Patent Application
20100168868
The present invention relates to a layered gradient material which comprises hydroxyapatite (hereinafter referred to as “HAp” and tribasic calcium phosphate (TCP) and is used as a biological material such as a bone substitute material, artificial tooth root or dental cement, and a method for producing the layered gradient material.
In the field of oral-maxilla surgery, loss of bones sometimes occurs due to external wound or surgical operation such as removal of tumors or cysts formed at maxilla bones. Furthermore, in implantation treatment or production of artificial tooth, there are many cases which need increase of alveolar bones, bone transplantation and implantation of bone substitute material. As transplantation materials used for these substitution and reconstruction, mostly fresh self bones are used because bones can be bonded thereto satisfactorily, surely and safely. However, fresh self bones for transplantation have problems that extra surgical invasion is required for gathering transplantation bones in addition to the operation of primary focuses and that there are limitations in supply and shape of bone tissue.
Under the circumstances, development of biological materials to be substituted for self bone transplantation materials has been made. W. R. Brown et al (Patent Document 1) developed self-curing cement comprising calcium phosphate (calcium phosphate cement which is hereinafter referred to as “CPC”), and Fukase et al (Non-Patent Document 1, Non-Patent Document 2, Non-Patent Document 3, Non-Patent Document 4, Non-Patent Document 5, and Non-Patent Document 6) reported that they made improvement in said cement to improve curing reaction and physical properties.
However, said bone substitute material in the form of cement is pasty until curing, and hence it has a degree of freeness in filling, but since strength cannot be assured for a period until curing (24 hours at 38° C.), there is restriction in reinforcement of the site which requires strength, such as body trunk bone. Therefore, block-like bone substitute materials are useful, and conventional dense body or porous body of HAp can ensure strength, but is low in reactivity with bone since it is excellent in biological affinity, and replacement for self bone can hardly be expected. When reactivity with bone is demanded, it is effective to use calcium phosphate such as β-phase TCP (hereinafter referred to as “β-TCP” or “βTCP” which is high in solubility, but β-TCP is inferior to HAp in mechanical strength. Thus, there has been no material which satisfies both strength and reactivity.
Real bones have such features that they comprise dense bone of outer side and have shell structure to exhibit strength and the inner side shows biological reactivity due to bone marrow tissue. Development of artificial materials which satisfy both the mechanical strength and biological reactivity has been desired.
Patent Document 1: U.S. Pat. No. 4,518,430
Non-Patent Document 1: Y. Fukase et al., J. Dent Res 1990; 69(12); 1852-1856
Non-Patent Document 2: Y. Fukase, “Nichidai Shika (Nihon University Dentistry)”, 1990; 64: 190-203
Non-Patent Document 3: M. Ogawa, “Nichidai Shika (Nihon University Dentistry)”, 1995; 69: 561-570
Non-Patent Document 4: H. Uehara, “Nichidai Shika (Nihon University Dentistry)”, 1995; 69: 728-744
Non-Patent Document 5: Y. Fukase et al., J. Oral Sci 1998; 40(2): 71-76
Non-Patent Document 6: S. Wada, “Nichidai Shika (Nihon University Dentistry)”, 1998; 72: 408-420
Accordingly, the object of the present invention is to provide an artificial material which have the same mechanical strength and biological reactivity as those of real bones, and a method for producing the same.
In an attempt to solve the above problems, the inventors have considered that according to spark plasma sintering method (SPS method), there can be produced a gradient functional material by changing compounding ratio of a plurality of materials or chemical properties of the materials, and hence there can be produced a material having functions of gradient in composition, biological reactivity and strength changing from the portion comprising 100% of HAp having strength to the portion comprising 100% of β-TCP having biological reactivity in the same specimen by changing the compounding ratio of HAp high in strength, but low in biological reactivity and β-TCP high in biological reactivity, but low in strength. Under the circumstances, they have conducted intensive research on production of gradient materials by SPS method. As a result, the present invention has been accomplished.
Thus, the present invention includes the following inventions (1) to (12).
(1) A layered gradient material for biological use which comprises hydroxyapatite (HAp) and tribasic calcium phosphate (TCP), the compounding ratio of HAp and TCP being changed stepwise or continuously;
(2) a layered gradient material for biological use described in the above (1), wherein TCP is β-phase TCP (β-TCP);
(3) a layered gradient material for biological use described in the above (2), wherein β-TCP has a particle size of 590 μm to 1000 μm;
(4) a layered gradient material for biological use described in any one of the above (1) to (3), wherein HAp has a particle size of 10 μm to 170 μm;
(5) a layered gradient material for biological use described in any one of the above (1) to (4) which is obtained by stacking a plurality of layers in which the compounding ratio of HAp and TCP is stepwise or continuously changed and then sintering the layered body by a pressure sintering method;
(6) a layered gradient material for biological use described in any one of the above (1) to (5) which is used as a bone substitute material;
(7) a method for producing a layered gradient material for biological use which comprises stacking a plurality of layers composed of a mixture of HAp and TCP in which the compounding ratio of HAp and TCP is stepwise or continuously changed and then sintering the resulting layered body by a pressure sintering method;
(8) a method for producing a layered gradient material for biological use described in the above (7), wherein the layered body is sintered by spark plasma sintering method (SPS method);
(9) a method for producing a layered gradient material for biological use described in the above (7) or (8), wherein a plurality of layers composed of a mixture of HAp and TCP in which the compounding ratio of HAp and TCP is stepwise or continuously changed are filled up in a frame and stacked in vertical direction and then the resulting layered body is sintered by SPS method;
(10) a method for producing a layered gradient material for biological use described in any one of the above (7) to (9), wherein TCP is β-TCP;
(11) a method for producing a layered gradient material for biological use described in the above (10), wherein β-TCP has a particle size of 590 μm to 1000 μm;
(12) a method for producing a layered gradient material for biological use described in any one of the above (7) to (11), wherein HAp has a particle size of 10 μm to 170 μm; and
(13) a method for producing a layered gradient material for biological use described in any one of the above (7) to (12) which is used as a bone substitute material.
The layered gradient material for biological use according to the present invention is excellent in mechanical strength such as compressive strength, bending strength and Young's modulus, and further, is excellent in biological reactivity such that in the case where it is used as a bone substitute material, bone destruction and new bone reconstruction actively occur. Accordingly, it is extremely useful as a biological material such as a bone substitute material, artificial tooth root and dental cement.
HAp used in the present invention is a compound represented by Ca10(PO4)6(OH)2 and is a main component constituting a bone. Particularly, HAp has a particle size of preferably 10 μm to 170 μm, more preferably 20 μm to 60 μm. The shape thereof is preferably spheroidal. As preferred examples of HAp, mention may be made of, for example, SHAp-100 (HAp prepared by spheroidizing HAp having the same component as of commercially available HAp-100 which has a particle size of 40 μm and is manufactured by Taihei Kagaku Kogyo Co., Ltd.).
TCP used in the present invention means tribasic calcium phosphate (Ca3(PO4)2), and is similar to HAp in physical properties, solubility and biological affinity. TCP includes high-temperature type α-phase (α-TCP), low-temperature type β-phase (β-TCP) and high-temperature and high-pressure type γ-phase (γ-TCP) which differ in crystal structure, and α-phase TCP and β-phase TCP are largely used as biological materials. α-TCP is high in solubility in water and is converted to HAp upon hydrolysis reaction. Furthermore, β-TCP is also relatively high in solubility in water and is superior in biological affinity, and is largely used for bone filling materials as biologically absorptive implantation materials. In the present invention, there may be used any of α-TCP, β-TCP and β-TCP, and β-TCP is particularly preferred. The particle size of β-TCP is preferably 590 μm to 1000 μm, and β-TCP is preferably in the shape of ground particles.
In the present invention, there may be optionally used apatite hydrogencarbonate, apatite fluoride, titanium, or the like in addition to HAp and TCP.
The layered gradient material for biological use of the present invention has such a construction that the compounding ratio of HAp and TCP is stepwise or continuously changed. More specifically, as shown in, for example,
As mentioned above, the gradient material of the present invention is obtained by stacking a plurality of layers composed of a mixture of preferably HAp and TCP in which the compounding ratio of HAp and TCP is stepwise or continuously changed to produce a layered body and then sintering the resulting layered body by a pressure sintering method. The number of the layers stacked is not limited to the above 5 layers, and the layered body may be composed of an optional number of layers, and, for example, it may be a layered body composed of a first layer comprising 100% of TCP and a second layer comprising 100% of HAp, a layered body composed of a first layer comprising 100% of TCP, a second layer comprising 50% of TCP and 50% of HAp in volume ratio and a third layer comprising 100% of HAp, a layered body composed of a first layer comprising 100% of TCP, a second layer comprising 80% of TCP and 20% of HAp in volume ratio, a third layer comprising 60% of TCP and 40% of HAp in volume ratio, a fourth layer comprising 40% of TCP and 60% of HAp in volume ratio, a fifth layer comprising 20% of TCP and 80% of Hap in volume ratio, and a sixth layer comprising 100% of HAp. Preferably, the layered body is composed of about 2-40 layers.
In order to form a layer comprising HAp and TCP, powdery mixtures of HAp and TCP may be mixed well using, if necessary, a pot mill rotating table. Then, layers having the respective compounding ratio are stacked and the resulting layered body is sintered by pressure sintering method. The pressure sintering method includes, for example, hot press (HP) method of carrying out the sintering while pressing the powders, hot hydrostatic pressing (HP) method of carrying out the sintering while omnidirectionally pressing the powders under equal pressure, normal pressure sintering method of carrying out the sintering under normal pressure a molded body previously formed by cold hydrostatic pressing (CIP), or the like, and in the present invention, SPS method (spark plasma sintering method) is preferred. This SPS (spark plasma sintering) method is a technology of synthesis and working of materials of the next generation type capable of performing the sintering in a short time of about 5-20 minutes including heating time and holding time in a region of from low temperature to ultra-high temperature of higher than 2000° C. by directly introducing electric energy in the form of pulse into voids of the pressed powder particles, whereby high energy of high-temperature plasma, namely, spark plasma generated in a moment by spark discharge is effectively applied to heat diffusion and electric field diffusion (Masao Tokita: Journal of Society of Particle Technology, Commentary 30[11] p. 790-804 (1993); New Ceramics, No. 10, p. 43-51, 1997). The spark plasma sintering method is a kind of pressure sintering method using ON-OFF direct current pulse passing method, by which dense sintered bodies can be obtained at a lower temperature and in a shorter time than in conventional hot press (HP) method and hot hydrostatic pressing (HP) method. Furthermore, the spark plasma sintering method is of a direct heat generation system utilizing discharge and Joule's heat at the time of passing great current pulse, and hence is extremely superior in thermal efficiency. Thus, uniform and high quality sintered bodies can be obtained by uniform heating caused by dispersion of the points of the discharge and Joule's heat.
In order to sinter the layered body by SPS method, mixtures of HAp and TCP at the respective compounding ratios are filled in a frame such as die•punch made of high-strength graphite to form layered bodies as shown in FIGS. 2,4 and 7. The shape of the frame may be selected according to the shape of gradient materials to be obtained, and it may be in the form of column, square pillow or the like. The frame is preferably a mold made of high-strength graphite. Then, the apparatus system is allowed to be in vacuous atmosphere or in non-oxidizing atmosphere such as nitrogen gas, argon or the like, and the layered bodies can be sintered by heating with passing pulsed direct current or direct current containing square wave or firstly passing pulsed direct current and then passing direct current containing square wave through the frame containing the layered body. As the spark plasma systems, there are two kinds of systems one of which passes only direct current (available mainly from Sumitomo Coal Mining Co., Ltd.) and another of which passes pulsed direct current for the first period of 0-750 seconds and thereafter passes direct current containing square wave (available mainly from Sodic Co., Ltd.). In the present invention, the spark plasma systems where heating is carried out by passing direct current are preferred.
As the sintering conditions in the present invention, preferred are, for example, a filling pressure in a frame of 5-20 MPa, a heating rate of 50-300° C./min, a sintering temperature of 700-1100° C., a sintering time of 2 minutes or longer, and sintering pressure of 20-75 MPa. After completion of the sintering, the layered body may be cooled at a controlled cooling speed of 80-150° C./min, or may be naturally cooled upon stopping operation of the apparatus. Thus, the layered gradient material for biological use of the present invention can be obtained.
The layered gradient material for biological use of the present invention can be applied to, for example, artificial bones, artificial tooth roots and artificial joints after being formed into suitable shapes depending on uses.
The present invention will be explained in detail below by the following examples, which should not be construed as limiting the invention.
As HAp, there was used SHAp-100 (having a particle size of 40 μm manufactured by Taihei Kagaku Kogyo Co., Ltd.) which was high in mechanical strength, and as β-TCP, there was used β-TCP(L) (β-TCP having a particle size of 590-1000 μm manufactured by Taihei Kagaku Kogyo Co., Ltd.) capable of realizing a porous material into which osteoblasts can enter.
The given amount of powders as shown in Table 1 were mixed using a pot mill rotating table (Model: AN3S manufactured by Nitto Kagaku Co., Ltd.). The volume ratio in Table 1 is assumed to be a volume ratio after sintering.
In this Example, the powders were not mixed using a mortar because β-TCP(L) having large particle diameter was not to be ground. The powders were filled in such a manner that those of the layer comprising 100% of β-TCP were filled as the uppermost layer and then those of the following layers were filled in succession as shown in
The section in longer direction of the specimen after sintered was photographed using SEM and shown in
(1) Compression Test
Columnar specimens were prepared using the same SHAp-100 and β-TCP(L) as used in Example 1 using a mold made of high-strength graphite as shown in
The specimen obtained by sintering was subjected to compression test according to JIS. The results obtained are shown in
(2) Bending Strength Test
A plate specimen was prepared using the same SHAp-100 and β-TCP(L) as used in Example 1 using a mold made of high-strength graphite shown in
The specimen obtained by sintering was subjected to bending strength test specified in JIS. The results obtained are shown in
(3) Young's Modulus Test
Young's modulus of the specimen used in the bending strength test was measured. The results obtained are shown in
(1) Production of Gradient Material
In the same manner as in Example 1, SHAp-100 (having a particle size of 40 μm manufactured by Taihei Kagaku Kogyo Co., Ltd.) was used as HAp, and β-TCP (L) (β-TCP having a particle size of 590-1000 μm manufactured by Taihei Kagaku Kogyo Co., Ltd.) was used as β-TCP, and these powders were filled in a die•punch made of high-strength graphite shown in
The specimen after sintered was formed into a square pillar of 5×5×20 mm as shown in
(2) Biological Test
1) Gradient Material
As shown in
As a control, a cylindrical SPS sintered body of 20 mm in diameter×20 mm in length comprising 100% of SHAp-100 was produced, and a square pillar of 5×5×20 mm was cut out from the sintered body and was subjected to dry air sterilization for implantation in a thighbone of house rabbit.
2) Experimental Animals
Five female house rabbits (Japanese White having a body weight of about 3.0 kg available from Sankyo Labo Service) which were bred for about two weeks in a feeding test facility were used as experimental animals.
3) Implantation in Bone
The house rabbit was subjected to intravenous injection with pentobarbital sodium (NEMBUTAL manufactured by Sankyo Co., Ltd.) from veins of ear. The thighbone part of the house rabbit was subjected to local anesthesia with lidocaine chloride (2% Xylocalne E, AstraZeneca). The part was dissected until reaching periosteum, and the skin periosteum was peeled with dull knife to expose the portion around the joint of thighbone. An insertion hollow of 5.0 mm square was made under pouring of water, and the SPS gradient functional material was inserted in bone marrow cavity. The periosteum was subjected to intradermal suturing with absorptive suturing yarns (VICRIL 3-0, Ethicon), and the flap was sutured with nylon yarns (NESCOSUTURE, Nippon Shoji Co., Ltd.). As the control group, a gradient material comprising only AHAp was inserted, the periosteum was subjected to intradermal suturing with absorptive suturing yarns, and the flap was sutured with nylon yarns.
After breeding for 1, 3 months from the implantation of the gradient material, excess pentobarbital sodium was intravenously injected to kill the house rabbits, and the gradient material was extracted including the bone around the filled-up part. The extracted material was fixed with 10% neutral formalin solution for 2 weeks and used for preparation of tissue slice. The thighbones extracted with lapse of time were subjected to decalcification for 2 days with a rapid decalcifying solution (Decalcifying Solution A, WAKO) of Plank Rychlo formulation, then neutralized for a half day with 5% aqueous sodium sulfate solution, and embedded in paraffin by usual method to prepare a thin slice, which was double-stained with hematoxylin and eosine and used for histopathological retrieval. The animal experiment was conducted in accordance with the guideline for animal experiments.
4) Results
4)-1
4)-2
4)-3
4)-4
4)-5
Outside the layer of fibrous connective tissue, there were bone marrow-derived tissue, and lamellar bone (d), osteoclast (e), fat cell (f) and the like were seen, and destruction and reconstruction of bone vigorously occurred. Particularly, since lamellar radius had irregular shape with many branches, it can be seen that reconstruction of bone vigorously occurred. There were recognized images of tissue higher in reactivity than that of the control.
4)-6
4)-7
The above results show that in the case of HAp used as control, there were no inflammatory cell infiltration, but reactivity in new bone reconstruction was very low. On the other hand, in the case of using the gradient material, the tissues contacting with test material differed depending on the difference in compounding ratio of HAp and β-TCP(L) granules. As in the case of the control, the portion contacting with HAp was encapsulated with fibrous connective tissue, but in the portion contacting with β-TCP(L) granules where β-TCP was contained in a large amount, there were recognized images showing vigorous destruction and reconstruction of bones. That is, many osteoclasts appeared, and osteoblasts were present near the osteoclasts and they destructed the bone. However, since there were distributed lamellar bones having irregular shape, it can be seen that bone reconstruction also occurred. Fat cells which were considered to be derived from bone marrow cells were juvenile, and hence were considered to be in the state high in reactivity before maturation.
As explained above, the layered gradient material for biological use according to the present invention is excellent in mechanical strength such as compressive strength, bending strength and Young's modulus, and further, is excellent in biological reactivity such that in the case where it is used as a bone substitute material, bone destruction and new bone reconstruction actively occur. Accordingly, it is extremely useful as a biological material such as a bone substitute material, artificial tooth root and dental cement.
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| Number | Date | Country | Kind |
|---|---|---|---|
| 2006-323181 | Nov 2006 | JP | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/JP2007/073040 | 11/29/2007 | WO | 00 | 12/30/2009 |
