JP2014046116A - Biologically absorbable composite material and method for producing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a composite biomaterial which has a mechanism to slowly release a chemical composition for effectively developing bone reconstruction ability and which has flexibility to improve fitting to an affected part, and to provide a method for producing the same.SOLUTION: A composite material includes a polyhydroxyalkanoate type copolymer and calcium carbonate mainly of a vaterite phase as principal ingredients, and a method for producing the composite material includes: a step to prepare the composite material by heating and kneading the polyhydroxyalkanoate and calcium carbonate; and a step to form a nonwoven fabric of the composite material by using an electrospinning method.

Description

The present invention relates to a bioactive material useful as a bone repair material, and more particularly to a bioabsorbable material containing calcium carbonate in order to increase affinity with bone.

A material that reacts with bone and chemically bonds directly when it is implanted in a bone defect is called a bioactive material. And it is divided into bioabsorbable materials that are gradually replaced by bone. Hydroxyapatite ceramics (for example, product name Apaceram manufactured by HOYA) have been put into practical use as surface active materials, and β-type tricalcium phosphate ceramics (for example, product name Osferion manufactured by Olympus Terumo Biomaterials) have been put to practical use as bioabsorbable materials. . As an evaluation of the bioactivity of the surface of these artificial materials, it is known that it is useful to observe the presence or absence of apatite generation ability by an immersion experiment using simulated body fluid (SBF). (Non-Patent Document 1)

Calcium carbonate (CaCO ₃ ) and gypsum (CaSO ₄ .2H ₂ O) are also known to be bioabsorbable. However, the strength and toughness are low and it is not easy to machine mechanically. On the other hand, biodegradable polymers such as polylactic acid, polyglycolic acid, copolymers thereof, and polyhydroxyalkanoates are easy to machine, but they are decomposed and discharged in vivo. It is resorbable and does not show osteogenic properties. In addition, some reports have been made that it may be acidified, such as lactic acid in the process of degradation, affecting the surrounding tissues.

Therefore, studies have been made to combine these inorganic compounds and organic compounds to provide bone formation and bioresorption properties, and to improve mechanical properties and machinability. For example, there is a method of producing a bioabsorbable material by combining polylactic acid and calcium carbonate (see Patent Document 1). There has been reported a method of synthesizing a bioabsorbable material by mixing a main component of calcium carbonate having a high solubility in water with a biodegradable polymer compound such as polylactic acid. Even if polylactic acid is decomposed and acidified, the calcium carbonate dissolves and exhibits a buffering effect. The pH is always kept in the vicinity of neutrality, and mechanical properties and processability are improved. However, it has been difficult to maintain the flexibility required for fitting with living tissue.

By the way, a polyhydroxyalkanoate species is a biodegradable polyester produced from a specific bacterial species, and includes a hundred polymers or more including a homopolymer and a copolymer. A typical example is poly (3-hydroxybutyrate), but the elongation at break in a tensile test is about 5%, and the flexibility is poor. Therefore, in order to make it more flexible, copolymers with other monomers have been produced. Among them, the poly (3-hydroxybutyrate-4-hydroxybutyrate) copolymer shows high flexibility, and when the 4-hydroxybutyrate content is 16% by weight, the elongation at break is about 450% (non-patented). Reference 2).

Maintaining chewing ability and exercise ability is extremely important in maintaining health in a super-aging society, and bone defects are desired to be cured as soon as possible. In order to improve the bone forming ability, there is an attempt to contain a bone-forming conductive agent (see Patent Document 2), a growth factor or a bone morphogenetic factor (see Patent Documents 3 and 4) in the bioabsorbable membrane. Dealing with factors is not easy. There is a need for the development of a bioabsorbable material with excellent bone remodeling ability that makes bone self-renewal faster and more reliable.

Looking at recent research trends in biomaterials, research has shifted from material design that combines materials and bone to material design for bone regeneration. The role of silicon on bone formation has attracted attention, and material design characterized by silicon content has been seen (see Non-Patent Document 3). For example, it has been reported that genetic action is performed on cells by slow release of silicon and bone formation is promoted (see Non-Patent Document 4). In addition, when a complex of three types of calcium carbonate (calcite, aragonite, and vaterite) and polylactic acid is immersed in simulated body fluid (SBF), hydroxyapatite with a composition and form similar to bone is obtained in the shortest time. It is shown that what is generated on the surface is a complex of vaterite and polylactic acid (see Non-Patent Document 5). From these facts, the use of a vaterite that releases silicon gradually is an important means for providing a material having a high bone reconstruction speed.

In using the bone defect filling material, a method of incising the affected area and directly embedding a dense or porous material large enough to fill the affected area, or filling a granular material is used. In order to ensure bone formation, it is desirable that the material is embedded without any gap in the affected area, but in the case of a dense or porous material, it is not easy to process according to the shape of the affected area, On the other hand, when a granular material is filled, it often drops off from the affected area after the operation, and measures have to be taken for each.

On the other hand, although it is not a method of filling the affected area, a shielding film that covers the defect part to prevent the invasion of cells and tissues that do not contribute to bone formation to the bone defect part and to utilize the self-regenerative ability of bone and rebuild the bone The bone regeneration induction method used is also known. This is to heal bone defects using the healing power inherent in the living body. For example, a nonwoven fabric layer mainly composed of silicon-eluting calcium carbonate (vaterite) and a biodegradable resin, A bone regeneration inducing membrane having a two-layer structure with a non-woven fabric layer containing a degradable resin as a main component and a method for producing the same are described (see Patent Document 5). In this membrane, the proliferation of mouse-derived osteoblast-like cells (MC3T3-E1) was good, and when bone defects on the skull were covered, vigorous bone formation was observed in the membrane Is reported (see Non-Patent Document 6), however, the elongation at break by the tensile test is small, and it is not easy to roll it into the affected part as a bone defect filling material or to fit it into a complicated shape.

JP 2001-294673 A JP-A-6-319794 Special table 2001-519210 gazette JP 2006-187303 A JP 2009-61109 A

T. Kokubo and H. Takadama, “How useful is SBF in predicting in vivo bone bioactivity”, Biomaterials, 27, 2907-2915 (2006) Y. Doi, A. Sefawa and M. Kunioka “Biosynthesis and characterization of poly (3-hudroxybutyrate-co-4-hydroxybutyrate) in Alcaligenes eutrophus”, Int. J. Biol. Macromol., 12 (1990) Koji Tsuru, Tetsuro Ogawa, Hajime Ogushi, “Research Technology and Standardization of Biomaterials”, Ceramics, 41, 549-553 (2006) H. Maeda, T. Kasuga and L. L. Hench, “Preparation of Poly (L-lactic acid) -Polysiloxane-Calcium Carbonate Hybrid Membranes for Guided Bone Regeneration”, Biomaterials, 27, 1216-1222 (2006) H. Maeda, T. Kasuga, M. Nogami and Y. Ota, “Preparation of Calcium Carbonate Composite and Their Apatite-Forming Ability in Simulated Body Fluid”, J. Ceram. Soc. Japan, 112, S804-808 (2004) T. Wakita, A. Obata and T. Kasuga, “New Fabrication Process of Layered Membranes Based on Poly (Lactic Acid) Fibers for Guided Bone Regeneration”, Materials Transactions, 50 [7], 1737-1741 (2009

SUMMARY OF THE INVENTION An object of the present invention is to provide a flexible biomaterial and a method for producing the same that can improve the fitting to an affected area based on a new mechanism for effectively deriving a bone reconstruction ability.

The present inventors have made use of a composite nonwoven fabric mainly composed of silicon-eluting calcium carbonate (Si—CaCo ₃ ) and a highly flexible biodegradable resin for supplying nutrients to cells. It has been found that the above-mentioned problems can be solved by securing the communication hole and improving the fitting property with the affected part. That is, according to the present invention, the following biocomposite material and a method for producing the same are provided.

[1] A biocomposite material mainly comprising a polyhydroxyalkanoate copolymer and calcium carbonate.

[2] The biocomposite material according to [1], wherein the copolymer of the polyhydroxyalkanoate species is poly (3-hydroxybutyrate-4-hydroxybutyrate).

The biocomposite material according to [2] above, wherein the content of 4-hydroxybutyrate in poly (3-hydroxybutyrate-4-hydroxybutyrate) is 10 to 40% by weight.
[3]

[4] The biocomposite material according to [1] or [2], wherein calcium carbonate is mainly a vaterite phase.

[5] The biocomposite material according to any one of [1] to [3], wherein the calcium carbonate contains silicon.

[6] The biocomposite material according to [4], wherein the calcium carbonate containing silicon is 10 to 40% by weight of the whole biocomposite material containing the calcium carbonate.

[7] The biocomposite material according to [5] or [6], wherein the silicon content in calcium carbonate is 1 to 10% by weight.

[8] The biocomposite material according to [1] to [7], wherein the biocomposite material is a nonwoven fabric.

[9] A step of preparing a composite material comprising a copolymer of polyhydroxyalkanoate species and calcium carbonate, and a non-woven fabric mainly comprising a copolymer of polyhydroxyalkanoate species and calcium carbonate using an electrospinning method. The manufacturing method of the biocomposite material including the process to form.

[10] The method for producing a biocomposite material according to [9], wherein the polyhydroxyalkanoate copolymer is poly (3-hydroxybutyrate-4-hydroxybutyrate).

[11] The method for producing a biocomposite material according to [9] or [10], wherein the calcium carbonate is mainly a vaterite phase.

[12] The method for producing a biocomposite material according to any one of [9] to [11], wherein the calcium carbonate contains silicon.

The biocomposite material according to the present invention is expected to be a biocomposite material exhibiting high elongation at break in mechanical property evaluation by a tensile test and having excellent flexibility. Moreover, according to the manufacturing method which concerns on this invention, the biomaterial with the above possibilities can be manufactured easily and efficiently.

_{3 is} a scanning electron micrograph of the surface of the Si—CaCO ₃ / P (3HB-4HB) composite nonwoven fabric of the present invention. 3 is a scanning electron micrograph of the surface of a P (3HB-4HB) nonwoven fabric as a comparative example. 2 is a stress-strain curve of the Si—CaCO ₃ / P (3HB-4HB) composite nonwoven fabric of the present invention and a P (3HB-4HB) nonwoven fabric as a comparative example. _{3 is} a scanning electron micrograph of the surface of the Si—CaCO ₃ / P (3HB-4HB) composite nonwoven fabric of the present invention after SBF immersion. 4 is a scanning electron micrograph of the surface of a comparative example P (3HB-4HB) non-woven fabric after SBF immersion. It is an X-ray diffraction pattern after SBF immersion of the Si—CaCO ₃ / P (3HB-4HB) composite nonwoven fabric of the present invention.

Hereinafter, embodiments of the present invention will be described with reference to the drawings. The present invention is not limited to the following embodiments, and changes, modifications, and improvements can be added without departing from the scope of the invention.

According to a preferred embodiment of the present invention, a nonwoven material is produced by using an electrospinning method in which a positive high voltage is applied to a polymer solution and fiberization occurs in the process of being sprayed on a negatively charged collector. Can do.

The present invention is a biocomposite material mainly comprising a copolymer of polyhydroxyalkanoate species and calcium carbonate. The total of the copolymer of polyhydroxyalkanoate species and calcium carbonate is 90% by weight of the composite material. % Or more, and more preferably 97% by weight. The copolymer of polyhydroxyalkanoate species is preferably poly (3-hydroxybutyrate-4-hydroxybutyrate), that is, P (3HB-4HB). In addition, P (3HB-4HB) copolymer of any composition has higher flexibility than poly (3-hydroxybutyrate) homopolymer and can be used for the production of flexible composite materials. However, it is preferable to use a 4-hydroxybutyrate content of 10 to 40% by weight, so that high flexibility necessary for fitting to the affected area can be expected. Calcium carbonate is preferably mainly in the vaterite phase, and preferably contains silicon. It is preferable that the compounding ratio of silicon with respect to calcium carbonate is 1 to 10% by weight. Silicon dissolves in the living body such as bone from calcium carbonate and contributes to bone repair or reconstruction. In addition, as a biocomposite material of this invention, a nonwoven fabric is more flexible and preferable compared with a film.

In producing the biocomposite material of the present invention, a spinning solution for producing a nonwoven material is prepared by dissolving P (3HB-4HB) in chloroform (CHCl ₃ ) or dichloromethane (DCM). In order to maintain a good spinning state, dimethylformamide (DMF) or methanol (CH ₃ OH) may be appropriately added up to 50% by weight based on CHCl ₃ or DCM. Next, Si-CaCO ₃ is added to the spinning solution of P (3HB-4HB) to prepare a spinning solution for producing a composite nonwoven fabric. When mixed so that the Si—CaCO ₃ content of the composite is 40% by weight, more preferably 35% by weight or less, high elongation at break can be exhibited in a tensile test. On the other hand, in order to promote good bone formation, the content is preferably 10% by weight, more preferably 15% by weight or more. In addition to the above spinning solution preparation method, a composite prepared by kneading a predetermined ratio of P (3HB-4HB) and Si—CaCO ₃ with a heating kneader in advance may be dissolved in a solvent to form a spinning solution. Si—CaCO ₃ is prepared using a method described in, for example, Japanese Patent Application Laid-Open No. 2006-161816. Si-CaCO ₃ / P (3HB-4HB) composite material has tricalcium phosphate, calcium sulfate, sodium phosphate, sodium hydrogen phosphate, calcium hydrogen phosphate, octacalcium phosphate, which are not problematic for biological use. Further, inorganic substances such as tetracalcium phosphate, calcium pyrophosphate, and calcium chloride may be contained.

By using an electrospinning device, the spinning solution for producing the charged Si-CaCO ₃ / P (3HB-4HB) composite nonwoven fabric discharged from the nozzle becomes a fiber while volatilizing the solvent in an electric field and minus. Facing the collector of the electrode, a layer of many fibers is formed on the collector. By changing the spinning conditions (concentration of spinning solution, type of solvent, supply speed, spinning time, applied voltage, distance between nozzle and collector, etc.), a desired nonwoven material can be obtained. Moreover, densification and a film thickness can also be adjusted by pressing the obtained nonwoven fabric. In addition, as a fiber diameter, it is more preferable that they are 1-50 micrometers and 5-20 micrometers.

Examples of the method for producing a Si—CaCO ₃ / P (3HB-4HB) composite nonwoven fabric according to the present invention will be described below. The following description of the examples is for a better understanding of the present invention, and the present invention is not limited to the following examples.

In each example, the following raw materials were used.
・ Silicon-eluting calcium carbonate (Si-CaCO ₃ ): Slaked lime (Microstar T purity 96% or more Yabashi Kogyo Co., Ltd.), methanol (special grade reagent purity 99.8% or more Kishida Chemical Co., Ltd.), γ-aminopropyltriethoxysilane ( TSL8331 purity 98% or higher GE Toshiba Silicone Co., Ltd.), carbon dioxide (high purity liquefied carbon dioxide, purity 99.9%, Taiyo Chemical Industry Co., Ltd.), a battery phase calcium carbonate poly (3) containing 2.9% by weight of Si -Hydroxybutyrate-4-hydroxybutyrate) (P (3HB-4HB): PURE PHA POWDER, PHA-0600, molecular weight about 1.1 million Da, G5 JAPAN Co., Ltd.)
・ Chloroform (CHCl ₃ ): Special grade reagent purity 99.0% or higher Kishida Chemical Co., Ltd.

(Example)
P (3HB-4HB) and Si-CaCO ₃ 110 ℃ in heating kneader, kneaded for 10 minutes containing Si-CaCO ₃ 30 _{wt% Si-CaCO 3 / P (} 3HB-4HB) and the complex was prepared. Then, a spinning solution was prepared by mixing Si—CaCO ₃ / P (3HB-4HB) complex and chloroform. As a comparative sample, P (3HB-4HB) was mixed with chloroform to prepare a spinning solution. In this example, a solution was prepared so that P (3HB-4HB) was 6% by weight with respect to chloroform for both the composite material sample and the comparative sample. The viscosity of the prepared solution was about 2500 [mPa · s] for the composite sample and about 3000 [mPa · s] for the comparative sample.

Using these spinning solutions, a non-woven fabric of Si-CaCO ₃ / P (3HB-4HB) composite and a non-woven fabric of P (3HB-4HB) were prepared by electrosping. Nonwoven fabric production conditions were as follows: Spinning solution supply speed was about 0.200 ml / min, applied voltage was 10 kV, nozzle-collector distance was 80 mm, nozzle was moved left and right (5-6 cm width) at 100 mm / min, collector Was a conveyor type (conveyor speed: 2 m / min, grounding), and the spinning time was 180 minutes.

Scanning electron microscope (SEM) photographs of the surface microstructures of the Si—CaCO ₃ / P (3HB-4HB) composite nonwoven fabric and the P (3HB-4HB) nonwoven fabric are shown in FIGS. 1 and 2, respectively. It can be confirmed that the Si-CaCO ₃ / P (3HB-4HB) composite nonwoven fabric contains granular Si-CaCO ₃ in the P (3HB-4HB) fiber.

Figure shows the stress-strain curve when a tensile test was performed at 5 mm / min on a Si-CaCO ₃ / P (3HB-4HB) composite nonwoven fabric cut into 5 mm × 40 mm size and P (3HB-4HB) nonwoven fabric. 3 shows. It can be said that the Si—CaCO ₃ / P (3HB-4HB) composite non-woven fabric has high flexibility compared with P (3HB-4HB) non-woven fabric and exhibits high tensile strength and break elongation exceeding 100%.

Si-CaCO ₃ / P (3HB-4HB) composite non-woven fabric is simulated body fluid (SBF: buffer solution with ion concentration similar to human body fluid (plasma) (Ca ²⁺ ; 2.5, Mg ²⁺ ; 1.5, Na ⁺ 142.0, K ⁺ ; 5.0, Cl ⁻ ; 148.8, HCO ³⁻ ; 4.2, HPO ₄ ²⁻ ; 1.0 mmol / L)), and a photograph taken by SEM after immersion (at 37 ° C. for 1 day) is shown in FIG. FIG. 6 shows X-ray diffraction patterns before and after SBF immersion. For comparison, FIG. 5 shows an SEM observation photograph after a P (3HB-4HB) nonwoven fabric is immersed in the simulated body fluid under the same conditions. FIG. 4 and FIG. 6 show that hydroxyapatite is generated on the surface of the composite material after immersion and has excellent bioactivity. On the other hand, with the P (3HB-4HB) non-woven fabric, formation of hydroxyapatite was not observed in the same test.

The composite material of the present invention can be used for living bodies, particularly for bone reconstruction.

Claims (12)

A biocomposite material comprising a polyhydroxyalkanoate copolymer and calcium carbonate as main components. The biocomposite material according to claim 1, wherein the copolymer of the polyhydroxyalkanoate species is poly (3-hydroxybutyrate-4-hydroxybutyrate). The biocomposite material according to claim 2, wherein the content of 4-hydroxybutyrate in poly (3-hydroxybutyrate-4-hydroxybutyrate) is 10 to 40% by weight. The biocomposite material according to any one of claims 1 to 3, wherein calcium carbonate is mainly a vaterite phase. The biocomposite material according to any one of claims 1 to 4, wherein the calcium carbonate contains silicon. The biocomposite material according to claim 5, wherein the silicon-containing calcium carbonate is 10 to 40% by weight of the whole biocomposite material containing the silicon-containing calcium carbonate. The biocomposite material according to claim 5 or 6, wherein the content of silicon in calcium carbonate is 1 to 10% by weight. The biocomposite material according to claim 1, wherein the biocomposite material is a nonwoven fabric. A step of preparing a composite material comprising a copolymer of polyhydroxyalkanoate species and calcium carbonate, and a step of forming a nonwoven fabric mainly comprising a copolymer of polyhydroxyalkanoate species and calcium carbonate using an electrospinning method. A method for producing a biocomposite material comprising: The method for producing a biocomposite material according to claim 9, wherein the copolymer of the polyhydroxyalkanoate species is poly (3-hydroxybutyrate-4-hydroxybutyrate). The method for producing a biocomposite material according to claim 9 or 10, wherein calcium carbonate is mainly a vaterite phase. The method for producing a biocomposite material according to any one of claims 9 to 11, wherein the calcium carbonate contains silicon.