mass%, the same applies hereinafter in the examples) of the 0.0001GO, 0.001GO, and 0.01GO scaffolds were 1.1%, 1.83%, and 2.22%, respectively, as a result of measurement. The surface structure of each scaffold of 0.001GO and 0.01GO was observed by SEM under conditions of platinum palladium deposition and acceleration voltage 10kV. As a control, the surface structure of col was also observed (FIGS. 1b to 1d).
[result]
In Col, a striated structure consistent with the collagen crosslinking cycle was observed in the collagen fibers. On the other hand, since 0.001GO and 0.01GO did not show a striated structure on the scaffold surface, it was considered that the scaffold surface was uniformly coated with GO. In addition, the GO-like structure was partially observed at 0.01GO. Although the SEM photograph of 0.0001GO is not shown, the scaffold surface was uniformly coated with GO as in the case of 0.001GO and 0.01GO.
[Conclusion]
The scaffold surface was coated with GO by immersing the scaffold in GO solution.

Example 1
Compressive strength test [materials and methods]
GO dispersion (1 wt%, nano GRAX (registered trademark), Mitsubishi Gas Chemical) was diluted with 1-methyl-2-pyrrolidone to prepare 0.0003, 0.001, 0.003, and 0.01 wt% GO solutions. Col (Terdermis (registered trademark), Olympus Terumo Biomaterial) molded to 6 x 6 x 3 mm is immersed in 0.1 mL of GO solution at various concentrations to coat the surface with GO, washed with ethanol, dehydrated, and dried in air 0.0003GO, 0.001GO, 0.003GO, and 0.01GO scaffolds, respectively. The GO contents (% by mass) of the 0.0003GO, 0.001GO, 0.003GO, and 0.01GO scaffolds are 1.42%, 1.83%, 2.06%, and 2.22%, respectively, as a result of measurement. The compressive strength of each scaffold at 25% and 50% displacement was measured at a crosshead speed of 0.5 mm / min using a universal testing machine. As a control, the compressive strength of col was measured in the same manner.

[result]
The compressive strength was improved to the concentration dependence of GO by GO coating for both 25% and 50% displacement. The measurement at 50% displacement was significantly higher even in the low concentration 0.0003GO scaffold. The largest was the 0.01GO scaffold, which was 1.7 times at 50% displacement and 1.8 times at 25% displacement compared to the control. (Figure 2)

[Conclusion]
Collagen scaffold GO coating increased the compressive strength in a concentration-dependent manner. Non-Patent Document 2 reports that the compressive strength at 50% displacement is significantly increased compared to the control in the GO scaffold using the GO coating solution having a concentration of 0.01 wt% or more. In particular, it was found that the compressive strength was significantly increased compared with col even in the GO scaffold having a GO content (mass%) of 1.42 to 2.22 mass%.

Example 2
Degradability test [materials and methods]
A GO dispersion (1 wt%, nano GRAX (registered trademark)) was diluted with 1-methyl 2-pyrrolidone to prepare 0.001, 0.01, 0.1 wt% GO solutions. Col (Terdermis (registered trademark)) molded to 6 x 6 x 3 mm is immersed in 0.1 mL of GO solution of each concentration, coated on the surface, washed with ethanol, dehydrated, dried in air, 0.001 GO each , 0.01GO and 0.1GO scaffolds. The GO content (mass%) of the scaffold is 1.83%, 2.22%, and 4.67%, respectively. After measuring the weight of each sample, it was immersed in 0.1 mg / ml collagenase at 37 ° C. for 3 hours. Thereafter, the sample was dried, and the amount of decomposition was calculated from the weight. As a control, the amount of col degradation was measured in the same manner.

[result]
Collagenase degradability was significantly lower than col for 0.001GO, 0.01GO and 0.1GO. 0.1GO was the lowest and significantly lower than 0.001GO and 0.01GO (Fig. 3). This indicates that the GO scaffold is difficult to be decomposed by an enzyme in the living body, and it is suggested that it is effective for securing a regenerating field in the living body together with the result of the compressive strength of Example 1.

[Conclusion]
Collagenase dissolution was significantly reduced in GO scaffolds with GO content (mass%) of 1.83 to 2.22 mass%.

Example 3
Cell proliferation test 1
[Materials and methods]
A GO dispersion (1 wt%, nano GRAX (registered trademark)) was diluted with 1-methyl 2-pyrrolidone to prepare 0.01, 0.025, 0.05, 0.1 wt% GO solutions. Col (Terdermis (registered trademark)) molded to 6 x 6 x 3 mm is immersed in 0.1 mL of GO solution of each concentration, coated on the surface, washed with ethanol, dehydrated, dried in air, 0.01 GO each 0.025GO, 0.05GO and 0.1GO scaffolds. As a result of measurement, the GO content (mass%) of the scaffold was 2.22%, 1.42%, 2.95%, and 4.67%, respectively. Each sample was seeded with 50000 osteoblast-like cells MC3T3-E1 and cultured under conditions of 37 ° C. and 5% CO ₂ . On the first, third, fifth and seventh days, cell proliferation was measured using cell counting kit-8 (CCK-8). As a control, the cell proliferation amount of col was measured in the same manner.

[result]
The cell growth amount of 0.01GO was the largest, and the higher the GO coating concentration, the more the cell growth was suppressed. 0.1GO significantly suppressed cell proliferation on day 1 and day 5 compared to col, and was significantly suppressed in all periods compared to 0.01GO. 0.025GO and 0.05GO also tended to suppress cell growth compared to 0.01GO, but no significant difference was observed on days 3, 5, and 7 (Fig. 4a).

[Conclusion]
In Non-Patent Document 2, the proliferation of cells (subcutaneous connective tissue cells) at 0.01 GO was equal to or less than that of the col group. However, in the results of osteoblast-like cells, unexpectedly, cell growth of 0.01 GO (GO content 2.22% by mass) was equal to or higher than that of the col group.

Cell proliferation test 2
[Materials and methods]
GO dispersion (1 wt%, nano GRAX (registered trademark)) was diluted with 1-methyl 2-pyrrolidone to prepare 0.0003, 0.001, 0.003, 0.01 wt% GO solutions. Col (Terdermis (registered trademark)) molded to 6 x 6 x 3 mm is immersed in 0.1 mL of GO solution at various concentrations to coat the surface, washed with ethanol, dehydrated, dried in air, and 0.0003 GO each. , 0.001GO, 0.003GO, 0.01GO scaffolds. As a result of measurement, the GO content of the scaffold was 1.42%, 1.83%, 2.06%, and 2.22%, respectively. Each sample was seeded with 50000 osteoblast-like cells MC3T3-E1 and cultured under conditions of 37 ° C. and 5% CO ₂ . On the first, third, and seventh days, the amount of cell proliferation was measured using CCK-8. As a control, the cell proliferation amount of col was measured in the same manner.

[result]
Each sample showed cell growth over time, but the GO coating increased cell proliferation on the 3rd and 7th days, and on the 7th day, GO content (% by mass) was 1.42 to 2.22% by mass GO. The coating scaffold was significantly higher than the control, and there was no significant difference between each group (Fig. 4b).

[Conclusion]
In the osteoblast-like cells, it was found that cell proliferation was significantly promoted compared with the col group in all GO-coated scaffolds having a GO content (mass%) of 1.42 to 2.22 mass%.

Example 4
Cell proliferation test 3 (Atelocollagen sponge mighty)
[Materials and methods]
A GO dispersion (1 wt%, nano GRAX (registered trademark)) was diluted with 1-methyl 2-pyrrolidone to prepare a 0.001, 0.01 wt% GO solution. Collagen sponge (φ5 × 3mm, Atelocollagen sponge mighty, Koken, cs) is immersed in 0.05mL of GO solution of each concentration, coated on the surface, washed with ethanol, dehydrated, dried in air, 0.001GO each 0.01GO sponge (Fig. 5a). As a result of measurement, the GO content (mass%) of the scaffold was 1.05% and 1.25%, respectively. Each sample was seeded with 10,000 osteoblast-like cells MC3T3-E1 and cultured under conditions of 37 ° C. and 5% CO ₂ . On the first, third, and seventh days, the amount of cell proliferation was measured using CCK-8. As a control, the amount of cs cell proliferation was measured in the same manner.

[result]
The amount of cell proliferation by GO coating tended to increase compared to cs. On the third day, a significant difference was observed between 0.01GO and cs. On day 7, cell proliferation was most increased at 0.01 GO (FIG. 5b).

[Conclusion]
It was also found that the proliferation of osteoblast-like cells in the atelocollagen sponge mighty increased with GO coating with a GO content (mass%) of 1.05% to 1.25% compared to the cs group.

Example 5
Bone augmentation in rat skull model [Materials and Methods]
GO dispersion (1 wt%, nano GRAX (registered trademark)) was diluted with 1-methyl 2-pyrrolidone to prepare 0.001 and 0.01 wt% GO solutions. A col (Terdermis (registered trademark)) molded to 6 x 6 x 3 mm is immersed in a GO solution of each concentration, coated on the surface, washed with ethanol, dehydrated, dried in air, 0.001GO, 0.01 GO scaffold. As a result of measurement, the GO content (mass%) of the scaffold was 1.83% and 2.22%, respectively.

  Wistar rats (10 weeks old) were subjected to general and local anesthesia to expose the skull, and corticoids in the range of 4 x 4 mm were removed with a round bar under water injection (decortication). Subsequently, the GO scaffold or col was placed in the removal site, the skin was repositioned and sutured. In addition, a control (ctrl) that does not transplant anything was set. The rats were euthanized on the 10th and 35th day after the operation, and the transplanted site was excised together with surrounding tissues, fixed with formalin, decalcified with EDTA, and a tissue specimen was prepared according to the usual method. Histological observation was performed with an optical microscope, images were taken into a personal computer, and the area of new bone on day 35 was calculated with NIH image software.

[result]
On the 10th day, the 0.01GO scaffold (GO content 2.22% by mass) maintained a three-dimensional space with a sponge network structure (Fig. 6a), but the col was compressed and the scaffold collapsed. Was observed (Figure 6b). It was considered that the GO scaffold can easily maintain the three-dimensional form in vivo.

  On day 35, bone augmentation was observed by 0.01GO scaffold transplantation (FIG. 7a). The new bone had bone marrow-like tissue inside, and macrophage-like cells (arrows) containing black aggregates that seemed to be GO were often observed around the bone and in the bone marrow (Fig. 7b). Bone augmentation was also observed at 0.001GO (GO content 1.83 mass%), but the augmentation was less than 0.01GO (Fig. 7c). In the col transplanted group, bone augmentation was negligible (Figure 8a). In the control, almost no bone formation was observed (FIG. 8b). The result of new bone area measurement is shown in FIG. 9c. The area of new bone showed the maximum value with a 0.01GO scaffold, 2.7 times that of the control and 2.2 times that of col, showing a statistically significant difference. The 0.001GO scaffold also promoted bone augmentation and was significantly greater than the control. In col, the area of new bone was small and almost equal to that of the control (FIG. 8c).

[Conclusion]
Transplanting GO scaffolds with a GO content (mass%) of 1.83% to 2.22% onto the skull promoted bone augmentation. In particular, in the 0.01GO scaffold (GO content 2.22% by mass), the effect of promoting bone growth was very remarkable.

Example 6
Immunohistochemical evaluation in rat skull model [Materials and Methods]
A GO dispersion (1 wt%, nano GRAX (registered trademark)) was diluted with 1-methyl 2-pyrrolidone to prepare a 0.01 wt% GO solution. A col (Terdermis (registered trademark)) molded into 6 × 6 × 3 mm was dipped in a GO solution, coated with GO on the surface, washed and dehydrated with ethanol, and dried in air to obtain a 0.01GO scaffold. As a result of measurement, the GO content (mass%) of the scaffold was 2.22%.

  Wistar rats (10 weeks old) were subjected to general and local anesthesia to expose the skull, and corticoids in the range of 4 x 4 mm were removed with a round bar under water injection (decortication). Subsequently, 0.01GO scaffold or col was transplanted. On day 14, samples were removed and serial frozen sections were prepared. The sections were stained with peroxidase to confirm the appearance of neutrophils, and the presence or absence of inflammation was confirmed. In addition, immunohistological observations were performed in order to identify cells that had ingress in the interior. The antibodies include anti-ED1 and anti-Galectin-3 antibodies as markers for macrophages and monocytes, anti-Smooth muscle actin antibodies as markers for vascular smooth muscle cells, anti-RECA-1 antibodies as fibroblasts, fibroblasts Anti-Prolyl-4-Hydroxylase beta antibody was selected as a cell marker.

[result]
As a result of peroxidase staining, neutrophils did not appear in both 0.01GO and col, indicating that biocompatibility was good (FIGS. 9a and b). In 0.01GO, many aggregates of GO were observed inside the scaffold. Moreover, as a result of immunostaining, many ED1-Galectin positive macrophages were identified in the GO scaffold (FIG. 10a). Therefore, it was speculated that the aggregation of GO occurred when macrophages entered 0.01GO and phagocytosed GO. Smooth muscle actin-positive smooth muscle cells (Fig. 10b) and RECA-1-positive vascular endothelial cells (Fig. 11a) are often observed in the GO scaffold, forming a blood vessel-like lumen structure. It was revealed that the construction of the vascular network was promoted. In the GO scaffold, a large amount of Prolyl-4-Hydroxylase beta-positive fibroblast ingrose was observed (FIG. 11b), indicating that self-organization may have progressed. On the other hand, in Col, there was little ingress into each cell (FIGS. 10a and b, and FIGS. 11a and b).

[Conclusion]
The 0.01GO scaffold (GO content 2.22% by mass) also had good biocompatibility in the regeneration of cranial cortical bone, and promoted cell and blood vessel ingrowth inside.

Example 7
Bone regeneration effect in canine tooth extraction socket [Materials and Methods]
A GO dispersion (1 wt%, nano GRAX (registered trademark)) was diluted with 1-methyl 2-pyrrolidone to prepare a 0.01 wt% GO solution. A col (Terdermis (registered trademark)) molded into 6 × 6 × 3 mm was dipped in a GO solution, coated with GO on the surface, washed and dehydrated with ethanol, and dried in air to obtain a 0.01GO scaffold. As a result of measurement, the GO content (mass%) of the scaffold was 2.22%. A beagle dog (female, 12-16 months old) was subjected to general and local anesthesia, and after extracting the maxillary third anterior molar, a GO scaffold or col was immediately implanted in the extraction socket and sutured. After 7 days, the thread was removed, and after 14 days, the experimental site was removed, and a tissue specimen was prepared for histological observation. In addition, the area of new bone was measured with software (NIH image).

[result]
When the GO scaffold was implanted, on the 14th day, many new bones were formed in the extraction socket (Fig. 12a), and osteoblasts and osteoclasts were arranged in the trabeculae, resembling normal bone tissue. (Fig. 12b, c). Connective tissue was formed in the central part of the extraction socket (Fig. 12d). Bone formation was observed in the Col group, but the amount was small, and most of the extraction fossa was filled with connective tissue (Fig. 13a). As a result of histological measurement at 2 weeks, the rate of new bone formation was 3.1% in the col group, while the 0.01GO scaffold was 37.2%, approximately 12 times that in the col group (FIG. 13b).

[Conclusion]
The 0.01GO scaffold (GO content 2.22% by mass) also showed good biocompatibility in the formation of new bone in the extraction socket and promoted very remarkable extraction socket bone formation.

Example 8
Skin regeneration in rat skin defect model [Materials and Methods]
A GO dispersion (1 wt%, nano GRAX (registered trademark)) was diluted with 1-methyl 2-pyrrolidone to prepare a 0.01 wt% GO solution. A col (Terdermis (registered trademark)) molded to 10 × 10 × 3 mm was immersed in a GO solution, coated with GO on the surface, washed with ethanol and dehydrated, and then dried in air to obtain a 0.01GO scaffold. As a result of measurement, the GO content (mass%) of the scaffold was 2.22%.

  Wistar rats (10 weeks old) were subjected to general and local anesthesia, and after shaving the back skin, a 10 × 10 mm skin defect was created with a scalpel. The defect was transplanted by attaching a GO coating scaffold to the defect, and pressed with a suture so as not to fall off. A col group and a control group (ctrl) in which nothing was transplanted were set. On the 10th day after the operation, the rats were euthanized, the transplanted site was removed together with surrounding tissues, fixed in formalin, and a tissue specimen was prepared according to the usual method. Histological observation with an optical microscope, images were taken into a personal computer, and the defect length of each specimen and the length of the epithelium extended to the defect were measured with NIH image software. (Defect / length of defect part) was calculated.

[result]
When 0.01GO was transplanted, the epithelium entered and proliferated directly under the scab of the defect, and most of the defect was covered by epithelial elongation, and the reduction of the defect was remarkable (FIG. 14a). Many cells of ingulose were observed inside the remaining GO scaffold (FIG. 14b). In addition, macrophage-like cells were observed, and black aggregates thought to be phagocytosed GO were often observed (FIG. 14b). As a characteristic finding, since the porous structure of the GO scaffold is observed directly under the new epithelium, it seems that the epithelium directly coats the GO scaffold that has advanced self-organization, and has high biocompatibility. It was assumed that there was (Fig. 14c, d).

In the col group, there was less epithelial elongation compared to 0.01GO (FIG. 15a). Cellular ingulose was observed inside the remaining col (FIG. 15b). Col was encapsulated with neoplastic connective tissue, and the epithelium extended to cover the connective tissue (FIGS. 15c and d).
In the control (FIG. 16a), inflammatory cell infiltration such as lymphocytes was observed in the connective tissue directly under the scab (FIG. 16b). The epithelial elongation (arrow) was small enough to be observed at both ends of the defect (FIGS. 16c and d). As a result of measuring the epithelial regeneration rate, the ctrl group was 8.4%, the col group was 26.3%, the 0.01GO group was 47.8%, and the 0.01GO epithelial regeneration rate was about 5 times that of the ctrl group and about 2 times that of the col group. There was a statistically significant difference compared to the ctrl and col groups (FIG. 17).

[Conclusion]
When 0.01GO scaffold (GO content 2.22% by mass) was applied to the skin defect, epithelial regeneration was promoted.

  The present invention is useful in the fields of bone regeneration therapy and skin regeneration therapy.

Claims (13)

A base material composed of a porous scaffold, and graphene oxide which is a coating on at least a part of the surface of the base material, and the coating amount of the graphene oxide is 0.1-2. Scaffold for bone regeneration or skin regeneration in the range of 5% by mass. The scaffold according to claim 1, wherein the porous scaffold is made of at least one material selected from the group consisting of collagen, gelatin, fibrin, chitosan, hyaluronic acid, polylactic acid, hydroxyapatite, and calcium triphosphate. The scaffold according to claim 1 or 2, wherein the porous scaffold has a porosity in the range of 50 to 99% and has a continuous pore structure. The scan according to any one of claims 1 to 3, wherein the compressive strength at 50% displacement (measurement condition: crosshead speed 0.5 mm / min) is 1.2 times or more that of the porous scaffold base material. Fold. The scaffold according to any one of claims 1 to 4, wherein the coating amount of graphene oxide is in the range of 1.0 to 2.3 mass% based on the mass of the substrate. 6. The graphene oxide coating according to claim 1, comprising a thin film graphene oxide particle having a thickness of 0.4 to 10 nm and a planar size of 20 nm or more. Scaffold. The scaffold according to any one of claims 1 to 6, which is used for bone regeneration. The scaffold according to claim 7, wherein the regeneration object is bone and cartilage. The scaffold according to any one of claims 1 to 6, which is for skin regeneration. The scaffold according to claim 9, wherein the regeneration object is epidermis and dermis.
It is a manufacturing method of the scaffold of any one of Claims 1-6,
The manufacturing method, comprising injecting a dispersion of graphene oxide particles into a substrate made of a porous scaffold, and coating at least a part of the surface of the substrate with graphene oxide. The manufacturing method according to claim 11, wherein the dispersion is a dispersion of graphene oxide particles in a mixed solution of water and N-methyl-2-pyrrolidone. The manufacturing method of Claim 11 or 12 whose density | concentration of the graphene oxide particle in the said dispersion liquid is the range of 0.0001-0.01 mass%.