JP2010046249A - Hard tissue filling material - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new hard tissue filling material having a filling property for a defective bone section, bone tissue regenerating capacity and the function of slow releasing a medicine component which accelerates it. <P>SOLUTION: The hard tissue filling material includes a composite comprising ceramic granules and crosslinked acid or basic gelatin hydrogel. The ceramic granules and the acid or basic gelatin hydrogel are mixed and freeze-dried and then the complex is formed by heat crosslinking reaction. Also, it is utilized as a medicine slow releasing base material. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a hard tissue filling material used as a filling material for restoring hard tissues such as teeth and bones.

  Tissue repair using a bone filling material is performed on bone defects such as long bones, jawbones, and skulls. Bone prosthetic materials are required to have excellent biocompatibility and osteoconductivity, so β-tricalcium phosphate (β-TCP), hydroxyapatite (HAp) and others Calcium phosphate ceramics are often used.

  β-Tricalcium Phosphate (β-TCP) has osteoconductivity, and when used as a bone filling material, β-TCP is absorbed by osteoclasts and osteogenesis by osteoblasts in vivo, so-called remodeling Is done. That is, it is characterized by being decomposed in vivo and gradually replaced with new bone.

  However, when these ceramics are embedded in a bone defect as a bulk mass formation, there is often a high risk of infection in the center, and the decomposition of the ceramics is slow, causing problems in terms of replacement with new bone. There are many cases. Therefore, it is considered to use ceramics in a small granular form instead of a lump. However, it is difficult to keep the granular material in the defective part, and there is still room for ingenuity in its handling.

  In order to solve this problem, it is necessary to use a granular ceramic together with a material that serves as a binder. That is, research and development of a material that can be formed into a hydrogel by mixing ceramic porous granules and a binder material are strongly desired.

  On the other hand, there has been proposed a bone filling material that forms new bone inside the pores of the porous body or between the granules by combining β-TCP porous granules with an organic material as a binder. In addition, a method for producing a graft material in which hydroxyapatite granules, sodium alginate, and platelet-rich plasma collected from a patient himself are mixed as a bone filler in the oral cavity has been reported (Patent Document 1). However, collection of platelet-rich plasma requires blood collection from the patient and complicated procedures, and the volume that can be used is limited, so that it is difficult to apply to large bone defects. In addition, sodium alginate is not bioabsorbable and remains in bone tissue.

  As a method for solving the problem of the complexity of the procedure for collecting platelet-rich plasma from patients, a method in which basic fibroblast growth factor (bFGF) is gradually released from calcium alginate has been studied (Patent Document 2). However, it is difficult for calcium alginate to regulate the degradability in the living body, and there is a concern that the entry of cells necessary for bone formation into the scaffold may be hindered and inflammation of the tissue due to residual may be caused.

On the other hand, as the binder, a material excellent in bioabsorbability and biocompatibility, particularly cell affinity is desired. As such materials, collagen, gelatin and the like are known (Patent Document 3). Furthermore, better osteogenesis can be expected if cell growth factors such as bFGF can be gradually released from the binder material. In this regard, gelatin is known to slow release growth factors such as bFGF, enhance its biological activity, and promote various tissue regeneration (Patent Document 4, Non-Patent Document 1). However, this document only uses gelatin as a sustained release substrate and does not describe or suggest the use of it as a binder.
JP 2004-201799 A JP 2007-203034 A JP-T-2002-501786 JP 2005-230372 A Y. TABATA, et al., Biomaterials 1999, 20; 2169-2175

  An object of the present invention is to provide a novel hard tissue filling material having a bone defect filling ability, a bone tissue regeneration ability, and a sustained release function of a medicinal component that promotes this.

  In order to solve the above-mentioned problems, the inventors have intensively studied and considered to use gelatin having bioabsorbability and high cell affinity as a binder. Then, a gelatin hydrogel aqueous solution is mixed with the ceramic granules, freeze-dried, and then subjected to a thermal crosslinking reaction to form a composite, thereby forming a hardened body with excellent defect filling and mechanical properties and sustained drug release. The present inventors have found that a tissue filling material can be obtained and completed the present invention.

  That is, the present invention is a hard tissue filling material comprising a composite comprising ceramic granules and a crosslinked acidic or basic gelatin hydrogel, wherein the composite is a mixture of ceramic granules and acidic or basic gelatin hydrogel. In addition, the present invention relates to a hard tissue filling material and a method for producing the same, which are formed by freeze-drying and then forming a composite by a thermal crosslinking reaction.

  The hard tissue filling material of the present invention may further contain at least one medicinal component. The medicinal component is incorporated into the composite by impregnating the composite constituting the hard tissue filling material. Since the gelatin hydrogel that constitutes the hard tissue filling material of the present invention releases the medicinal component gradually as it decomposes in vivo, the effect of the medicinal component can be maintained over a desired period.

  As the medicinal component, a bone morphogenetic factor or a bone growth factor is preferable. For example, FGF, BMP, TGF-β, IGF and PDGF can be used.

  Examples of the ceramic include hydroxyapatite, carbonate apatite, fluorapatite, chlorapatite, β-TCP, α-TCP, calcium metaphosphate, tetracalcium phosphate, calcium hydrogen phosphate, dicalcium phosphate, octacalcium phosphate, hydrogen phosphate. Calcium dihydrate, calcium phosphate glass, a mixture of these calcium phosphates, alumina, and at least one selected from zirconia can be used. Of these, β-TCP having osteoconductivity is preferable.

The ceramic granules preferably have an average diameter of about 1 μm to 3 mm.
The ceramic granules and the acidic or basic gelatin hydrogel are preferably contained in a volume ratio of 1/1000 to 1/10, particularly 1/600 to 1/60.

  The hard tissue prosthetic material of the present invention prevents leakage from the transplant site when using ceramic granules as a prosthetic material for a living body, and can also gradually release medicinal components such as bone forming factors and bone growth factors. The hard tissue prosthetic material of the present invention has a gap between ceramics secured by gelatin hydrogel, and keeps blood flow and cells easily entering, while releasing medicinal components such as growth factors along with its decomposition. , Promote bone regeneration.

  The composite of the ceramic granule and the gelatin hydrogel aqueous solution of the present invention has a sustained release effect and a stabilization effect such as a bone formation factor and a bone growth factor, and can exert its function for a long time in a small amount. Therefore, the angiogenesis promoting function and the osteogenic function of these drugs are effectively exhibited at the site of local administration.

1. “Ceramics” according to the present invention is a sintered inorganic solid material, specifically, hydroxyapatite, carbonate apatite, fluorapatite, chlorapatite, β-TCP, α-TCP, calcium metaphosphate, Examples include tetracalcium phosphate, calcium hydrogen phosphate, dicalcium phosphate, octacalcium phosphate, calcium hydrogen phosphate dihydrate, calcium phosphate glass, a mixture of these calcium phosphates, alumina, zirconia, and the like.

  The “ceramic granule” according to the present invention is not limited to the term “granule”, but includes all small bodies such as grains, small blocks, and pulverized bodies, each of which may be uniform or non-uniform. Means a thing. In addition, it is preferable that the granules have a shape close to a spherical shape in terms of manufacturing, adjustment of strength, filling rate, reproducibility, operability, and the like.

  In the present invention, the ceramic granule is formed into a composite with gelatin hydrogel and used as a hard tissue filling material such as a bone defect or a drug sustained-release base. Therefore, it is desirable that the composite is formed so that cells necessary for osteogenesis can easily enter. Therefore, the gap between the ceramic granules is 3 to 350 μm, preferably 100 to 300 μm. The diameter is also selected. That is, the size of one granule is an average particle size of 3 μm to 5 mm, preferably 500 to 850 μm.

2. Seratin hydrogel The “gelatin hydrogel” according to the present invention is a hydrogel obtained by forming a cross-link between gelatin molecules by giving a thermal reaction or the like to gelatin.
Here, “gelatin” means denatured collagen that has been irreversibly changed to a water-soluble protein by cleaving a salt bond or a hydrogen bond between peptide chains of collagen by hot water treatment or the like.

  The gelatin used in the present invention may be either acidic gelatin or basic gelatin. In the present specification, “acidic gelatin” means gelatin having an isoelectric point prepared by alkali treatment of collagen and having an isoelectric point of less than 7.0 and not less than 2.0, preferably not more than 6.5 and not less than 4.0, more preferably 5. 5 or more and 4.5 or more are intended. The “basic gelatin” is gelatin having an isoelectric point of 7.0 or more and 13.0 or less prepared by acid treatment of collagen, preferably 7.5 or more and 10.0 or less, more preferably 8.5 or more. Less than 9.5 is contemplated. For example, Nitta Gelatin's sample isoelectric point (IEP) 5.0 or the like can be used as “acidic gelatin”, and Nitta Gelatin's sample IEP 9.0 or the like can be used as basic gelatin. can do.

  Which gelatin is used is appropriately selected according to the medicinal component to be blended and the use. For example, bFGF is stable under acidic conditions, so acidic gelatin is used when formulating such drugs. On the other hand, when gelatin is formulated that is stable under basic conditions, basic gelatin is used.

  The degree of crosslinking of gelatin can be appropriately selected according to the desired water content, that is, the level of bioabsorbability of the hydrogel. The cross-linking may be any part of the collagen constituting the gelatin, but it is particularly preferable to cross-link the carboxyl group and the hydroxyl group, the carboxyl group and the ε-amino group, or the ε-amino group. By introducing cross-linking in this way, the composite has the desired mechanical strength properties. Further, the degradation rate (remaining period) in the living body can also be controlled by the introduction rate of crosslinking. In general, as the concentration of gelatin and the crosslinking agent and the crosslinking time increase, the degree of crosslinking of the hydrogel increases and the bioabsorbability decreases.

  The degree of crosslinking of gelatin can be evaluated using the water content as an index. The water content is the weight percent of water in the hydrogel relative to the weight of the swollen hydrogel. If the water content is large, the degree of crosslinking of the hydrogel is low and it is easily decomposed. The water content showing a preferable sustained-release effect is about 80 to 99 w / w%, and more preferably about 95 to 98 w / w%.

  Crosslinking can be performed by a method such as thermal reaction, chemical crosslinking using a crosslinking agent or a condensing agent, physical crosslinking using γ rays, ultraviolet rays, electron beams, or the like. In the case of chemical crosslinking, examples of the crosslinking agent used include aldehyde-based crosslinking agents such as glutaraldehyde and formaldehyde; isocyanate-based crosslinking agents such as hexamethylene diisocyanate; 1-ethyl-3- (3-dimethylaminopropyl) Examples thereof include carbozide-based crosslinking agents such as carbodiimide hydrochloride; polyepoxy-based crosslinking agents such as ethylene glycol diethyl ether; transglutaminase and the like. The amount of the crosslinking agent to be added is appropriately set depending on the crosslinking agent to be used.

  When the crosslink is formed by a thermal reaction, specifically, it can be carried out under a vacuum condition at 140 ° C. to 160 ° C. for 6 hours to 72 hours. It is possible to control the degradation rate (remaining period) in the body.

3. Method for Producing Composite The hard tissue filling material according to the present invention is composed of a composite containing an acidic or basic gelatin hydrogel crosslinked with the above-described ceramic granules. In the composite, the gelatin hydrogel functions as a binder for ceramic granules and also functions as a sustained-release base for medicinal components.

  The composite is prepared by mixing an aqueous gelatin hydrogel solution with ceramic granules, and performing a crosslinking reaction after lyophilization.

  First, ceramic granules and an acidic or basic gelatin hydrogel aqueous solution are mixed. At this time, the final mixing ratio of the two is such that the volume ratio of ceramics / gelatin aqueous solution is 1/1000 to 1/10, preferably 1/600 to 1/60.

  Next, the mixture is freeze-dried according to a conventional method. By this lyophilization process, countless pores are formed in the composite, and a porous composite having a desired porosity and pore diameter is obtained.

  The lyophilized composite is then crosslinked. As described above, the crosslinking can be performed by a method such as a thermal reaction, chemical crosslinking using a crosslinking agent or a condensing agent, physical crosslinking using γ rays, ultraviolet rays, electron beams, etc. Is preferably subjected to thermal crosslinking. This is because when the chemical crosslinking is performed, the crosslinking agent may remain attached to the ceramic surface, or only the surface of the composite may be crosslinked and uniform crosslinking may not be performed. On the other hand, in the case of thermal crosslinking, uniform crosslinking is formed in the entire gelatin hydrogel, and a desired sustained release effect is achieved.

  The degradability of gelatin hydrogel is controlled by the degree of crosslinking. Its degradability can be controlled within the range of 3 days to 4 months in the animal body, for example, mouse subcutaneous tissue. In the present invention, if the degradability is too fast, the role as a filling agent cannot be fulfilled, and unexpected tissues and soft tissues invade and proliferate (because the regeneration speed is higher than that of bone tissues). On the other hand, if it is slow, its remaining physically interferes with tissue regeneration. Therefore, the degradability was set to 1 to 6 weeks, preferably 2 to 4 weeks.

  The composite is formed into a certain shape by the freeze-drying and crosslinking steps. Further, since the gelatin hydrogel is in a dried state, it can be stored for a long time. The shape of the composite is not particularly limited, and examples thereof include a columnar shape, a prismatic shape, a sheet shape, a disk shape, a spherical shape, and a particle shape. A cylindrical shape, a prismatic shape, a sheet shape, or a disk shape is suitable for use as an embedded piece, or can be finely pulverized into particles. In addition, spherical, particulate, and paste-like materials can be administered by injection.

4). Hard tissue prosthetic material The composite obtained by the above method has communication holes, and each constituent component is biodegradable, so that the invasion property of the cells is good and the bone conductivity is excellent. In addition, mechanical strength characteristics and in vivo retention (appropriate in vivo degradation rate) can be controlled by crosslinking. Therefore, it is suitable for a hard tissue filling material (implant) used in an orthopedic region such as a bone filler or a dental region.

The hard tissue filling material of the present invention can contain other components as necessary. Examples of such components include inorganic salts such as St, Mg and CO ₃ , organic substances such as citric acid and phospholipids, and medicinal components such as bone morphogenetic factors, bone growth factors and anticancer agents. As will be described later, when a medicinal component is contained, the composite constituting the hard tissue filling material functions as a sustained release substrate.

In addition, depending on the purpose such as the stability of gelatin hydrogel and the sustained release of medicinal components, amino sugar or its high molecular weight or chitosan oligomer, basic amino acid or its oligomer or high molecular weight, polyallylamine, polydiethylaminoethyl Basic polymers such as acrylamide and polyethyleneimine may be added.
The hard tissue prosthetic material of the present invention is a bone filler filled in a bone defect part, dental implant placement, and other cases where excessive damage to the jaw bone at the time of surgery related to hard tissue is performed, or emergency treatment It may be used as a temporary prosthetic material used when it is necessary.

5). Sustained drug release substrate The composite of the ceramic granule obtained in the present invention and the gelatin hydrogel aqueous solution is freeze-dried and then subjected to a thermal crosslinking reaction, whereby the gelatin hydrogel is dried. Long-term storage is possible. By again impregnating medicinal components such as bone formation factors and bone growth factors from the back of the syringe, it is possible to obtain a filling material imparted with a sustained drug release effect.

  Examples of the medicinal component in the present invention include an antitumor agent, an antibacterial agent, an anti-inflammatory agent, an antiviral agent, an anti-AIDS agent, a low molecular drug such as a hormone, a bioactive peptide or protein containing a bone morphogenetic factor or a bone growth factor. , Glycoproteins, polysaccharides, nucleic acids and the like. Particularly preferred are those having a molecular weight of about 50,000 or less. These medicinal ingredients may be natural substances or synthetically produced substances.

  Specifically, epidermal growth factor (EGF), fibroblast growth factor (FGF), platelet derived growth factor (PDGF), hepatocyte growth factor (HGF), transforming growth factor (TGF), insulin-like growth factor ( BGF-2, BMP-4, BMP-5, BMP-6, BMP-7 (OP-1) and BMP-8 (OP-2) and other bone growth factors such as IGF) Forming protein (BMP), glial-induced neurotrophic factor (GDNF), neurotrophic factor (NF), platelet-rich plasma (PRP) containing many cell growth factors widely used in dentistry, interferon, interleukin-2 , Anticancer drugs such as ifosfamide, antibiotics such as streptomycin, gentamicin, gatifloxacin, atorvastatin, Cholesterol-lowering agents such as pravastatin, simvastatin, lidocaine, protamine sulfate, sodium iodohypurate, iodosulfopromophthalein, heparin sodium, glucose, norepinephrine, dextran, thiopental sodium, sodium chromate injection Xylitol, procaine hydrochloride, tetracaine hydrochloride, tubocurarine chloride, squismethonium chloride, amorphous insulin zinc, amyl nitrite, azimarin and the like.

  Of these, bone morphogenetic or bone growth factors including FGF, BMP, TGF-β, IGF and PDGF are preferred.

  The medicinal component may be impregnated in the composite after forming a composite composed of ceramic granules and an aqueous gelatin hydrogel solution. Drop the solution containing the medicinal component from one side of the complex or use a device such as a diffusion chamber to place the complex between cells containing medicinal component solutions of different concentrations and impregnate the drug. Then, a concentration gradient of medicinal components can be formed in the complex.

The compounding ratio of the medicinal component to the gelatin hydrogel (composite) is preferably about 5 times or less in terms of molar ratio. More preferably, the molar ratio is about 5 to about 1/10 ⁴ times. This impregnation operation is usually completed at 4-37 ° C. for 15 minutes to 1 hour, preferably at 4-25 ° C. for 15-30 minutes, during which time the gelatin hydrogel swells with a solution containing a medicinal component, It is combined with the gel by physicochemical interaction and fixed in the hydrogel. In addition to the physical interaction such as Coulomb force, hydroxyl bond strength, and hydrophobic interaction, the medicinal component and gelatin hydrogel can be bonded between the functional group of the drug or the metal and the functional group on the hydrogel. It is considered that a coordination bond of is involved alone or in combination.

  The medicinal component is gradually released out of the complex as the gelatin hydrogel is degraded in vivo. This release rate is determined by the degree of degradation and absorption in the living body of the gelatin hydrogel used, and the degree and stability of the bond strength between the medicinal ingredient and the gelatin hydrogel in the complex. The degree of degradation and absorption of gelatin hydrogel in the living body can be adjusted by adjusting the degree of crosslinking during the preparation of the hydrogel.

  In the present invention, when a negatively charged substance such as nucleic acid is used as the medicinal component, the gelatin hydrogel is positively charged so that a stable complex of the medicinal component and the gelatin hydrogel is formed. Is preferred. A stable gelatin hydrogel complex is formed by a strong bond (ionic bond) between the negative charge of the medicinal component and the positive charge of the gelatin hydrogel. In order to positively charge the gelatin hydrogel, it can be cationized by previously introducing an amino group or the like into the gelatin hydrogel. This increases the binding force between the gelatin hydrogel and the medicinal component, and a more stable gelatin hydrogel complex can be formed.

  The cationization step is not particularly limited as long as it is a method capable of introducing a functional group that can be cationized under physiological conditions, but a 1, 2 or tertiary amino group or ammonium group is added to the hydroxyl group or carboxyl group of gelatin. A method of introducing under mild conditions is preferred. For example, alkyldiamines such as ethylenediamine, N, N-dimethyl-1,3-diaminopropane, trimethylammonium acetohydrazide, spermine, spermidine, diethylamide chloride, and the like can be used with various condensing agents such as 1-ethyl-3- (3 -Dimethylaminopropyl) carbodiimide hydrochloride, cyanuric chloride, N, N'-carbodiimidazole, cyanogen bromide, diepoxy compound, tosyl chloride, diethyltriamine-N, N, N ', N ", N" -pentane There exists the method of making it react using dianhydride compounds, such as an acid dianhydride, trisyl chloride, etc. Of these, the method of reacting ethylenediamine is preferred because it is simple and versatile.

  The medicinal component-containing hard tissue filling material of the present invention can be administered to a living body by an arbitrary method, but local administration is particularly preferable in order to continuously release the medicinal component at a specific target site with directionality. If necessary, it may be mixed with a pharmaceutically acceptable carrier (stabilizer, preservative, solubilizer, pH adjuster, thickener, etc.). Further, various additives for adjusting the sustained release effect may be included. In addition, it is desirable to pass through sterilization processes, such as sterilization filtration, in formulation.

  Since the hard tissue filling material of the present invention has a sustained release effect and a stabilization effect of a medicinal component, the medicinal component can be released at a desired site with a controlled direction for a long time. Therefore, the action of the medicinal component is effectively exhibited within the lesion site.

  The hard tissue prosthetic material of the present invention is highly useful as a biological implant base material for sustained drug release, while having a simple manufacturing method and strength sufficient to be prosthetic to a biological hard tissue. The hard tissue filling material of the present invention is a mixture of ceramic granules and gelatin, and optionally contains medicinal ingredients such as bone formation factors and bone growth factors. Reproduction can be achieved. Furthermore, cross-linked gelatin hydrogel functions as a carrier material for sustained release of medicinal ingredients, stabilizes growth factors, releases bioactive factors over a long period of time, and promotes the growth of biological tissue cells can do. Thereby, rapid regeneration of a living tissue having a larger volume and maintenance of the regenerated tissue can be realized.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited by these Examples.

Example 1: Hard tissue filling material composed of granular ceramic and gelatin hydrogel (1) As the granular ceramic composed of β-TCP, Osferion G1 (registered trademark) having a diameter of 0.5 to 1.5 mm was used. The tip of the syringe was cut and covered with a thin sheet material such as Parafilm (registered trademark) so that the filled granular ceramic material did not leak. Further, in order to discharge the excess gelatin hydrogel aqueous solution, a hole was formed in the sheet material to produce a molding container.
(2) Acidic gelatin (isoelectric point 5.0) was obtained from Nitta Gelatin (Osaka). 0.5 g of gelatin and 4.5 g of ultrapure water (Milli Q) were placed in a beaker and allowed to stand at room temperature for 30 minutes. Then, it stirred for 30 minutes, heating at 40 degreeC, and produced 10 wt% gelatin aqueous solution.
(3) 0.2 g of granular ceramics is filled in a molding container, 300 μL of 10% gelatin hydrogel aqueous solution is injected from the rear of the piston and stirred, and then the gelatin hydrogel is prevented from leaking from the tip. Extrude only the aqueous solution. At that time, it is suppressed by using a means such as holding by a hand or fixing with a fixing band or the like so that the sheet material is not pushed out together. The mixture was allowed to stand at room temperature for 30 minutes.
(4) Freeze-drying The produced composite was put into a plastic tray as a molded container and frozen in a freezer at -80 ° C. Thereafter, the frozen complex was lyophilized according to a conventional method.
(5) Thermal cross-linking Gelatin molecules were cross-linked by heating at 140 ° C. for 36 hours in a vacuum state and obtained in a state of being molded into a cylindrical shape (FIG. 1).

Example 2: Structure of hard tissue filling material composed of granular ceramic and gelatin hydrogel Scanning electron microscope SEM of the surface and fractured surface of the hard tissue filling material composed of granular ceramic and gelatin hydrogel prepared in Example 1 Observed. As a result, a network-like gelatin structure having pores in the gaps between the β-TCP granules in the hard tissue filling material was observed, and the average pore diameter was 340 μm. From this, it was shown that the composite of the ceramic granule of the present invention and the gelatin hydrogel aqueous solution has a continuous pore structure and a structure advantageous for cell proliferation and subsequent bone tissue formation (FIG. 2). .

Example 3: Cell adhesion ability of hard tissue filling material comprising granular ceramic and gelatin hydrogel The cell adhesion ability of the hard tissue filling material comprising granular ceramic and gelatin hydrogel prepared in Example 1 was examined. As a comparison object, the difference in cell adhesion ability between each material was examined by measuring the cell proliferation activity (MTT assay) using a spherical ceramic composite using calcium alginate.

  A spherical ceramic composite using calcium alginate was prepared as follows. Specifically, a spherically shaped ceramic made of β-TCP having a diameter of 500 to 700 μm was immersed in a 1% calcium chloride aqueous solution for 30 seconds in a beaker, and water was evaporated in a dryer at 60 ° C. for coating. Load the coated ceramics into a syringe with a cut end, and cover with a thin sheet material such as Parafilm (registered trademark) so that the ceramic material does not leak out and a gap that allows sodium alginate aqueous solution to pass through. After that, 1% sodium alginate aqueous solution was poured from the piston side of the syringe and only the sodium alginate aqueous solution was extruded to gel the sodium alginate with calcium chloride, thereby producing a spherical ceramic / alginate composite molded into a cylindrical shape. .

  Each complex was pressed onto a cell culture substrate, seeded with mouse calvaria-derived osteoblast-like cells at a concentration of 30,000 cells / ml, and cell proliferation activity after 3 hours of culture at 37 ° C. was determined by MTT assay. Measured and taken as the number of adherent cells.

  The results are shown in FIG. In FIG. 3, control represents a culture dish group, gelatin represents a hard tissue filling material group composed of granular ceramics and gelatin hydrogel, and alginate represents a spherical ceramic composite = alginic acid group using calcium alginate. As a result, the initial cell adhesion number after 3 hours was significantly higher than that of the alginic acid group. From this, it was shown that the present invention is a bone grafting material having higher cell adhesion ability and biocompatibility than granular ceramic composites using calcium alginate.

Example 4: Examination of degradation of binder material and sustained release of drug-Sustained release test in mice Subcutaneous study of sustained release of hard tissue filling material made of granular ceramic and gelatin hydrogel prepared in Example 1 did.

Method (1) Chloramine T method was used for radiolabeling. That is, 190 μl of 0.5 M pottasium phosphate buffer (KPB: pH 7.5) was added to 10 μl of 1 mg / ml bFGF aqueous solution, and 5 μl of Na ¹²⁵ I (740 Mbq / ml in 0.1 N NaOH) was mixed. Subsequently, 100 μl of a 0.2 mg / ml chloramine T solution was added and allowed to react with stirring for 2 minutes, and then the reaction was stopped with 100 μl of 4 mg / ml sodium disulfite. The reaction product was added to the column, 1500 μl of PBS was added from above, and 500 μl of PBS was added dropwise to the column to recover the radiolabeled bFGF aqueous solution.
(2) The hard tissue filling material composed of the granular ceramic and gelatin hydrogel prepared above was impregnated with 20 μl of the radiolabeled bFGF aqueous solution obtained in (1) at 4 ° C. overnight.
(3) After measuring the γ dose of the hard tissue filling material composed of granular ceramics and gelatin hydrogel containing radiolabeled bFGF aqueous solution, 3 mice per group (ddy male mice (Japan SLC, Inc., Kyoto), 6 weeks old, body weight 22-24 g) was implanted subcutaneously in the back. Subcutaneous gelatin hydrogels were collected after 1, 4, 7, and 10 days to measure the γ dose, and the residual rate of the bFGF aqueous solution was calculated by comparing with the γ dose before implantation. The γ dose was measured with a gamma counter (ARC-301B, Aloka Co., Ltd).

Results The results are shown in FIG. The dose value decreased over time with respect to the radiation dose at the time of implantation. This confirms the release of the bFGF aqueous solution to the living tissue accompanying the degradation of gelatin by the enzyme in the living body, that is, the sustained release of bFGF from the hard tissue filling material. From this, it was confirmed that the present invention can be used as a sustained-release drug-filling material having the property of allowing sustained release of growth factors, drugs and the like that promote bone formation consisting of proteins.

Example 5: Examination of biocompatibility and degradability of binder material The biocompatibility and resolution of the hard tissue filler composed of granular ceramics and gelatin hydrogel prepared in Example 1 were examined in a rabbit ulna defect model. .

Method (1) Create a 8.0 mm full-thickness bone defect using a steel bar at the center of the ulna of a New Zealand White rabbit (male, 3.0-4.0 kg, Shimizu Experimental Materials Co., Ltd., Kyoto) The defect portion was filled with a hard tissue filling material impregnated with a bFGF aqueous solution. Thereafter, the periosteum and the skin were sutured with silk thread (mani). In addition, as a control, a defect part was prepared in the ulna, and a hard tissue filling material not containing bFGF was filled and sutured. Three rabbits were used per group.
(2) The healing period was 4 weeks, and the tissue was collected. The collected tissue was subjected to HE staining by preparing a tissue section.

Results As shown in FIG. 5, no gelatin hydrogel remained and no inflammatory changes were observed in the gap between the granular ceramics, and the formation of new bone accompanying the decomposition of the hard tissue filling material was observed to be in contact with the ceramic granules. . In FIG. 5, TCP represents β-TCP granules, and NB represents new bone.
From this result, it became clear that the present invention is a bone prosthetic material having biocompatibility and bioresorbability, which can promote tissue formation ability by rapidly decomposing in living tissue.

  INDUSTRIAL APPLICABILITY The present invention can be used as a hard tissue filling material in orthopedics and dentistry, especially as a bone filling material used for bone defects, and as a sustained drug release carrier (base material) used by being implanted in bone or subcutaneously. It is.

Photograph showing the macro structure of the material of the present invention SEM image showing the microstructure of the material of the present invention The figure which shows the difference of the cell adhesive ability of the present invention and the granular ceramic composite using calcium alginate The figure which shows the result of the sustained-release test in the mouse | mouth subcutaneous of this invention The figure which shows the degradability of gelatin and the result of bone formation in the rabbit ulna defect model of the present invention

Claims (10)

  A hard tissue filling material comprising a composite comprising a ceramic granule and a crosslinked acidic or basic gelatin hydrogel, wherein the composite is mixed with a ceramic granule and an acidic or basic gelatin hydrogel and lyophilized. Thereafter, a hard tissue filling material formed by forming a composite by a thermal crosslinking reaction.   The hard tissue filling material according to claim 1, further comprising at least one medicinal ingredient.   The hard tissue filling material according to claim 2, wherein the medicinal component is selected from a bone formation factor or a bone growth factor.   The hard tissue filling material according to claim 3, wherein the bone morphogenetic factor or bone growth factor is at least one selected from FGF, BMP, TGF-β, IGF, and PDGF.   The ceramic is hydroxyapatite, carbonate apatite, fluorapatite, chlorapatite, β-TCP, α-TCP, calcium metaphosphate, tetracalcium phosphate, calcium hydrogen phosphate, dicalcium phosphate, octacalcium phosphate, calcium hydrogen phosphate. The hard tissue filling material according to any one of claims 1 to 4, which is at least one selected from dihydrate, calcium phosphate glass, a mixture of these calcium phosphates, alumina, and zirconia.   The hard tissue filling material according to claim 5, wherein the ceramic is β-TCP.   The hard tissue filling material according to any one of claims 1 to 6, wherein the ceramic granules have an average diameter of 1 µm to 3 mm.   The ceramic granule and the acidic or basic gelatin hydrogel are contained as a volume ratio in a ratio of ceramic / gelatin aqueous solution = 1/1000 to 1/10, according to any one of claims 1 to 7. Hard tissue filling material.   A method for producing a hard tissue filling material, comprising mixing ceramic granules and acidic or basic gelatin hydrogel, freeze-drying, and then forming a composite by a thermal crosslinking reaction.   Furthermore, the manufacturing method of the hard tissue filling material of Claim 9 characterized by impregnating the said composite_body | complex with at least 1 sort (s) of medicinal component.