JP6230332B2 - Non-woven fabric containing bone filling material - Google Patents

Description

  The present invention relates to a nonwoven fabric containing a bone prosthetic material.

  In recent years, when teeth are lost due to aging, periodontal disease, etc., a treatment (so-called “implant treatment”) in which an artificial dental root is embedded in the alveolar bone and an artificial dental crown and superstructure are mounted thereon has been widely performed. It has become.

  When a tooth is lost (that is, when a tooth is lost), the alveolar bone that previously supported the tooth is rapidly absorbed and reduced. Therefore, in implant treatment, a situation often occurs where the thickness of the alveolar bone is insufficient to embed the artificial dental root in the alveolar bone. When the thickness of the alveolar bone is insufficient, there is a high possibility that it will not be stable even if an artificial tooth root is embedded. In such a case, treatments such as transplantation of bone or regeneration of bone are taken.

  For example, as one of the methods frequently used for alveolar bone regeneration, there is a GBR method (guided bone regeneration method). Specifically, in this method, a crushed autologous bone or bone grafting material is placed on a portion where the alveolar bone is deficient (affected part), and a membrane (also referred to as a shielding film or GBR film) is placed thereon ( That is, it is a method of promoting the regeneration of the alveolar bone while embedding the affected part supplemented with the bone prosthetic material and preventing the mixture of gingival tissues. However, the bone filling materials currently used are not sufficient in cell adhesion and proliferation, and it takes a long time to regenerate alveolar bone. Moreover, it cannot be said that the adhesiveness to the alveolar bone and the indwelling property to the affected part are good, and even if the bone filling material is embedded with the GBR film, it may leak from the affected part.

  Moreover, although bone cement may be used, there exists a fault that a cell cannot infiltrate into bone cement.

  As described above, the bone regeneration material currently used has a problem that cell adhesion and proliferation are not sufficient.

  In order to improve such a situation, research and development of a bone regeneration material suitable for bone (especially alveolar bone) regeneration has been continued (for example, Patent Document 1 and Patent Document 2).

WO2007 / 132186 JP 2007-325543 A

  An object of the present invention is to provide a bone regeneration material suitable for bone (especially alveolar bone) regeneration.

  The inventors of the present invention are non-woven fabrics containing a bone prosthetic material, wherein the bone prosthetic material is contained between the fibers constituting the non-woven fabric, and the non-woven fabric that is a biocompatible fiber is a very non-woven fabric. In particular, it has been found that the bone regeneration material has a high proliferation efficiency (high cell proliferation ability), and the nonwoven fabric contains a water-soluble polymer so that the bone filling material is strongly adsorbed by the nonwoven fabric. (That is, the bone-retaining material retention of the nonwoven fabric is improved). It has also been found that the bone-retaining material retention of the nonwoven fabric is improved by coating the bone-implanting material with a biocompatible polymer that is a component of the biocompatible fiber in advance. And further improvements were made to complete the present invention.

That is, the present invention includes, for example, the subject matters described in the following sections.
Item 1a.
A non-woven fabric containing a bone filling material,
Bone prosthetic material is included between the fibers that make up the nonwoven fabric,
The fibers constituting the nonwoven fabric are biocompatible fibers,
Non-woven fabric.
Item 2a.
The nonwoven fabric according to Item 1a, wherein the biocompatible fiber is a fiber comprising a biocompatible polymer.
Item 3a.
The biocompatible polymer is selected from the group consisting of polylactic acid, polyglycolic acid, polylactic acid-polyglycolic acid copolymer, polycaprolactone, chitin, collagen, polylysine, polyarginine, hyaluronic acid, sericin, cellulose, dextran, and pullulan. The nonwoven fabric according to Item 2a, which is at least one selected.
Item 4a.
Bone prosthesis materials are β-TCP (β-tricalcium phosphate), α-TCP (α-tricalcium phosphate), HA (hydroxyapatite), DCPD (dicalcium phosphate), OCP (octacalcium phosphate), 4CP (tetracalcium phosphate), alumina, zirconia, calcium aluminate (CaO—Al ₂ O ₃ ), aluminosilicate (Na ₂ O—Al ₂ O ₃ —SiO ₂ ), bioactivated glass, quartz, and calcium carbonate The nonwoven fabric according to any one of Items 1a to 3a, which is at least one selected from the group consisting of:
Item 5a.
The nonwoven fabric according to any one of Items 1a to 4a, wherein the particle diameter of the bone grafting material is about 50 to 5000 μm.
Item 6a.
The nonwoven fabric in any one of Claims 1a-5a whose porosity of a nonwoven fabric is 78.5-97%.
Item 7a.
The non-woven fabric according to any one of Items 1a to 6a, wherein the fiber portion has a porosity of 80 to 99.99%.
Item 8a.
The nonwoven fabric according to any one of Items 1a to 7a, wherein the bulk density (g / cm ³ ) of the nonwoven fabric is 0.1 to 0.6.
Item 9a.
The nonwoven fabric according to any one of Items 1a to 8a, wherein the bone filling material is coated with a biocompatible polymer.
Item 10a.
The biocompatible polymer coating the bone grafting material is polylactic acid, polyglycolic acid, polylactic acid-polyglycolic acid copolymer, polycaprolactone, chitin, collagen, polylysine, polyarginine, hyaluronic acid, sericin, cellulose, The nonwoven fabric according to Item 9a, which is at least one selected from the group consisting of dextran and pullulan.
Item 11a.
The non-woven fabric according to Item 9a or 10a, wherein the biocompatible polymer contained in the biocompatible fiber is the same as the biocompatible polymer coating the bone grafting material.
Item 12a.
The bone regeneration material containing the nonwoven fabric in any one of claim | item 1a-11a.
Item 13a.
The osteoblast culture scaffold containing the nonwoven fabric in any one of claim | item 1a-11a.

Item 1b.
A non-woven fabric containing a bone filling material,
Bone prosthetic material is included between the fibers that make up the nonwoven fabric,
The fiber constituting the nonwoven fabric is a biocompatible fiber, and the biocompatible fiber is a fiber comprising a biocompatible polymer.
Furthermore, a nonwoven fabric containing a water-soluble polymer.
Term 2b
A non-woven fabric containing a bone filling material,
Bone prosthetic material is included between the fibers that make up the nonwoven fabric,
The fiber constituting the nonwoven fabric is a biocompatible fiber, and the biocompatible fiber is a fiber comprising a biocompatible polymer and a water-soluble polymer.
The nonwoven fabric according to Item 1b.
Item 3b.
Item 3. The nonwoven fabric according to Item 1b or 2b, wherein the water-soluble polymer is attached to the fibers constituting the nonwoven fabric.
Item 4b.
The biocompatible polymer is selected from the group consisting of polylactic acid, polyglycolic acid, polylactic acid-polyglycolic acid copolymer, polycaprolactone, chitin, collagen, polylysine, polyarginine, hyaluronic acid, sericin, cellulose, dextran, and pullulan. The nonwoven fabric according to any one of Items 1b to 3b, which is at least one selected.
Item 5b.
Bone prosthesis materials are β-TCP (β-tricalcium phosphate), α-TCP (α-tricalcium phosphate), HA (hydroxyapatite), DCPD (dicalcium phosphate), OCP (octacalcium phosphate), 4CP (tetracalcium phosphate), alumina, zirconia, calcium aluminate (CaO—Al ₂ O ₃ ), aluminosilicate (Na ₂ O—Al ₂ O ₃ —SiO ₂ ), bioactivated glass, quartz, and calcium carbonate The nonwoven fabric according to any one of Items 1b to 4b, which is at least one selected from the group consisting of:
Item 6b.
The non-woven fabric according to any one of Items 1b to 5b, wherein the water-soluble polymer is at least one selected from the group consisting of hydroxypropylcellulose, polyethylene glycol, carboxymethylcellulose, polyvinyl alcohol, hydroxypropylmethylcellulose, and carboxyvinyl polymer. .
Item 7b.
Item 6. The nonwoven fabric according to any one of Items 1b to 6b, wherein the bone grafting material has a particle size of about 50 to 5000 μm.
Item 8b.
Item 9. The nonwoven fabric according to any one of Items 1b to 7b, wherein the nonwoven fabric has a porosity of 78.5 to 97%. Item 9b.
The non-woven fabric according to any one of Items 1b to 8b, wherein the fiber portion has a porosity of 80 to 99.99%.
Item 10b.
The nonwoven fabric in any one of claim | item 1b-9b whose bulk density (g / cm < ³ >) of a nonwoven fabric is 0.1-0.6.
Item 11b.
A non-woven fabric containing a bone filling material,
Bone prosthetic material is included between the fibers that make up the nonwoven fabric,
The fiber constituting the nonwoven fabric is a biocompatible fiber, and the biocompatible fiber is a fiber comprising a biocompatible polymer.
The bone grafting material is a non-woven fabric coated with a biocompatible polymer.
Item 12b.
The nonwoven fabric according to any one of Items 1b to 10b, wherein the bone grafting material is coated with a biocompatible polymer.
Item 13b.
The nonwoven fabric according to Item 11b or 12b, wherein the bone grafting material coated with the biocompatible polymer is a bone grafting material having a biocompatible polymer attached to 30% or more of the surface thereof.
Item 14b.
Item 14. The nonwoven fabric according to any one of Items 11b to 13b, wherein the biocompatible polymer contained in the biocompatible fiber is the same as the biocompatible polymer coating the bone grafting material.
Item 15b
The bone regeneration material containing the nonwoven fabric in any one of claim | item 1b-14b.
Item 16.
An osteoblast culture scaffold comprising the nonwoven fabric according to any one of Items 1b to 14b.

Term A-1.
Item 11. A method for regenerating bone, comprising the step of applying the nonwoven fabric according to any one of 1a to 11a and 1b to 14b to a site where bone is to be regenerated.
Term A-2.
The method according to Item A-1, wherein the bone is alveolar bone.
Term B-1.
The nonwoven fabric according to any one of Items 1a to 11a and 1b to 14b for use in bone regeneration.
Term B-2.
The nonwoven fabric according to Item B-1, wherein the bone is alveolar bone.
Term C-1.
Use of the nonwoven fabric according to any one of Items 1a to 11a and 1b to 14b in production of a preparation for bone regeneration.
Term C-2.
The use according to Item C-1, wherein the bone regeneration preparation is an preparation for alveolar bone regeneration.
Term C-3.
Use of the nonwoven fabric according to any one of Items 1a to 11a and 1b to 14b as an in vitro cell scaffold.

  When the nonwoven fabric containing the bone grafting material of the present invention is used as a scaffold for cell culture, the proliferation efficiency of cells (particularly osteoblasts) is greatly increased (that is, the proliferation ability of the cells can be enhanced). Moreover, since the said nonwoven fabric contains a bone grafting material, when used as a scaffold for culture of an osteoblast, bone regeneration efficiency can also be improved. For this reason, the said nonwoven fabric can be used suitably as a bone regeneration material. Specifically, when the bone is damaged due to an external factor (for example, an accident), or when the bone is reduced or lost due to an internal factor (for example, osteoporosis, periodontal disease), the nonwoven fabric of the present invention is applied. , (Specifically, by embedding or sticking to the affected area), early bone regeneration can be achieved.

  In particular, among the non-woven fabrics containing the bone prosthetic material of the present invention, those containing a water-soluble polymer have improved adhesion of the bone prosthetic material to the non-woven fabric (that is, the non-woven fabric retains the bone prosthetic material). And preferred. Due to this property, for example, the possibility that the bone prosthetic material may fall off the nonwoven fabric due to vibration or the like during transportation or during surgery for application to an affected area (such as a bone damage site or a bone loss site) can be further reduced.

A brief outline of a method for producing a nonwoven fabric by electrospinning is shown. An example of the earth electrode used when manufacturing the nonwoven fabric of this invention is shown. Sectional drawing of the nonwoven fabric (and the normal nonwoven fabric manufactured by the electrospinning method) of this invention is shown. Sectional drawing (by a scanning electron microscope) of the nonwoven fabric of this invention is shown. The result of having examined the cell growth ability of the samples 1-3 which are examples of the nonwoven fabric of this invention is shown. It is a tissue slice observation image which shows the grade of the connective tissue infiltration to a block-shaped bone grafting material when a block-shaped bone grafting material (block of male ferion) is transplanted into a rat. The outer broken line shows the outline of the transplanted bone graft material, and the inner dotted line shows the tip of the tissue infiltrated into the bone graft material. It is a tissue slice observation image which shows the grade of connective tissue infiltration to the nonwoven fabric at the time of transplanting the nonwoven fabric of this invention to a rat. The outer broken line shows the contour of the transplanted nonwoven fabric, and the inner dotted line shows the tip of the tissue infiltrated into the nonwoven fabric. An image obtained by HE staining after culturing cells using each nonwoven fabric as a scaffold, and a cell infiltration distance measured from the image are shown. The pore size measurement result of each nonwoven fabric and an image obtained by culturing cells and staining with HE using each nonwoven fabric as a scaffold are shown. The outline | summary of the measuring method of the distance (fiber distance) between the fibers of a nonwoven fabric is shown. The pore size measurement result of each nonwoven fabric, an image obtained by culturing cells using each nonwoven fabric as a scaffold and staining with HE, and the cell infiltration distance measured from the image are shown. It is the graph which showed the fall rate of the bone grafting material in each nonwoven fabric. It is a graph which shows the result of having investigated the bone grafting material fall rate at the time of changing content of the water-soluble polymer (HPC) in a nonwoven fabric. It is a photograph which shows that the bone grafting material retention property of a nonwoven fabric improves when the bone grafting material coated with the biocompatible polymer is used. When the biocompatible polymer (polylactic acid) is sprayed onto the bone grafting material by electrospinning (spraying condition 1), many bone grafting materials are peeled off, whereas when using a polylactic acid-coated bone grafting material ( The spray condition 2) shows that the bone grafting material hardly peels off. In addition, spraying condition 3 sprayed before and after adding the bone grafting material to form a sandwich, and the bone grafting material hardly peeled off.

  Hereinafter, the present invention will be described in more detail. “Mass” may be read as “weight”.

  The present invention relates to a nonwoven fabric containing a bone grafting material. In the nonwoven fabric, the bone filling material is included between the fibers constituting the nonwoven fabric. Moreover, the fiber which comprises a nonwoven fabric is a biocompatible fiber. Furthermore, preferably, the nonwoven fabric contains a water-soluble polymer.

A well-known thing can be used as a bone grafting material contained in a nonwoven fabric. Known bone grafting materials include, for example, β-TCP (β-tricalcium phosphate), α-TCP (α-tricalcium phosphate), HA (hydroxyapatite), DCPD (dicalcium phosphate), OCP (octa Calcium phosphate), 4CP (tetracalcium phosphate), alumina, zirconia, calcium aluminate (CaO—Al ₂ O ₃ ), aluminosilicate (Na ₂ O—Al ₂ O ₃ —SiO ₂ ), bioactivated glass, Examples include quartz and calcium carbonate. Specifically, a piece comprising these components (preferably a piece comprising these components) can be used. The bone grafting material can be used alone or in combination of two or more. When two or more types are used in combination, two or more types of bone prosthetic material 1 pieces may be used in combination, or two or more pieces consisting of only one component may be used in combination. .

  Moreover, the size of the bone grafting material 1 piece should just be below the magnitude | size of the grade embedded and contained in a nonwoven fabric. Further, the shape is not particularly limited, and may be, for example, a particle shape, a block shape, or a cylindrical shape.

  The particle diameter of the bone grafting material 1 piece is preferably shorter than the thickness of the nonwoven fabric, more preferably about 50 to 5000 μm, more preferably about 75 to 5000 μm, still more preferably about 150 to 3000 μm, and particularly preferably 500 to 1500 μm. Degree. The said particle size is the value calculated | required with the dry-type sieve method. Specifically, it is a value obtained using a low-tap shaker with a sieve defined in JIS8801. By the dry sieving method, a bone filling material having the particle size can be obtained. Moreover, the grade of the particle size of the bone grafting material whose particle size is unknown can be measured by the dry sieving method. The “particle diameter” here is not intended to limit the shape of the bone grafting material to a granular shape, but merely indicates a value obtained by the above method. Even if it is a bone prosthetic material having a non-granular shape (for example, a block shape or a cylindrical shape), the value of “particle size” here can be obtained.

  In addition, you may purchase and use what is marketed as a bone grafting material for the nonwoven fabric of this invention. For example, Osferion (Olympus Terumo Biomaterials Co., Ltd.), Bone Serum (Olympus Terumo Biomaterials Co., Ltd.), Neoborn (MMT Co., Ltd.), Osteograft-S (Nippon Medical Materials Co., Ltd.), Apaceram (Pentax Co., Ltd.) And the like.

  In the nonwoven fabric of the present invention, the bone grafting material is present between a plurality of (many) fibers constituting the nonwoven fabric. That is, the bone grafting material is included between the fibers constituting the nonwoven fabric. It is not contained by being buried in one fiber. It can be said that the fibers constituting the nonwoven fabric exist so as to entangle the bone grafting material.

  The fibers constituting the nonwoven fabric of the present invention are biocompatible fibers. A biocompatible fiber refers to a fiber comprising a biocompatible polymer. Those having biodegradability in vivo are preferred. The content of the biocompatible polymer in the fiber is usually larger than 50% by mass, for example, 60% by mass or more, 65% by mass or more, 70% by mass or more, 75% by mass or more, 80% by mass or more, 85% by mass or more, It may be 90% by weight or more, 95% by weight or more, or substantially 100%.

  A biocompatible polymer means that there is no or little foreign body reaction when adhered to or embedded in a living body (it does not give adverse effects or strong stimulation to the living body over a long period of time, and can coexist peacefully with the living body while performing its original function. ) A polymer. Examples thereof include bioabsorbable polymers and biodegradable polymers.

  More specifically, biocompatible polymers include polylactic acid, polyglycolic acid, polylactic acid-polyglycolic acid copolymer, polycaprolactone, polybutylene succinate, polyethylene succinate, polystyrene, polycarbonate, polyhexamethylene carbonate. , Polyarylate, polyvinyl isocyanate, polybutyl isocyanate, polymethyl methacrylate, polyethyl methacrylate, polynormal propyl methacrylate, polynormal butyl methacrylate, polymethyl acrylate, polyethyl acrylate, polybutyl acrylate, polyacrylonitrile, polyvinyl acetate, polyvinyl methyl ether , Polyvinyl ethyl ether, polyvinyl normal propyl ether, polyvinyl isopropyl ether , Polyvinyl normal butyl ether, polyvinyl isobutyl ether, polyvinyl tertiary butyl ether, polyvinyl chloride, polyvinylidene chloride, poly (N-vinyl pyrrolidone), poly (N-vinyl carbazole), poly (4-vinyl pyridine), polyvinyl methyl ketone, Polymethylisopropenyl ketone, polyethylene oxide, polypropylene oxide, polycyclopentene oxide, polystyrene sulfone, Teflon (registered trademark) (polytetrafluoroethylene), polycyanoacrylate, polyetheretherketone, polyurethane, polyimide, polyvinyl chloride, polyethylene (Including ultra-high molecular weight polyethylene), polypropylene, polyethylene terephthalate, polyvinylidene fluoride (polyvinylidene difur) Ride), synthetic polymers such as polysulfone, polyethersulfone and copolymers thereof, regenerated cellulose, cellulose diacetate, cellulose triacetate, methylcellulose, propylcellulose, benzylcellulose, fibroin, natural rubber and other biopolymers and their derivatives Illustrated. Examples also include chitin, gelatin, collagen, polyamino acids (polylysine, polyarginine), hyaluronic acid, sericin, dextran, pullulan and the like.

  Among these, polylactic acid, polyglycolic acid, polylactic acid-polyglycolic acid copolymer, polyhydroxybutyric acid, polycaprolactone, polyethylene adipate, polybutylene adipate, polybutylene succinate, polyethylene succinate and polycyanoacrylate, and these Aliphatic polyesters such as copolymers, aliphatic polycarbonates such as polybutylene carbonate and polyethylene carbonate can be mentioned as preferred examples, more preferably polylactic acid, polyglycolic acid, polylactic acid-polyglycolic acid copolymer, Polycaprolactone is mentioned. Of these, polylactic acid is particularly preferred. A biocompatible polymer can be used individually by 1 type or in combination of 2 or more types.

In addition, you may use together other polymers and compounds (For example, a polymer copolymer, a polymer blend, a phospholipid, another compound, etc., and mixtures thereof) in the range which does not impair the effect of this invention. Furthermore, the fibers (biocompatible fibers) constituting the nonwoven fabric of the present invention may contain a water-soluble polymer. (This corresponds to the form [1] when the nonwoven fabric described below contains a water-soluble polymer.)
The nonwoven fabric of the present invention preferably further contains a water-soluble polymer. The water-soluble polymer may be contained in any form. For example, [1] By using a biocompatible polymer and a fiber containing the water-soluble polymer as a biocompatible fiber, this is used as a fiber constituting the nonwoven fabric. A form in which the non-woven fabric contains a water-soluble polymer and [2] a form in which the non-woven fabric contains the water-soluble polymer by attaching the water-soluble polymer to the surface of the biocompatible fiber are preferable. [3] The water-soluble polymer may be contained in the nonwoven fabric as a fiber different from the biocompatible fiber constituting the nonwoven fabric. In the case of [3], not only biocompatible fibers but also water-soluble polymer fibers can be referred to as fibers constituting the nonwoven fabric. In this specification, the fibers constituting the nonwoven fabric are biocompatible fibers. The water-soluble polymer is described as being contained in the nonwoven fabric in the form of fibers.

  When the nonwoven fabric of the present invention contains a water-soluble polymer, the amount of the water-soluble polymer contained in the nonwoven fabric is not particularly limited and can be appropriately set according to the type of the water-soluble polymer used. In particular, in the case of [1] above, the content ratio of the biocompatible polymer and the water-soluble polymer contained in the biocompatible fiber is not particularly limited, but when the biocompatible polymer content is 10 parts by mass, The content of the functional polymer is preferably 0.5 to 10 parts by mass, more preferably 1 to 8 parts by mass, further preferably 3 to 6 parts by mass, and still more preferably 2 to 5 parts by mass.

  Furthermore, when the nonwoven fabric of this invention contains a water-soluble polymer, you may provide multiple types of inclusion form. That is, the biocompatible fiber constituting the nonwoven fabric may contain a biocompatible polymer and a water-soluble polymer, and the water-soluble polymer may adhere to the surface of the biocompatible fiber. (This is a non-woven fabric having the water-soluble polymer-containing form of [1] and [2].) As the water-soluble polymer, sodium alginate, propylene glycol alginate, ethyl cellulose, carboxymethyl cellulose, xanthan gum, chondroitin sulfate sodium , Hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, polyvinyl alcohol, polyethylene glycol (eg, macrogol 1500, macrogol 4000, macrogol 6000, macrogol 20000, etc.), methylcellulose, acrylic resin alkanolamine solution, carboxy Vinyl polymer, gelatin, dextran (eg dextran 70: molecular weight about 70 00 dextran), dextrin, potato starch, sodium hyaluronate, hydroxyethyl methylcellulose, sodium polyacrylate, partially neutralized polyacrylic acid, polyoxyethylene polyoxypropylene glycol (eg, polyoxyethylene (160) polyoxypropylene (30) glycol, polyoxyethylene (200) polyoxypropylene (70) glycol etc.), polysorbate (eg polysorbate 80 etc.), water candy, sodium metaphosphate, methyl vinyl ether / maleic anhydride copolymer, etc. . Of these, hydroxypropylcellulose, polyethylene glycol, carboxymethylcellulose, polyvinyl alcohol, hydroxypropylmethylcellulose, and carboxyvinyl polymer are preferable. A water-soluble polymer can be used individually by 1 type or in combination of 2 or more types.

  Furthermore, the bone grafting material used in the present invention is more preferably coated with a biocompatible polymer. The bone grafting material coated with the biocompatible polymer is strongly adsorbed by the nonwoven fabric (that is, the bone grafting material retention property of the nonwoven fabric is improved).

  The biocompatible polymer used for the coating is the same as described for the biocompatible polymer contained in the biocompatible fiber. That is, bioabsorbable polymers and biodegradable polymers can be exemplified. More specifically, examples of biocompatible polymers include polylactic acid, polyglycolic acid, polylactic acid-polyglycolic acid copolymer, polycaprolactone, polybutylene. Succinate, polyethylene succinate, polystyrene, polycarbonate, polyhexamethylene carbonate, polyarylate, polyvinyl isocyanate, polybutyl isocyanate, polymethyl methacrylate, polyethyl methacrylate, polynormal propyl methacrylate, polynormal butyl methacrylate, polymethyl acrylate, polyethyl Acrylate, polybutyl acrylate, polyacrylonitrile, polyvinyl acetate, polyvinyl methyl ether, polyvinyl ethyl ether, poly Nil normal propyl ether, polyvinyl isopropyl ether, polyvinyl normal butyl ether, polyvinyl isobutyl ether, polyvinyl tertiary butyl ether, polyvinyl chloride, polyvinylidene chloride, poly (N-vinylpyrrolidone), poly (N-vinylcarbazole), poly (4- Vinyl pyridine), polyvinyl methyl ketone, polymethyl isopropenyl ketone, polyethylene oxide, polypropylene oxide, polycyclopentene oxide, polystyrene sulfone, Teflon (registered trademark) (polytetrafluoroethylene), polycyanoacrylate, polyether ether ketone, polyurethane , Polyimide, polyvinyl chloride, polyethylene (including ultra-high molecular weight polyethylene), polypropylene, polyethylene Synthetic polymers such as terephthalate, polyvinylidene fluoride (polyvinylidene difluoride), polysulfone, polyethersulfone and copolymers thereof, regenerated cellulose, cellulose diacetate, cellulose triacetate, methylcellulose, propylcellulose, benzylcellulose, fibroin, natural Examples include biopolymers such as rubber and derivatives thereof. Examples also include chitin, gelatin, collagen, polyamino acids (polylysine, polyarginine), hyaluronic acid, sericin, dextran, pullulan and the like.

  Among these, polylactic acid, polyglycolic acid, polylactic acid-polyglycolic acid copolymer, polyhydroxybutyric acid, polycaprolactone, polyethylene adipate, polybutylene adipate, polybutylene succinate, polyethylene succinate and polycyanoacrylate, and these Aliphatic polyesters such as copolymers, aliphatic polycarbonates such as polybutylene carbonate and polyethylene carbonate can be mentioned as preferred examples, more preferably polylactic acid, polyglycolic acid, polylactic acid-polyglycolic acid copolymer, Polycaprolactone is mentioned. Of these, polylactic acid is particularly preferred. The biocompatible polymer for coating the bone grafting material can be used alone or in combination of two or more.

  In addition, you may use together other polymers and compounds (For example, a polymer copolymer, a polymer blend, a phospholipid, another compound, etc., and mixtures thereof) in the range which does not impair the effect of this invention.

  In particular, since the biocompatible polymer contained in the biocompatible fiber and the biocompatible polymer coated with the bone grafting material are the same, the bone grafting material retention of the nonwoven fabric is further improved, which is preferable. . As an example of a specific optimum mode, a biocompatible polymer contained in a biocompatible fiber and a biocompatible polymer coated with a bone grafting material are all polylactic acid, polyglycolic acid, polylactic acid-poly Examples include glycolic acid copolymers or polycaprolactone.

  “Coating” here does not only refer to the situation where 100% of the surface of the bone grafting material is covered with the biocompatible polymer, but some biocompatible polymer is adhered to the surface of the bone grafting material. Including such a situation. For example, the situation where the biocompatible polymer is attached to 30% or more (preferably 50% or more, more preferably 70% or more, and further preferably 90% or more) of the surface of the bone grafting material is also included.

  The method for coating the bone grafting material with the biocompatible polymer is not particularly limited. For example, a method of immersing the bone filling material in a solution in which the biocompatible polymer is dissolved and taking it out and drying it as necessary is exemplified. Moreover, for example, a method in which a solution in which a biocompatible polymer is dissolved is sprayed on a bone grafting material. The solvent for dissolving the biocompatible polymer is not particularly limited, and a known solvent can be appropriately selected and used according to the type of the biocompatible polymer.

  The average fiber diameter of the fibers constituting the nonwoven fabric is preferably about 0.05 to 20 μm, more preferably about 0.1 to 5 μm, and still more preferably about 0.1 to 3 μm. The average fiber diameter is advantageous because osteoblasts are particularly easy to adhere and the bone regeneration efficiency is improved. In addition, the fiber diameter here means the diameter of a fiber. The average fiber diameter is an average value calculated from the diameters of 50 fibers selected at random from the electron microscope image of the nonwoven fabric.

  The thickness of the nonwoven fabric can be appropriately set according to the size of the affected part (bone defect site) to which the nonwoven fabric is applied. Preferably it is about 0.1-5 cm, More preferably, it is about 0.1-1 cm, More preferably, it is about 0.1-0.5 cm. Here, the “thickness” of the nonwoven fabric refers to a thickness measured without applying pressure in the thickness direction of the nonwoven fabric. For the measurement, a thickness gauge (Digital thickness gauge, Ozaki Seisakusho, DG-205M) or the like can be used.

The bulk density ((g / cm ³ ) of the nonwoven fabric of the present invention, ie, {nonwoven fabric weight (g) / nonwoven fabric volume (cm ³ )}) is preferably about 0.1 to 0.6, preferably 0.1 to 0 About 0.1 to 0.4, more preferably about 0.1 to 0.4, still more preferably about 0.1 to 0.3, still more preferably about 0.15 to 0.25, and 0.19 to 0.00. About 24 is particularly preferable. When the bone filling material to be contained is β-TCP or α-TCP, it is particularly preferable that the bulk density is in this range. In addition, the nonwoven fabric volume here cuts a nonwoven fabric into a rectangle (about 4 cm < ² >), measures the length of length, width, and thickness with the above-mentioned thickness meter, and multiplies the length of length, width, and thickness. The volume (cm ³ ) determined in this way.

The porosity of the nonwoven fabric of the present invention is preferably about 78.5 to 97%, about 80 to 97%, about 85 to 97%, about 90 to 97%, about 90 to 95%, about 91 to 95%, 91 About 5 to 95% and about 92 to 95% are preferable in this order. The porosity (%) can be determined from the density (true density) of the fibers constituting the nonwoven fabric and the bone filling material itself. That is, by dividing the weight of the fiber and the weight of the bone prosthetic material contained in 1 cm ³ of the nonwoven fabric of the present invention by the respective true density, the volume occupied by each of the fiber and the bone prosthetic material can be obtained. Therefore, by subtracting the total value of these volumes (cm ³ ) from 1 (cm ³ ) and multiplying this by 100, the porosity (%) of the nonwoven fabric can be obtained.

In addition, when the nonwoven fabric of this invention contains a water-soluble polymer, the porosity (%) of a nonwoven fabric is the value (volume) which remove | divided the weight of the water-soluble polymer contained per 1 cm < ³ > of nonwoven fabric of this invention by the density of the said polymer. ) May be calculated after further subtracting from 1 (cm ³ ).
The formula for obtaining the porosity (%) of the nonwoven fabric is as follows.

[1-{(Fiber weight / Fiber true density) + (Bone prosthesis weight / Bone prosthetic density)}] × 100

However, if it contains a water-soluble polymer,
[1-{(Fiber weight / Fiber true density) + (Bone filler weight / Bone filler true density) + (Water-soluble polymer weight / Water-soluble polymer density)}] × 100

The volume of the fibers occupy (cm ^3), the volume of the bone substitute material occupies (cm ³⁾ divided by the value obtained by subtracting from 1 (cm ^3), by multiplying 100 to the fiber portion of the non-woven fabric It is also possible to obtain the porosity (%) of

In the case where the nonwoven fabric of the present invention contains a water-soluble polymer, the porosity of the fibrous portion of the nonwoven fabric (%), the volume occupied by the fibers and the water-soluble polymer (cm ³⁾ occupied volume (cm ^3), The volume (cm ³ ) occupied by the bone prosthetic material is divided by a value obtained by subtracting from 1 (cm ³ ) and multiplied by 100.
The formula for determining the porosity (%) of the fiber portion of the nonwoven fabric is as follows.

[1- (Fiber weight / Fiber true density) / {1- (Bone prosthetic material weight / Bone prosthetic material true density)}] × 100

However, if it contains a water-soluble polymer,
[1-{(Fiber weight / Fiber true density) + (Water-soluble polymer weight / Water-soluble polymer density)} / {1- (Bone prosthetic material weight / Bone prosthetic material true density)}] × 100

  The porosity of the fiber portion of the nonwoven fabric is preferably about 85 to 99.99%, more preferably about 90 to 99.99%, further preferably about 97.5 to 99.99%, and about 98 to 99.8%. Is even more preferable.

  In the present specification, the true density is a value obtained by the constant volume expansion method. For the measurement of the true density, for example, a wet / dry automatic densimeter (Acpic 1330; Shimadzu Corporation) can be used.

  When the porosity of the nonwoven fabric and the porosity of the fiber portion of the nonwoven fabric are within the above ranges, cells are particularly liable to infiltrate, and fluid permeability such as body fluids and blood is high, so that new blood vessels easily invade during tissue regeneration. This is advantageous. In addition, the porosity of the fiber part of the nonwoven fabric of this invention is a high porosity compared with a normal nonwoven fabric. Although a limited interpretation is not desired, it is believed that the inclusion of a bone prosthetic material between the fibers results in a high porosity. Despite the high porosity of the fiber portion, the nonwoven fabric of the present invention can be recovered to some extent even if pressure is applied or the pressure is removed (for example, if the hand is pressed or the hand is subsequently removed). This is also considered to be an effect produced by including the bone grafting material between the fibers.

  The pore size of the nonwoven fabric of the present invention is preferably about 0.5 to 500 μm, more preferably about 1 to 100 μm, further preferably about 2 to 50 μm, still more preferably about 3 to 30 μm, and particularly preferably about 6 to 20 μm.

  The pore size of the nonwoven fabric here is the mode value obtained by the half-dry method (ASTM E1294-89) using perfluoropolyester, using the surface layer of the nonwoven fabric of the present invention as a measurement sample. (Class width 1 μm). For the pore size measurement, a capillary flow porometer (CFP-1200-AEL, Porous Materials Inc) can be used.

  Moreover, in the nonwoven fabric of this invention, a fiber is sparse or dense depending on a part (namely, the sparse / dense structure of fiber distribution exists). The fiber sensitive distance of the portion where the fibers are densely present is preferably about 5 to 40 μm, more preferably about 10 to 30 μm, and further preferably about 15 to 25 μm. Moreover, the interfiber distance of the part where a fiber exists sparsely is about 50-100 micrometers. The inter-fiber distance of the nonwoven fabric here is a value obtained by detecting the fiber from an image obtained by observing a frozen block section of the nonwoven fabric with a microscope and using the detected fiber data by the inter-centroid distance method.

  The bone filling material ratio of the nonwoven fabric of the present invention is preferably about 10 to 98%, more preferably about 50 to 98%, and still more preferably about 80 to 98%.

In addition, the bone prosthetic material ratio here is
{Bone prosthetic material contained in nonwoven fabric (g) / nonwoven fabric (g)} x 100 (%)
This is the value obtained by. The bone prosthetic material (g) contained in the non-woven fabric is obtained by dissolving the fibers constituting the non-woven fabric of the present invention (and optionally a water-soluble polymer) with a solvent (dichloromethane, ethanol, etc.) (that is, the fiber portion of the non-woven fabric). This is a value obtained by measuring the weight of the residue after the dissolution.

  In addition, when using a bone grafting material coated with a biocompatible polymer as the bone grafting material, the numerical values related to the bone grafting material (the bone grafting material weight, the bone grafting material true density, the bone grafting material ratio, the bone grafting material The volume occupied) is calculated as a “bone replacement material” including the coating portion of the biocompatible polymer.

  The nonwoven fabric of the present invention can be produced by an electrospinning method. The electrospinning method is a well-known method as one of methods for producing a nonwoven fabric. Specifically, a solution in which a polymer (and a dispersion aid if necessary) is dissolved in a volatile solvent (for example, chloroform, dichloromethane, hexafluoroisopropyl alcohol, or a mixed solution thereof) is formed between the electrodes. This is a method for producing a fibrous substance by discharging the solution into an electrostatic field and spinning the solution toward an electrode (ground electrode). A very brief overview of the electrospinning method is shown in FIG. FIG. 1 is an example. If it is a well-known electrospinning method and can manufacture the nonwoven fabric of this invention, it can be used for manufacture of the nonwoven fabric of this invention. FIG. 1 will be described briefly. When a high voltage is applied to the polymer solution in the syringe (nozzle attached to the tip), the polymer solution drop becomes a sharp cone. When the voltage is further increased, the solution flies (sprays) toward the ground electrode (eg, copper, aluminum, etc.), and forms a thin fiber film (ie, non-woven fabric) on the ground electrode. In other words, in FIG. 1, the ground electrode also serves as the collector.

  In the present invention, the concentration of the biocompatible polymer in the biocompatible polymer solution used for the electrospinning method can be appropriately set, but is usually about 1 to 30% by mass, preferably about 2 to 25% by mass, and more preferably. Is about 3 to 20% by mass.

  Further, the distance between the electrodes (the distance between the syringe and the ground electrode in FIG. 1) depends on the charge amount, the nozzle size, the spinning solution flow rate, the spinning solution concentration, etc., and can be set as appropriate. When the voltage is about 10 kV, about 5 to 50 cm is preferable, and about 10 to 30 cm is more preferable. The applied electrostatic potential is usually about 3 to 100 kV, preferably about 5 to 50 kV, and more preferably about 5 to 30 kV.

  In the process for producing the nonwoven fabric of the present invention, the bone grafting material is supplied during the production of the nonwoven fabric by the electrospinning method. Specifically, for example, by electrospinning, a small amount of biocompatible polymer solution is sprayed to produce a non-woven fabric, an appropriate amount of bone filling material is dispersed on the non-woven fabric, and then the biocompatible polymer is dissolved. The nonwoven fabric of this invention can be manufactured by the process of spraying a solution. Preferably, the nonwoven fabric of the present invention is produced by repeating the process several times to several tens of times (specifically, about 2 to 50 times, preferably about 5 to 10 times). In other words, the method for producing a nonwoven fabric of the present invention includes the step, and preferably includes repetition of the step several times to several tens of times. As one aspect of a preferred production method, a method in which a biocompatible polymer solution is sprayed at a rate of 0.5 to 1.5 μL / sec and 0.1 to 0.2 g of bone grafting material is added every 15 minutes. Can be illustrated. In the exemplified method, the bone grafting material is added until the total addition amount is about 1 to 2 g.

  Further, for example, after a small amount of biocompatible polymer solution is sprayed to produce a nonwoven fabric, an appropriate amount of bone grafting material is dispersed on the nonwoven fabric, and before spraying the biocompatible polymer solution, It is also possible to spray the solution in which the biocompatible polymer is dissolved in the bone grafting material and coat the bone grafting material with the biocompatible polymer.

  In addition, in the electrospinning method, as the thickness of the nonwoven fabric to be manufactured increases, the ground electrode becomes difficult to be negatively charged due to the presence of the nonwoven fabric, or the nonwoven fabric itself is positively charged. To do.

  When a positive voltage is applied to the polymer solution and a negative voltage is applied to the spray destination electrode, and the polymer solution is sprayed by these potential differences, the ground electrode is less likely to be negatively charged, or If the nonwoven fabric itself is positively charged, the biocompatible polymer solution is difficult to be sprayed. For this reason, it has been difficult to obtain a relatively thick nonwoven fabric by the conventional electrospinning method. In the present invention, in order to produce a relatively thick nonwoven fabric, it is preferable to make the following measures.

As the device, for example, it is preferable not only to use a metal plate (for example, an aluminum or copper plate) as the ground electrode but also to have a protrusion (preferably a columnar shape or a conical shape) on the plate. Furthermore, it is preferable that the protrusion is movable up and down. If the ground electrode is provided with such a protrusion, when the thickness of the nonwoven fabric increases and the ground electrode becomes difficult to be negatively charged, the ground electrode is further negatively charged by raising the protrusion. It becomes possible. The protrusions are preferably provided on the metal plate in a grid pattern with an interval of about 1 to 3 cm, for example. Further, the cross-sectional area of the projections is preferably about 0.001～0.5Cm ^2, about 0.01～0.1Cm ² is more preferable. The present invention also includes a ground electrode for electrospinning having such a configuration. As will be described later, FIG. 2 shows one embodiment of an electrospinning ground electrode (a protruding ground electrode) having such a configuration.

  Further, for example, it is also an effective device to prevent the positively charged non-woven fabric that has been spun from being neutralized. The neutralization process can be performed by using, for example, an ionizer (static neutralizer). Further, for example, the static elimination treatment can be performed by spraying ethanol. This is because when ethanol is sprayed on a non-woven fabric having a positive charge, ethanol evaporates in a state of being positively charged, and thus there is an effect of removing the positive charge charged on the non-woven fabric.

  Moreover, as a manufacturing method when the nonwoven fabric of the present invention further contains a water-soluble polymer, a method of immersing the bone filling material-containing nonwoven fabric manufactured as described above in a solution in which the water-soluble polymer is dissolved (solution immersion method), In addition to spraying a solution in which a water-soluble polymer is dissolved in a bone filler-containing non-woven fabric (solution spray method), the water-soluble polymer is also dissolved in a biocompatible polymer solution used in electrospinning. Examples include a method of mixing (solution spinning method), a method of spraying a water-soluble polymer solution at the same time when spraying a biocompatible polymer solution by electrospinning (solution simultaneous spinning method), etc. The Here, the method names shown in parentheses are named by the inventors of the present invention.

  Among the forms when the above-described nonwoven fabric contains a water-soluble polymer, for example, the nonwoven fabric of form [1] is formed by a mixed solution spinning method, and the nonwoven fabric of form [2] is formed by a solution dipping method or a solution spray method. 3] can be produced by a solution co-spinning method.

  In addition, when using the mixed solution spinning method, an appropriate amount of ethanol (which may be water-containing ethanol) is sprayed after spinning, and the water-soluble polymer is moistened to further improve the adhesion (that is, the bone-retaining material retention of the nonwoven fabric). Can be improved), which is preferable.

  In addition, when the solution simultaneous spinning method is used, both the biocompatible polymer solution and the water-soluble polymer solution are repulsively charged because a positive voltage is applied. Is preferably traversed (meaning horizontal movement of the nozzle). By traverse, a nonwoven fabric in which fibers of both polymers are mixed evenly can be obtained.

  Among these methods, a mixed solution spinning method, a solution dipping method, and a solution spray method are preferable.

  The nonwoven fabric of the present invention, when used as a scaffold for cell culture, greatly enhances the proliferation efficiency of cells (particularly osteoblasts) (enhances cell proliferation ability). Can be suitably used. Specifically, when the bone is damaged due to an external factor (for example, an accident), or when the bone is reduced or lost due to an internal factor (for example, osteoporosis, periodontal disease), the nonwoven fabric of the present invention is applied. , (Specifically, by embedding or sticking to the affected area), early bone regeneration can be achieved. In particular, since the nonwoven fabric of the present invention can have a thickness that was impossible with conventional nonwoven fabrics, as with conventional bone prosthetic materials, by embedding in a site (affected area) where bone regeneration should be performed, It can be used to promote bone regeneration.

  In particular, in the nonwoven fabric of the present invention, since the bone grafting material is entangled and held in the nonwoven fabric fiber, the bone grafting material has a high indwellability and has an appropriate toughness, so that the affected part has a complicated shape. Can also be easily applied (embedded). Moreover, it is excellent in cell permeability and liquid permeability.

  Although not limited, the nonwoven fabric of the present invention can be preferably used for regeneration of alveolar bone, particularly in implant treatment.

  Conventionally, in the GBR method (Guided Bone Regeneration method), after filling the bone replacement material into the area where the alveolar bone is regenerated, gingival tissue and epithelial tissue that impede bone tissue regeneration infiltrate into the area. In order to suppress this, a shielding film had to be applied (ie, the bone grafting material and the shielding film had to be applied “sequentially” and installed together). For this reason, since the bone filling material is first filled in the area, and then a shielding film is applied, the labor of the practitioner is large, and the practitioner is required to have advanced techniques. On the other hand, when the non-woven fabric of the present invention is used in place of the bone grafting material, it can be applied "at once" together with the shielding film, so that the labor of the practitioner is reduced and technically compared to the conventional technique. Can also be applied easily. In particular, the conventional bone prosthetic material often leaks from the application site, but such a problem does not occur if the nonwoven fabric of the present invention is used instead of the bone prosthetic material. Moreover, since the nonwoven fabric of this invention is excellent in a softness | flexibility, it can be applied after changing according to the shape of an affected part, or can be applied after cut | disconnecting according to the shape of an affected part.

  Furthermore, in the nonwoven fabric of the present invention, by increasing the length from the bone filling material contained inside to the outside (that is, increasing the fiber layer on the surface of the nonwoven fabric of the present invention that contacts the gingival tissue or epithelial tissue) Therefore, infiltration of the gingival tissue or epithelial tissue into the regeneration area can be suppressed (that is, the function of a shielding film can be added). In this case, only the nonwoven fabric of the present invention can be used as a substitute for the shielding film and the bone grafting material.

  In addition, as described above, the nonwoven fabric of the present invention is manufactured by spraying a small amount of a biocompatible polymer solution using an electrospinning method, and then dispersing an appropriate amount of bone grafting material on the nonwoven fabric. Furthermore, it can be produced by repeating the process of “spraying a biocompatible polymer solution”, but various forms of useful nonwoven fabrics can be produced by slightly changing the production process. For example, a large thick non-woven fabric is produced by first spraying a large amount of a biocompatible polymer solution, and then a bone filling material is added to only a relatively narrow portion of the nonwoven fabric, and then the biocompatible polymer solution is sprayed. When the operation is repeated, a silk hat-shaped nonwoven fabric is obtained in which a wide and thick nonwoven fabric is used as a base, and a nonwoven fabric containing a bone grafting material is raised in part. When considered as a top hat, the brim portion is a wide and thick base non-woven fabric, and the crown portion is a non-woven fabric containing bone prosthetic material. In this top hat-shaped non-woven fabric, the crown portion can be embedded in the area where the alveolar bone is to be regenerated, and further the gingival tissue and epithelial tissue can be prevented from infiltrating into the area at the brim portion. That is, the top hat-shaped nonwoven fabric functions as both a shielding film and a bone grafting material.

  Thus, the nonwoven fabric of the present invention can be used as a bone regeneration material. Furthermore, what added or added the osteoblast etc. to the nonwoven fabric of this invention is also included by this invention. That is, the present invention includes a bone regeneration material including the nonwoven fabric, and the bone regeneration material may be composed of the nonwoven fabric or the nonwoven fabric further including osteoblasts. . In order to include osteoblasts, for example, cells may be cultured for a short period of time using the nonwoven fabric as a scaffold.

  Moreover, the nonwoven fabric of this invention can be used as an osteoblast culture scaffold. In this case, a non-woven fabric having the same structure as that of the bone regeneration material can be used as an osteoblast culture scaffold.

Furthermore, since the nonwoven fabric of the present invention can be used as a bone regeneration material, it can be used, for example, in the following treatments, surgical procedures or uses.
<Periodontal tissue regeneration and oral surgery>
Tissue regeneration induction method for subbony defects, class II root bifurcation lesions, retraction defects, dehiscence defects; bone regenerative surgery for alveolar ridge, alveolar ridge augmentation, bone regeneration guidance for implants around bone implants; Clinicoplasty; sinus lift method in maxillary sinus floor elevation; socket preservation method in preservation of extraction socket; nasal floor elevation; bone extension surgery, bone filling after curettage of osteonecrosis, cancer of bone tissue Bone filling after lesion curettage, bone reconstruction for bone filling to treat fractures due to trauma; gingival enlargement under the bridge, root coverage for gingival recession, interdental papillary reconstruction, and other aesthetic treatments Etc. <Orthopedics>
Bone extension surgery: After curettage of osteonecrotic part, after cancer lesion curettage of bone tissue, fracture treatment by trauma, spinal compression fracture, bone reconstruction in treatment of pseudo-joint; bone extension surgery: carrier of medicinal ingredients in the treatment of osteoporosis Use as material, etc.

  In addition, this invention also includes the method of applying the nonwoven fabric of the said invention to the site | part which should reproduce | regenerate a bone (preferably alveolar bone), and reproducing a bone. This method can be used, for example, in the above-mentioned treatment and surgical procedure.

  Hereinafter, the present invention will be specifically described, but the present invention is not limited to the following examples. In the experiment, textbooks in the relevant technical field (for example, Molecular Cloning: A Laboratory Manual (3 Vol. Set); Cold Spring Harbor Laboratory Press) may be referred to as appropriate.

Nonwoven manufacturing 1
A polylactic acid solution was obtained by adding 43 g of a mixed solution of hexafluoroisopropyl alcohol: dichloromethane = 8: 2 (mass ratio) to 7 g of polylactic acid (Mitsui Chemicals, LACEA, H-400) (14 w / w%). ). The polylactic acid solution was filled in a syringe (Henke SASS WOLF, 5 mL), and a needle (Terumo, non-bevel needle 21G1.1 / 2) was attached to the syringe and set in an electrospinning apparatus. The distance from the syringe to the target ground was 8 cm, and the spraying amount and spraying time were changed and sprayed at an applied voltage of 10 kV under the conditions shown in Table 1. During the spraying, the bone filling material (Osferion / Olympel Termo Biomaterial Co., Ltd.) was added as evenly as possible every 15 minutes. The addition was performed until the total addition amount reached 2 g. In addition, the particle size of the used bone grafting material (male ferion) is 0.5 to 1.5 mm (standard value). After the addition of the total amount of the bone grafting material, the polylactic acid solution was sprayed for another 15 minutes. Thus, four types of nonwoven fabrics shown in Table 1 were produced.

  In manufacturing, an aluminum plate provided with a copper protrusion that was movable up and down was used as a ground electrode (also serving as a collector). An outline of the ground electrode is shown in FIG. During the spraying, the copper protrusions were raised by 0.5 mm every 15 minutes.

Physical property evaluation of nonwoven fabric 1
By the following procedures, the thickness, bulk density, bone filling material ratio, and fiber diameter of the fibers constituting the nonwoven fabric were measured for the above four types of nonwoven fabrics (samples 0, 1, 2, and 3).

Each sample was cut into rectangles (about 4 cm ² ) and the weight was measured. The length, width, and thickness of the measurement sample were measured with a thickness gauge (Digital thickness gauge, Ozaki Seisakusho, DG-205M), and the volume (cm ³ ) was obtained by multiplying the length, width, and thickness. . In addition, thickness was made into the average value of the measured value of 20 places.

From the sample weight and volume, the bulk density was determined by the following equation.
Bulk density (g / cm ³ ) = sample weight (g) / sample volume (cm ³ )
Further, each cut sample was put in a conical tube, and 50 mL of dichloromethane was added to dissolve the polylactic acid in the sample. Next, the precipitate was left in the conical tube, and the supernatant was removed. The dichloromethane of the precipitate was evaporated, and the weight of the remaining precipitate was measured and used as the weight of the bone grafting material. And the bone grafting material ratio was calculated | required by the following formula.
Bone prosthetic material ratio (%) = (dichloromethane insoluble matter (precipitation) / sample weight used) × 100

  Moreover, each sample cross-section was image | photographed using the scanning electron microscope (Hitachi High Technology Co., Ltd., S-3400N), Image J (ver.1.43u) (image processing developed by NIH) The fiber diameter was measured by a soft fair. The average value of the fiber diameters of 50 fibers was defined as the fiber diameter of each sample.

  In addition, the cross-sectional photograph of each sample is shown in FIG. In FIG. 3, A shows sample 0, B shows sample 1, C shows sample 2, and D shows sample 3. From FIG. 3, it was confirmed that Samples 1 to 3 were thick nonwoven fabrics by incorporating the bone grafting material as compared with Sample 0. Further, FIG. 4 shows a photograph of a cross section of the sample 3 using a scanning electron microscope. FIG. 4 also shows a schematic diagram.

  The results of the above physical property evaluation are shown in Table 2.

Furthermore, for samples 1 to 3, the porosity (%) of the nonwoven fabric and the porosity (%) of the fiber portion of the nonwoven fabric were calculated. For the calculation, values of 1.26 g / cm ³ as the true density of polylactic acid and 3.17 g / cm ³ as the true density of the bone grafting material (osferin) were used. The results are shown in Table 3. In addition, the said true density value is a value calculated | required with the wet / dry automatic densimeter (Acupic 1330; Shimadzu Corporation).

  Polylactic acid non-woven fabric (sample 0), which does not contain bone grafting material, is as thin as 0.163mm, especially as a material for bone regeneration used by filling the affected area (bone defect). The polylactic acid nonwoven fabric (samples 1 to 3) to which bone grafting material was added was a relatively thick nonwoven fabric with a thickness of 2 to 3 mm. It recovered, and it was considered suitable as a bone regeneration material.

Evaluation of cell proliferation ability of nonwoven fabric The cell proliferation ability of each nonwoven fabric (samples 1 to 3) was examined by the following procedure. Specifically, the cell proliferation ability was examined by measuring the amount of DNA of cells grown by each sample.

<Cell culture>
Each nonwoven fabric sample (samples 1 to 3) was cut into the same size as the bottom of a 48-hole petri dish (Sumitomo Bakelite Co., Ltd., SUMILON, MS-80480) and placed on the bottom of the 48-hole petri dish. These include about 50 mg of bone grafting material. Further, as a control sample, about 50 mg of a bone filling material (male ferion) was placed on the bottom of a 48-well petri dish as it was.

A stainless steel tube (penicillin cup) is placed on each evaluation sample, and 10% FBS / MEM medium (10% FBS / MEM medium supplemented with antibiotics and glutamic acid: “10% FBS / MEM medium” below) is also the same. ) Was added. The plate was centrifuged for 5 minutes in a plate centrifuge (2500 rpm, room temperature), degassed under reduced pressure, and further centrifuged for 5 minutes (2500 rpm, room temperature). Then, 200 μL of 10% FBS / MEM medium was added and incubated in a 37 ° C., 5% CO ₂ incubator for 1 hr or longer. 500 μL of the medium was sucked and removed, and human osteosarcoma-derived cells MG-63 were suspended in 10% FBS / MEM medium to 1.6 × 10 ⁵ cells / mL, and 100 μL each was seeded in each well ( 1.6 × 10 ⁴ cells / well). After incubating for 5 hours to attach the cells to the evaluation sample, 200 μL of 10% FBS / MEM medium was added and cultured. Samples after 1 day, 3 days and 8 days of culture were used for evaluation of cell proliferation ability.

<Measurement of cell proliferation ability>
After the culture, each evaluation sample (nonwoven fabric) to which the cells adhered was taken out and added to a petri dish containing PBS (phosphate buffered saline). Measure the weight of the sample containing PBS and absorb the amount of water absorbed from the dry weight and the sample weight of the sample containing PBS (from the sample weight of sample sample containing PBS, the dry weight before being used for the evaluation sample experiment) The amount obtained by subtracting (measured when cut to the same size as the bottom of the petri dish) was determined.

  The TE buffer solution was added to each petri dish so that the total amount of the water absorption of the sample and the TE buffer solution (Tris / Tris-HCl 10 mM, EDTA 1 mM) was 1200 μL. Freeze-thaw (freezing at −80 ° C. and thawing at room temperature twice) was performed twice, followed by sonication for 30 minutes to disrupt the cells. Measurement was performed by adding 100 μL of cell lysate (cell lysate obtained by freeze-thawing and sonication) in which DNA was eluted in TE buffer to a 96-well fluorescence measurement plate (Nunc black microwell, cat. 137101). A sample was used.

  Picogreen (Invitrogen) was diluted with TE buffer (100 μL was diluted to 20 mL), 100 μL was added to the measurement sample, and incubated at room temperature for 5 minutes. Using a fluorescent plate reader (Molecular devices spectra Max gemin XPS), the fluorescence intensity was measured at excitation light of 480 nm and measurement wavelength of 520 nm. Since pico green is a double-stranded DNA-specific stain, the obtained fluorescence intensity reflects the amount of DNA (and hence the number of cells). The results are shown in FIG. Compared to the case where only the bone grafting material (control) was used, it was confirmed that a large number of cells adhered and proliferated on the thick polylactic acid nonwoven fabric (samples 1 to 3) containing the bone grafting material. Therefore, it was found that these nonwoven fabrics are excellent as bone regeneration materials.

Measurement of pore size of nonwoven fabric The pore size (mode) of the nonwoven fabric was measured by a half dry method (ASTM E1294-89) using perfluoropolyester (using a circular measurement adapter having a diameter of 7 mm). As a measuring instrument, a capillary flow porometer (CFP-1200-AEL, Porous Materials Inc) was used. The class width when determining the mode value was 1 μm.

Production of nonwoven fabric 2
45 g of a mixed solution of hexafluoroisopropyl alcohol: dichloromethane = 8: 2 (mass ratio) is added to 5 g of polylactic acid (Evonik Degussa Japan, RESOMER (registered trademark), L 206S) and dissolved to obtain a polylactic acid solution. (10% by weight). The polylactic acid solution was filled in a syringe (Henke SASS WOLF, 5 mL) and set in an electrospinning apparatus (MEC, NF-103A). The distance from the syringe to the target ground electrode (4 x 4 cm aluminum block: the same mechanism as shown in Fig. 2) is 22 cm, the applied voltage is 15 kV, the spray amount is 1 ml / hour, and the total spray time is 90 min. Sprayed. During spraying, 0.033 g of bone filling material (Olympel Terumo Biomaterials Co., Ltd., Osferion G1) and the bone filling material were further finely crushed and the particle size adjusted with a sieve was added evenly every 3 minutes. . The addition was performed 30 times until the total addition amount was about 1 g. In this way, five types of nonwoven fabrics (A, B, C, D and E) shown in Table 4 were produced.

  Moreover, the nonwoven fabric was similarly produced using the block-shaped (20 mm x 10 mm x 3.5mm rectangular parallelepiped-shaped) male ferion (male ferion A1) as a bone grafting material. The nonwoven fabric is designated as nonwoven fabric F (Table 4).

Physical property evaluation of nonwoven fabric 2
By the following procedures, the thicknesses of the nonwoven fabrics A to F, the bulk density, the bone filling material ratio, and the fiber diameter of the fibers constituting the nonwoven fabric were measured and calculated.

  Each sample was cut into a 4 × 4 cm square and weighed. The thickness of the sample was measured with a Digimatic micrometer (Mitutoyo Corporation, CLM1-15QM). The thickness was an average value of measured values at 20 locations.

Next, in the same manner as in “Nonwoven fabric physical property evaluation 1”, the bulk density, the bone filling material ratio, and the fiber diameter of the fibers constituting the nonwoven fabric were measured and calculated. However, the fiber diameter was determined using an electron microscopic image of 2000 times instead of 500 times.
The results of the above physical property evaluation are shown in Table 5. In addition, since the nonwoven fabric F was manufactured using the block-shaped bone grafting material, the bulk density is larger than other nonwoven fabrics.

Furthermore, the porosity (%) of each nonwoven fabric and the porosity (%) of the fiber portion of each nonwoven fabric were calculated. Specifically, the calculation, 1.26 g / cm ³ as the true density of the polylactic acid, using 3.17 g / cm ³ as the true density of the bone prosthetic material (Osuferion), in the same manner as the "Characterization first nonwoven fabric" Calculated. However, since the non-woven fabric F has a lot of block-shaped bone grafting material itself and contains voids, 0.7065 g / cm ³ was used as the true density of the bone grafting material only when calculating the porosity of the fiber portion of the nonwoven fabric F. The results are shown in Table 6.

  Since the nonwoven fabric F was manufactured using the block-shaped bone filling material, the porosity of the fiber part of a nonwoven fabric is low compared with another nonwoven fabric. For this reason, it was a slightly harder nonwoven fabric than other nonwoven fabrics.

Nonwoven manufacturing 3
The concentration of polylactic acid solution used is 5% by weight, the spraying condition from the electrospinning device is 1 ml / hour, the total spraying time is 195 min. After spraying for 15 min for the first time, 0.03 g of bone filling material every 6 min A nonwoven fabric α was produced in the same manner as in “Nonwoven Fabric Production 2”, except that was added until the total addition amount was about 1 g.

Physical property evaluation of nonwoven fabric 3
In the same manner as in “Nonwoven fabric physical property evaluation 2”, the thickness, the bulk density, the bone filling material ratio, and the fiber diameter of the fibers constituting the three-dimensional nonwoven fabric were measured and calculated, and the porosity (% ), And the porosity (%) of the fiber portion of the three-dimensional nonwoven fabric. The results are shown in Table 7 and Table 8.

Production of nonwoven fabric 4
The ground electrode is a 6 x 25 cm aluminum block, the spraying condition is 16 cm wide, the spraying amount is 1 ml / hour, the total spraying time is 360 min, and during the spraying, 0.135 g of bone filling material (Osferion) is applied every 6 min. The nonwoven fabric β was produced in the same manner as in “Production of nonwoven fabric 2” except that 59 × was added evenly to 4 × 16 cm until the total addition amount was about 8 g.

Nonwoven fabric property evaluation 4
In the same manner as in “Nonwoven fabric physical property evaluation 2”, the thickness of the nonwoven fabric β, the bulk density, the bone grafting material ratio, and the fiber diameter of the fibers constituting the three-dimensional nonwoven fabric were measured and calculated, and the porosity (% ), And the porosity (%) of the fiber portion of the three-dimensional nonwoven fabric. The results are shown in Table 9 and Table 10.

Nonwoven Transplant Nonwoven β was transplanted into rats as follows, and the degree of connective tissue infiltration was examined. In addition, the block-shaped bone filling material itself (Osferion A1) was also transplanted into rats in the same manner as a control.

  SD male rats (8 weeks old, approximately 200 g) were purchased and used as experimental animals. The rat was subjected to 2.5% isoflurane inhalation anesthesia, the back, which was the surgical site, was shaved and further disinfected with isodine and rubbing alcohol. The skin on the back was incised to create a void in the loose connective tissue. A non-woven fabric β or a block of bone filling material (10 × 10 × 5 mm, cut out from Olympus Terumo Biomaterials, Osferion A1) was implanted in the void and closed with a suture. Two weeks after implantation, blood was discharged from the abdominal aorta under inhalation anesthesia with 2.5% isoflurane, and the transplanted rats were euthanized. After confirming the lethality of the rat, the implanted specimen was collected including the peripheral part. The collected sample was immersed and fixed in a 10% neutral buffered formalin solution (mildform (registered trademark), manufactured by Wako Pure Chemical Industries, Ltd.). Thereafter, frozen non-decalcified tissue sections were prepared from the samples and stained with hematoxylin-eosin. The obtained tissue slice specimen was observed with an optical microscope. The results are shown in FIGS. 6a and 6b. The connective tissue infiltration into the non-woven fabric β was better than that of the control block bone substitute.

Examination of cell invasion 1
<Cell culture>
Nonwoven fabrics A to D were cut to a size of about 1 cm in diameter and placed on the bottom of a 24-hole petri dish (Sumitomo Bakelite Co., Ltd., SUMILON, MS-80480). 10% FBS / MEM medium with antibiotics and glutamic acid added to the evaluation sample held with a penicillin cup (stainless steel tube) (unless otherwise indicated, “10% FBS / MEM medium”) ) Was added, moistened and degassed under reduced pressure. Incubation was continued for 1 hr or more in a 37 ° C., 5% CO ₂ incubator. Pre-cultured MG-63 (derived from human osteosarcoma, Human Science Research Resource Bank, Lot. 05262004) is suspended in 10% FBS / MEM medium to 3.2 × 10 ⁵ cells / mL, and 100 μL each. Each well was seeded (3.2 × 10 ⁴ cells / well). Cells cultured overnight were used as evaluation samples.
<Evaluation of cell invasiveness>
The cells were fixed with 4% paraformaldehyde solution for 1 hr and washed with PBS. Thereafter, the sample was frozen with hexane under cooling with dry ice and embedded in 4% CMC. The frozen sample was sliced into 30 μL and hematoxylin / eosin staining (HE staining) was performed. The sliced sample was observed with an upright microscope (Olympus Corporation, BH-2). In addition, the maximum cell infiltration distance was measured using Image J ver 1.44. The results are shown in FIG. It was confirmed that the invasiveness of the cells became higher as the porosity and the porosity of the fiber portion increased.

Production of nonwoven fabric 5
Total spraying time was 120 min. During spraying, 0.135 g of bone grafting material (Osferion G1) was added 39 times, evenly to 4 x 16 cm until the total addition amount was about 5.3 g. Produced a nonwoven fabric γ in the same manner as in “Nonwoven Fabric Production 4”.

Examination of cell invasion 2
Cell invasiveness of the nonwoven fabric β and the nonwoven fabric γ was examined. Nonwoven fabrics β and γ were cultured as follows. That is, it was cut into a size of about 1 cm in diameter and degassed under reduced pressure in a 10% FBS / MEM medium to be completely moistened. Then, 37 ° C., in a 5% CO ₂ incubator, and incubated over 1hr. Pre-cultured MG-63 (derived from human osteosarcoma, Human Science Research Resource Bank, Lot. 05262004) was suspended in 10% FBS / MEM medium to 1.6 × 10 ⁵ cells / mL and evaluated. The sample was immersed in 10 mL of cell solution for 60 minutes. Samples were gently agitated in the solution every 15 minutes. A sample was taken out from the cell solution and placed on the bottom of a 24-well petri dish (Sumitomo Bakelite Co., Ltd., SUMILON, MS-80240). 1 mL of the medium was added, the sample was pressed with a penicillin cup (stainless steel tube), and cultured overnight. A non-woven fabric in which the cells were cultured was used as a sample, and the cells were fixed with a 4% paraformaldehyde solution for 1 hr and washed with PBS. Thereafter, the sample was frozen with hexane under cooling with dry ice and embedded in 4% CMC. The frozen sample was sliced at a thickness of 30 μL and subjected to HE staining. Sliced samples were observed with a microscope (Olympus Corporation, BH-2) to evaluate cell invasiveness.

  Further, the pore size of each nonwoven fabric was measured in the same manner as the above-mentioned “Measurement of pore size of nonwoven fabric”. However, when measuring the pore sizes of the nonwoven fabrics β and γ, the surface layer of the nonwoven fabric was peeled off and used for the measurement.

  The above results are shown in FIG. The pore size in FIG. 8 indicates the mode value.

  Furthermore, the distance between the fibers of each nonwoven fabric (interfiber distance) was measured as follows. That is, the sample (nonwoven fabric) to be measured is immersed in PBS and degassed under reduced pressure. A sample infiltrated with PBS was immersed in a 4% CMC (carboxymethylcellulose) gel to prepare a frozen block. A sample having a thickness of 2 μm was prepared from the prepared block, and sealed between a slide glass and a cover glass with a resin. Sliced samples were photographed with a phase contrast microscope. The cross section of the fiber was detected from the photographed image, and the distance between the fibers was measured by the center-of-gravity distance method. The analysis of the center-of-gravity distance method was performed using "A image-kun" (Asahi Kasei Engineering, ver 2.20). The outline of the analysis is shown in FIG. The inter-fiber distance measured in this manner was 18.4 μm for the nonwoven fabric β, 27.2 μm for the nonwoven fabric γ, and 33.3 μm for the nonwoven fabric sample 2.

Examination of cell invasiveness 3 (reference example)
In order to analyze the relationship between the cell invasiveness and the pore size of the nonwoven fabric, ordinary nonwoven fabrics (planar nonwoven fabrics (i) to (iii): Table 11) were manufactured, and the cell invasiveness was examined.

  Specifically, 45 g of a mixed solution of hexafluoroisopropyl alcohol: dichloromethane = 8: 2 (mass ratio) is dissolved in 5 g of polylactic acid polylactic acid (Evonik Degussa Japan Co., Ltd., RESOMER (registered trademark), L 206S). To obtain a polylactic acid solution (10% by weight). The polylactic acid solution was filled in a syringe (Henke SASS WOLF, 5 mL) and set in an electrospinning apparatus (MEC, NF-103A). A non-woven fabric (i) was produced by spraying at a distance of 25 cm from the syringe to the ground serving as a target (3 × 3 cm aluminum block) at an applied voltage of 15 kV under a spraying amount of 1 ml / hour and a spraying total time of 60 min. Nonwoven fabrics (ii) and (iii) were produced in the same manner as the nonwoven fabric (i) except that different types of rotary drums were used for grounding. The nonwoven fabrics (i) to (iii) were examined for cell invasiveness in the same manner as in “Study 2 on cell invasiveness”. However, only in the pore size measurement of the nonwoven fabric (i), the class width when determining the mode value was set to 0.1 μm. The results are shown in FIG. The pore size in FIG. 10 indicates the mode value. In addition, the “maximum infiltration distance” in FIG. 10 was obtained from the observation image of the HE-stained tissue section. The interfiber distance of each nonwoven fabric was 7.3 μm for the nonwoven fabric (i), 13.4 μm for the nonwoven fabric (ii), and 15.8 μm for the nonwoven fabric (iii).

Examination of non- woven fabric containing water-soluble polymer Preparation of non-woven fabric was examined how the adhesiveness of the bone prosthetic material was changed by adding a water-soluble polymer to the non-woven fabric.

  In the following examination, RESOMER, L206S manufactured by Evonik Degussa Japan Co., Ltd. was used as polylactic acid (PLLA).

<Water-soluble polymer>
Moreover, the polymer listed below was used as a water-soluble polymer.
HPC: Hydroxypropyl cellulose (Nippon Soda Co., Ltd., NISSO-HPC, L)
PEG1500: Polyethylene glycol (Sanyo Chemical Co., Ltd., Macrogol 1500)
PEG6000: Polyethylene glycol (Sanyo Chemical Co., Ltd., Macrogol 6000)
CMC: Carboxymethylcellulose (Daiichi Kogyo Seiyaku Co., Ltd., Serogen, PR-S)
PVA: Polyvinyl alcohol (Nippon Gosei Co., Ltd., Gohsenol, EG-05)
HPMC: Hydroxypropyl methylcellulose (Shin-Etsu Chemical Co., Ltd., TC-5, E)
CVP: Carboxyvinyl polymer (Lubrizol, Carbopol, 71GNF)

<Solvent>
Moreover, what was described below was used as a solvent.
DCM: Dichloromethane (Wako Pure Chemicals, special grade)
HFIP / DCM: Mixed solvent of hexafluoroisopropanol (Central Glass, HFIP) and dichloromethane (Wako Pure Chemicals, special grade) (mass ratio 8/2)

<Bone prosthetic material>
In addition, “TPC grains” produced as follows were used as bone prosthetic materials.
10 g of βTCP (Taihei Chemical Industry Co., Ltd., βTCP-100) was added with 10 g of ammonium polyacrylate (Toa Gosei, Aron A-30SL) and sonicated for 20 minutes. Add 2 g of polyoxyethylene stearyl ether (Nikko Chemicals, NIKKOL BS-20), disperse it while foaming with a mixer and let it stand at 40 ° C overnight, then heat to 1000 ° C over 3 hours and 1000 ° C For 40 minutes, and then allowed to cool naturally to obtain a TCP porous body. The porous body was finely crushed using a pestle and mortar, and those having a particle size of 500-1500 mm were selected by sieving to obtain TCP particles.

Examination of the effect of various water-soluble polymers to improve the adhesiveness of bone filling materials
36 g of HFIP / DCM was added to 4 g of PLLA and dissolved to obtain a PLLA solution (10 w / w%).
HPC was obtained by adding 9.8 g of water to 0.2 g of HPC and dissolving it (2 w / w%). Similarly, CMC solution (2 w / w%), PEG (PEG1500) solution (2 w / w%), PVA solution (2 w / w%), HPMC solution (2 w / w%), CVP solution (2 w / w%) was prepared.

  The PLLA solution was filled in a syringe (Henke SASS WOLF, 5 mL) and set in an electrospinning apparatus (MEC, NF-103A). The distance from the syringe to the target ground electrode was 30 cm, and sprayed at an applied voltage of 20 kV under the conditions of a spray amount of 1 ml / hour, a total spray time of 120 min, and a traverse distance of 20 cm, to produce a PLLA nonwoven fabric. . What cut | disconnected this to 4 cm x 4 cm was used for examination.

  Place 50 mg of TCP particles on the PLLA non-woven fabric (4 cm x 4 cm), add 2 mL of HPC solution from above with a micropipette, apply evenly at the tip of the pipette, and apply at room temperature. Dried overnight. It was examined whether or not the TCP particles dropped when the nonwoven fabric was inverted (that is, when the surface on which the TCP particles were placed was turned down). CMC solution, PEG solution, PVA solution, HPMC solution, and CVP solution were performed in the same manner, and water was used as a control.

  In the case where water was applied, all the granules dropped, whereas in the case where each polymer solution was applied, no single drop was observed. The results are shown in Table 12 below.

  From the results, it was confirmed that the water-soluble polymer greatly improved the adhesion between the bone filling material and the nonwoven fabric.

Production of Non- woven Fabric Containing Bone Prosthetic Material and Water-Soluble Polymer Non-woven fabrics containing a water-soluble polymer in addition to the bone prosthetic material were produced using various production methods as described below. In addition, PEG6000 was used as PEG.

<Production of nonwoven fabric by mixed solution spinning method>
25.5 g of HFIP / DCM was added to 3 g of PLLA and 1.5 g of HPC and dissolved to obtain a PLLA / HPC mixed solution (10% / 5%). PLLA / PEG mixed solution (10% / 2%) was obtained by adding and dissolving 26.4 g of HFIP / DCM in 3 g of PLLA and 0.6 g of PEG. Similarly, a PLLA / PEG6000 mixed solution (10% / 2%) was obtained.

  A syringe (Henke SASS WOLF, 5 mL) was filled with a PLLA / HPC mixed solution or PLLA / PEG mixed solution, and set in an electrospinning apparatus (MEC, NF-103A). The distance from the syringe to the target ground electrode (4 × 4 cm aluminum block) was 27 cm, sprayed at an applied voltage of 15 kV under the conditions of a spray amount of 1 ml / hour and a total spray time of 30 min. During spraying, 55.6 mg of TCP grains were added evenly every 3 minutes. The addition was performed nine times in a static elimination environment with an ionizer (Kasuga Denki Co., Ltd., KD-750BB) until the total addition amount reached about 500 mg. Finally, an appropriate amount of ethanol (SIGMA, special grade) or 30% ethanol aqueous solution was sprayed and then dried to prepare two types of nonwoven fabrics.

<Production of nonwoven fabric by solution spray method>
36 g of HFIP / DCM was added to 4 g of PLLA and dissolved to obtain a PLLA solution (10 w / w%). Ethanol 199.8 g was added to HPC 0.2 g and dissolved to obtain an HPC solution (0.1 w / w%). A CMC solution was obtained by adding 199.8 g of a 30% aqueous ethanol solution to 0.2 g of CMC to obtain a CMC solution (0.1 w / w%).

  The PLLA solution was filled in a syringe (Henke SASS WOLF, 5 mL) and set in an electrospinning apparatus (MEC, NF-103A). The distance from the syringe to the target ground electrode (4 × 4 cm aluminum block) was 27 cm, sprayed at an applied voltage of 15 kV under the conditions of a spray amount of 1 ml / hour and a total spray time of 30 min. During spraying, 55.6 mg of TCP grains were added evenly every 3 minutes. The addition was performed nine times until the total addition amount was about 500 mg. An appropriate amount of HPC solution or CMC solution was sprayed when adding the TCP grains. In this way, two types of nonwoven fabrics were produced.

<Production of nonwoven fabric by solution dipping method>
36 g of HFIP / DCM was added to 4 g of PLLA and dissolved to obtain a PLLA solution (10 w / w%). 49.5 g of ethanol was added to 0.5 g of HPC and dissolved to obtain an HPC solution (1 w / w%). CMC solution (1 w / w%) was obtained by adding 49.5 g of 30% ethanol aqueous solution to 0.5 g of CMC and dissolving.

  The PLLA solution was filled in a syringe (Henke SASS WOLF, 5 mL) and set in an electrospinning apparatus (MEC, NF-103A). The distance from the syringe to the target ground electrode (4 × 4 cm aluminum block) was 27 cm, sprayed at an applied voltage of 15 kV under the conditions of a spray amount of 1 ml / hour and a total spray time of 30 min. During spraying, 55.6 mg of TCP grains were added evenly every 3 minutes. The addition was performed nine times in a static elimination environment with an ionizer (Kasuga Denki Co., Ltd., KD-750BB) until the total addition amount reached about 500 mg. Two types of nonwoven fabrics were prepared by immersing in 20 mL of HPC solution or CMC solution for 1 minute and drying.

<Production of nonwoven fabric by solution simultaneous spinning method>
36 g of HFIP / DCM was added to 4 g of PLLA and dissolved to obtain a PLLA solution (10 wt%). 17 g of ethanol was added to 3 g of HPC and dissolved to obtain an HPC solution (15 wt%).

  The PLLA solution and the HPC solution were filled in a syringe (Henke SASS WOLF, 5 mL) and set in an electrospinning apparatus (MEC, NF-103A). The distance from the syringe to the target ground electrode (4 x 4 cm aluminum block) is 27 cm, the applied voltage is 25 kV, the spray amount is 1 ml / hour, the total spray time is 20 min, and the traverse distance is 14 cm. Sprayed with. During spraying, 55.6 mg of TCP grains were added evenly every 2 minutes. The addition was performed nine times in a static elimination environment with an ionizer (Kasuga Denki Co., Ltd., KD-750BB) until the total addition amount reached about 500 mg.

<Preparation of non-woven fabric without water-soluble polymer>
36 g of HFIP / DCM was added to 4 g of PLLA and dissolved to obtain a PLLA solution (10 w / w%).

  The PLLA solution was filled in a syringe (Henke SASS WOLF, 5 mL) and set in an electrospinning apparatus (MEC, NF-103A). The distance from the syringe to the target ground electrode (4 × 4 cm aluminum block) was 27 cm, sprayed at an applied voltage of 15 kV under the conditions of a spray amount of 1 ml / hour and a total spray time of 30 min. During spraying, 55.6 mg of TCP grains were added evenly every 3 minutes. The addition was performed nine times in a static elimination environment with an ionizer (Kasuga Denki Co., Ltd., KD-750BB) until the total addition amount reached about 500 mg.

  As understood from the above, in the nonwoven fabric obtained by the mixed solution spinning method, the water-soluble polymer is present in the fibers of the nonwoven fabric (coexists with the polylactic acid which is a biocompatible polymer to form the fibers of the nonwoven fabric). . In the solution spray method and the solution dipping method, the water-soluble polymer is present outside the fibers of the nonwoven fabric (is present on the surface of the polylactic acid fiber, which is a biocompatible polymer). In the solution co-spinning method, the water-soluble polymer is contained in the nonwoven fabric as a fiber different from the fiber of the biocompatible polymer.

  The manufacturing method of each of the above nonwoven fabrics and the polymers used are shown in Table 13 below.

  In these nonwoven fabric manufacturing methods, the protruding ground electrode (FIG. 2) is not used, but in the mixed solution spinning method, solution dipping method, and solution simultaneous spinning method, the nonwoven fabric is positively charged using an ionizer. In addition, in the solution spray method, since the HPC solution or CMC solution sprayed at the time of adding the TCP particles is an ethanol solution, the ethanol is evaporated in a state of being positively charged, and slow charging is performed. . For this reason, it was possible to manufacture a thick (three-dimensional) nonwoven fabric by any method.

Evaluation of non-woven fabric containing bone prosthetic material and water-soluble polymer Using a micrometer (Mitutoyo Co., Ltd., CLM1-15QM), the thickness of 10 points was measured randomly on each non-woven fabric, and the average value was taken as the thickness of the non-woven fabric. . The nonwoven fabric was cut into a size of 4 × 1.5 cm, placed in a conical tube (Becton Dickinson, 50 mL), and shaken (1500 rpm × 1 min) with a high-speed shaker (Tokyo Science Instrument, CM-1000). (Thus, the TCP particles were dropped.) The mass of the TCP particles dropped at the time of cutting and shaking was measured, and the drop rate was calculated.

  A polymer other than PLLA (water-soluble polymer) in the sample was dissolved in 30 mL of ethanol or 30% ethanol. Next, the precipitate was left in the conical tube, and the supernatant was removed. The ethanol in the precipitate was evaporated, the weight of the remaining precipitate was measured, and PLLA was dissolved with DCM. After leaving the precipitate and removing the supernatant, the DCM of the precipitate was evaporated, and the weight of the remaining precipitate was measured to obtain the weight of the bone grafting material (TCP particles).

  Moreover, each nonwoven fabric was image | photographed using the scanning electron microscope (Hitachi High-Technology Co., Ltd., S-3400N), and the fiber diameter was measured by Image J (ver. 1.43u) from a 2000 times electron microscope image. The average value of the fiber diameters of 50 fibers was defined as the fiber diameter of the fibers constituting each nonwoven fabric.

The following values were used for the true density (g / mL). (The values of TCP and PLLA are values obtained by a wet / dry automatic densimeter (Acupic 1330; Shimadzu Corporation), and the values of HPC, CMC and PEG are the values described in the respective MSDS.)
TCP: 3.17, PLLA: 1.26
HPC: 1.22, CMC: 1.60, PEG: 1.20

  The calculation methods for each evaluation item are summarized in Table 14 below. “Sample weight” in Table 14 means “weight of nonwoven fabric”. The calculation method of the bulk density, the porosity (of the nonwoven fabric), and the porosity of the fiber part (of the nonwoven fabric) is basically the same as the above-described calculation method.

  The results are shown in Table 15 and Table 16 below. “Additional polymer ratio” in Table 16 represents a water-soluble polymer ratio. Further, the “drop rate (%)” in Table 16 is shown as a graph in FIG. In these charts, the nonwoven fabric that did not use the water-soluble polymer is marked as “PLLA only”. Moreover, the nonwoven fabric using a water-soluble polymer represents the abbreviated name of the water-soluble polymer used and the nonwoven fabric manufacturing method side by side.

  From the results, it was confirmed that the adhesion of the bone prosthetic material to the nonwoven fabric was improved (that is, the bone prosthetic retainability of the nonwoven fabric was improved) by including a water-soluble polymer. Further, among them, a non-woven fabric containing a water-soluble polymer by a mixed solution spinning method or a solution dipping method is preferable for adhesion, and a non-woven fabric containing a water-soluble polymer by a mixed solution spinning method is most preferable. all right.

Examination of the amount of water-soluble polymer A PLLA / 5 having an HPC concentration of 5%, 1%, or 0% in the same manner as in the above mixed solution spinning method, except that the distance from the syringe to the target ground electrode was 22 cm. Three types of nonwoven fabrics were obtained using HPC mixed solution (10% / 5%, 10% / 1%, or 10% / 0%). And the fall rate of the bone grafting material was investigated like the method as described in the above-mentioned "evaluation of the nonwoven fabric containing a bone grafting material and a water-soluble polymer." In this study, the amount of bone prosthetic material dropped when the nonwoven fabric was cut into a size of 4 × 1.5 cm was also examined. The results are shown in FIG. In FIG. 12, the obtained three types of nonwoven fabrics are denoted as “HPC 5%”, “HPC 1%”, and “HPC 0%”, respectively. In addition, “before” indicates the bone grafting material falling rate at the time of nonwoven fabric processing (when cutting into a size of 4 × 1.5 cm), and “after” indicates the bone grafting material falling rate at the time of shaking.

Evaluation of non-woven fabric using bone filling material coated with biocompatible polymer Polylactic acid (Evonik Degussa Japan Co., Ltd., RESOMER (registered trademark), L 206S) 5 g, hexafluoroisopropyl alcohol: dichloromethane = 8: 2 (mass) Ratio) was added and dissolved to obtain a polylactic acid solution (10% by weight). The polylactic acid solution was filled in a syringe (Henke SASS WOLF, 5 mL) and set in an electrospinning apparatus (MEC, NF-103A). The distance from the syringe to the target ground electrode (4 x 16 cm aluminum block: the same mechanism as shown in Fig. 2) is 22 cm, the applied voltage is 15 kV, the spray amount is 1 ml / hour, and the total spray time is 90 min. It sprayed and the bone filling material was added on each of the following conditions, and the nonwoven fabric was manufactured. The bone prosthetic material is obtained by immersing Osferion (Olympus Terumo Biomaterial Co., Ltd.) as it is or by dipping it in the polylactic acid solution (10% by weight) (hereinafter also referred to as “polylactic acid-coated bone prosthetic material”). Using.

<Spray condition 1>
First, 500 mg of the bone grafting material was spread on the ground electrode (aluminum block) and sprayed thereon for 10 minutes to produce a nonwoven fabric.
<Spraying condition 2>
A non-woven fabric was produced in the same manner as in the above <spraying condition 1> except that a polylactic acid-coated bone filling material was used as the bone filling material.
<Spraying condition 3>
First, spraying was performed on the ground electrode for 10 minutes, 500 mg of the bone grafting material was sprinkled thereon, and further spraying was performed for 10 minutes thereon to produce a nonwoven fabric. (As can be seen from this, in the nonwoven fabric, biocompatible polymer fibers produced by spraying are present above and below the bone grafting material and are sandwiched.)
Each nonwoven fabric produced under the above spraying conditions 1 to 3 was placed in a transparent plastic bag, tilted and lightly shaken, and then the amount of bone grafting material peeled off from the nonwoven fabric was compared. The results are shown in FIG. As shown in FIG. 13, the non-woven fabric manufactured in the sandwich condition under the spray condition 3 hardly peeled off the bone prosthetic material, whereas the non-woven fabric manufactured under the spray condition 1 peeled off a large amount of the bone prosthetic material. fell. However, when the polylactic acid-coated bone grafting material was used, the bone grafting material hardly peeled off even if it was not a sandwich-like nonwoven fabric (spraying condition 2). From this, it was found that the adhesiveness to the nonwoven fabric can be improved by coating the bone grafting material with a biocompatible polymer.

Claims (16)

A non-woven fabric containing a bone filling material,
Bone prosthetic material is included between the fibers that make up the nonwoven fabric,
The fiber constituting the nonwoven fabric is a biocompatible fiber, and the biocompatible fiber is a fiber comprising a biocompatible polymer.
Furthermore, the nonwoven fabric which the water-soluble polymer adhered to the fiber which comprises a nonwoven fabric. A non-woven fabric containing a bone filling material,
Bone prosthetic material is included between the fibers that make up the nonwoven fabric,
The bone grafting material has a particle size of about 50 to 5000 μm ,
The fibers constituting the nonwoven fabric is biocompatible fibers and water-soluble polymer fibers, water-soluble polymer fibers contained in the nonwoven fabric as a separate fibers biocompatible fibers,
Non-woven fabric. A non-woven fabric containing a bone filling material,
Bone prosthetic material is included between the fibers that make up the nonwoven fabric,
The fiber constituting the nonwoven fabric is a biocompatible fiber, and the biocompatible fiber is a fiber comprising a biocompatible polymer and a water-soluble polymer,
The nonwoven fabric according to claim 1, further comprising a water-soluble polymer attached to fibers constituting the nonwoven fabric. The nonwoven fabric according to claim 1 or 3, wherein the bone filling material has a particle size of about 50 to 5000 µm. Furthermore, the nonwoven fabric of Claim 2 which the water-soluble polymer adhered to the fiber which comprises a nonwoven fabric. The biocompatible polymer is selected from the group consisting of polylactic acid, polyglycolic acid, polylactic acid-polyglycolic acid copolymer, polycaprolactone, chitin, collagen, polylysine, polyarginine, hyaluronic acid, sericin, cellulose, dextran, and pullulan. The nonwoven fabric according to any one of claims 1 to 5, which is at least one selected. Bone prosthesis materials are β-TCP (β-tricalcium phosphate), α-TCP (α-tricalcium phosphate), HA (hydroxyapatite), DCPD (dicalcium phosphate), OCP (octacalcium phosphate), 4CP (tetracalcium phosphate), alumina, zirconia, calcium aluminate (CaO—Al ₂ O ₃ ), aluminosilicate (Na ₂ O—Al ₂ O ₃ —SiO ₂ ), bioactivated glass, quartz, and calcium carbonate The nonwoven fabric in any one of Claims 1-6 which is at least 1 sort (s) selected from the group which consists of. The water-soluble polymer is at least one selected from the group consisting of hydroxypropylcellulose, polyethylene glycol, carboxymethylcellulose, polyvinyl alcohol, hydroxypropylmethylcellulose, and carboxyvinyl polymer, according to any one of claims 1 to 7. Non-woven fabric. The nonwoven fabric in any one of Claims 1-8 whose porosity of a nonwoven fabric is 78.5-97%. The nonwoven fabric in any one of Claims 1-9 whose porosity of the fiber part of a nonwoven fabric is 80-99.99%. The nonwoven fabric in any one of Claims 1-10 whose bulk density (g / cm < ³ >) of a nonwoven fabric is 0.1-0.6. The bone grafting material is coated with a biocompatible polymer,
The bone filling material coated with the biocompatible polymer is a bone filling material in which the biocompatible polymer is attached to 50% or more of the surface.
The nonwoven fabric in any one of Claims 1-11. The nonwoven fabric according to claim 12, wherein the bone filling material coated with a biocompatible polymer is a bone filling material having a biocompatible polymer attached to 90% or more of the surface thereof. The nonwoven fabric according to claim 12 or 13, wherein the biocompatible polymer contained in the biocompatible fiber is the same as the biocompatible polymer coating the bone grafting material. The material for bone regeneration containing the nonwoven fabric in any one of Claims 1-14. An osteoblast culture scaffold comprising the nonwoven fabric according to any one of claims 1 to 14.

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