JP2001333974A - New method of implanting artificial bone to combine control of biocompatibility in surface of biomaterial by polishing surface and sticking of cultured osteoblasts - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an artificial bone material that is highly biocompatible by sticking cultured osteoblasts to its surface. SOLUTION: The artificial bone material is highly biocompatible by sticking cultured osteoblasts to its surface. Feature of the artificial bone material is to be stuck with the osteoblasts to surface of the material by impregnating the culture medium of the osteoblasts obtained from cultured bony tissue into a material that mainly consists of calcium phosphate ceramic glassily polished by polishing surface.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a highly biocompatible artificial bone material having osteoblasts adhered to its surface, and more specifically, to engraft and grow cultured osteoblasts on the surface of the material. According to the differentiated material, osteoblasts adhere to the surface of the existing biomaterial by mechanical polishing,
The present invention relates to a material whose biocompatibility has been improved by controlling it to a state in which it is easy to grow, and a new artificial bone graft technique using the same. The present invention provides a new artificial bone material to which autologous tissue is adhered so as to minimize complications due to immunorejection and resorption when implanting an artificial bone in orthopedic treatment.

[0002]

2. Description of the Related Art Bone transplantation has been performed as an orthopedic treatment for a wide range of bone defects associated with high-level trauma or bone resection due to a bone tumor in a living body. Currently, autogenous bone, allogeneic bone, xenogeneic bone, and artificial bone are being transplanted as a bone transplantation method. Further, cases of using artificial bone materials such as calcium phosphate-based ceramics, polymers, metals and the like which are safe in vivo as artificial bones are increasing year by year. Generally, autograft bone transplantation is desirable, but the amount of bone that can be collected is limited, and normal tissue must be invaded during transplantation. In addition, allogeneic / xenogeneic bone transplantation has immunological rejection and complications. On the other hand, ceramics, polymers,
There is a problem in that bone formation can be obtained or not obtained by implanting only an artificial bone material such as metal, and the treatment results are not uniform. Therefore, when artificial bone is used, it is expected that the healing results can be improved by intentionally attaching osteoblasts to the surface of the material and differentiating the bone cells on the surface as in the case of autologous bone. For that purpose, it is necessary to establish a method for efficiently attaching osteoblasts to the material and improving the biocompatibility of the material by forming a surface on which the cells can easily grow.

Conventionally, when a known artificial bone material is used for treatment, surface coating with highly biocompatible hydroxyapatite or the like, smoothing of joint movable parts by friction, etc.
Except for partial surface roughening for the purpose of increasing the specific surface area for enhancing the adhesiveness to the tissue, the surface treatment of the manufactured artificial bone material is hardly performed. Therefore, if various cells involved in bone regeneration in the living body can be efficiently engrafted on the surface of the material and transplanted in an easily proliferating state, the self-regenerating action of bone can be promoted, and artificial bone material in vivo , Which can be expected to result in a shorter treatment time.

[0004] However, no knowledge has been obtained as to what surface condition of each biomaterial is an environment suitable for cell proliferation and cell proliferation. If the surface of the artificial bone material can be controlled to a surface state on which cells can easily engraft and proliferate, new osteoblasts can be proliferated on the surface of the artificial bone material, and a new bone treatment using it can be performed.
Thereby, various current problems can be overcome. For that purpose, it is necessary to develop a material suitable for allowing the actually cultured osteoblasts to engraft on the surface of the artificial bone material, and to optimize the surface condition. Furthermore, since the polishing treatment of the material surface can be easily incorporated as a process in the manufacturing process, it is an inexpensive and safe control method with few problems such as delamination deterioration at the interface after coating such as coating treatment. , Its advantages are great.

[0005]

Under such circumstances, the present inventors, in view of the above-mentioned prior art, efficiently attach osteoblasts to the surface of a material of an artificial bone and facilitate cell proliferation. As a result of intensive research aimed at developing a new biocompatible material that can control the surface state, it has been optimized using materials such as calcium phosphate-based ceramic sintered compacts and hardened products with good osteoblast proliferation. The inventor obtained the knowledge that the intended purpose can be attained by achieving the objective, and further conducted further research, thereby completing the present invention. The present invention
An object is to provide a highly biocompatible artificial bone material having osteoblasts attached to the surface.

[0006]

The present invention for solving the above problems comprises the following technical means. (1) A highly biocompatible artificial bone material having osteoblasts adhered to its surface, and a culture solution of osteoblasts obtained by culturing bone tissue was polished to a mirror surface by surface polishing treatment. An artificial bone material characterized by impregnating a material containing calcium phosphate ceramics as a main component and causing osteoblasts to adhere to the surface of the material. (2) The calcium phosphate ceramic is α-Ca ₃
(PO ₄ ) ₂ , β-Ca ₃ (PO ₄ ) ₂ , Ca ₈ (HP
_{O 4) 4 5H 2 O,} Ca 10 (OH) 2 (PO 4) 6 1
The artificial bone material according to the above (1), comprising at least one species. (3) Materials mainly composed of calcium phosphate ceramics are α-Ca ₃ (PO ₄ ) ₂ , β-Ca ₃ (P
_{O 4) 2, Ca 8 (} HPO 4) 4 5H 2 O, Ca 10 (O
The composition according to the above (1), comprising a calcium phosphate-based ceramics sintered body or hardened body containing at least one of H) ₂ (PO ₄ ) ₆ and at least one of biopolymers such as soda glass and lactic acid resin, and metals. Artificial bone material. (4) Bone tissue collected from between the trochanters of the femur at the time of total hip arthroplasty was tissue-cultured for 5 to 7 weeks, and the resulting culture solution containing human osteoblasts was surface-polished to a calcium phosphate ceramic material. Is immersed for a predetermined time to allow osteoblasts to adhere to the surface.

[0007]

Next, the present invention will be described in more detail.
explain. In the present invention, (1) artificial bones currently used
Focusing on the relationship between the surface roughness of the material and the affinity with osteoblasts,
Cell production from existing artificial bone material by mechanical surface polishing
(2) To control the surface of osteoblasts
Calcium phosphate ceramic sintered body with good growth
And optimization of materials such as
are doing. Sintering and hydration hardening are used to control surface roughness.
Calcium phosphate ceramic material, that is, α
-Ca_Three(PO_Four)_Two, Β-Ca_Three(PO_Four )_Two , Ca
₈ (HPO_Four )_Four 5H_Two O, Ca_Ten(OH)_Two (PO
_Four )₆ Material containing at least one or more of
Biopolymers such as glass and lactic acid resin and metals (titanium and
Artificial bone material consisting of at least one of titanium alloys)
No. 200 or less diamond embedded polishing plate or
Rough surface polished with paper, and average 0.3μm particle size
Buffing with diamond or alumina slurry,
A smooth surface with a mirror-polished surface is used.

[0008] In general, it is important for bone growth after bone transplantation to be in an environment in which osteoblasts are likely to proliferate after cell engraftment.
Generally, it is said that the larger the specific surface area is, the larger the contact interface between the tissue fluid and the cell is, and the more likely the cell engraftment occurs. However, in practice, osteoblast engraftment occurs on the rough surface having dents of several tens to several hundreds of μm, but contact between cells after engraftment is hindered, proliferation does not proceed, and cells decrease. Was observed. In addition, on a smooth surface that has been paste-polished, large osteoblasts of several hundred μm adhere to the surface of the material from the culture solution and further proliferate in spindle and polygonal cell forms to cover the surface, increasing the number of cells. Was observed. For this reason, after the cells have adhered to the surface, a network between the cells is formed in the growth process and adheres well, so that the cells are less likely to peel off than the rough surface. From these results, it is considered that a smooth surface that does not hinder the networking of cells is more suitable for transplanting the cultured osteoblasts in a state of engraftment and proliferation in a biological material.

As a substrate for cultured osteoblasts, a hydroxyapatite (HA) and tricalcium phosphate (β-TCP) sintered body are used as artificial bone materials such as a calcium phosphate ceramic sintered body and a hardened body. In comparison, on the smooth surface by polishing, hydroxyapatite showed more proliferation after cell engraftment. This is considered to be because hydroxyapatite is similar to the component of natural bone hard tissue and hardly dissolves, so that it easily contacts cells and has high affinity with osteoblasts. In addition, tricalcium phosphate is known to have better reactivity with bone and osteoclasts than hydroxyapatite. It is thought that networking of engrafted cells is likely to be suppressed on the surface.

[0010] On the other hand, the number of osteoblasts was significantly reduced on the surface of the octacalcium phosphate (OCP) hardened material as compared with the surface of the HA or TCP sintered body. This is due to the fact that the crystals grow as disc-shaped crystal particles from the observation of the substrate surface, and even after polishing, there are many gaps on the surface and it is difficult for a smooth surface to appear, so it is difficult for cells to engraft, and osteoblast network It is considered that the cause is that it is hard to occur. From the above results, when the cultured osteoblasts are attached and proliferated and used for bone transplantation, it is better to finish the surface smoothly with HA or TCP sintered body, and rough surfaces with many uneven surfaces and scratches remain. Osteoblasts are more likely to engraft and proliferate compared to, so cultured osteoblasts engraft on the material surface,
It was found to be suitable for growing and transplanting.

In the present invention, a sintered body and a hardened body such as a calcium phosphate ceramic having a bone-like composition, and an artificial bone material currently used for medical treatment are used as a material for treating a damaged or defective bone. In this case, in order to increase the biocompatibility of the surface and further improve the healing effect, the surface of the material is controlled by a surface treatment by easy polishing so that osteoblasts involved in bone formation can be easily engrafted and proliferated. Furthermore, in order to minimize complications due to immune rejection and resorption, autologous osteoblasts are engrafted on the artificial bone material, and the artificial bone material grown on the surface is applied as a new artificial bone transplantation technology. is there.

[0012] Until now, in a sintered body or a hardened body of calcium phosphate or the like, which is expected to be used for treatment as an artificial bone, it is necessary to actively control cell engraftment on the material surface and the presence or absence of the growth state. Almost no surface treatment is performed. In the present invention, using a variety of calcium phosphate sintered bodies and hardened bodies subjected to polishing treatment with cultured osteoblasts, a material to which osteoblasts easily adhere, and surface polishing treatment conditions, for example, an average of 0.3 μm particles By using the smooth surface of a hydroxyapatite sintered body buffed with a slurry of diamond or alumina of diameter, osteoblasts are grown in close contact with the surface, and cultured autologous osteoblasts are attached, Used as artificial bone material.

In the present invention, as autologous osteoblasts, human femoral neck, trabecular bone or compact bone at the proximal end of the humerus are used. These bone tissues were cultured in a medium containing inorganic salts, amino acids, vitamins, nutrients of saccharides, antibiotics and serum at a constant temperature of about 30 ° C. in a flask and allowed to stand. Prepare a cell solution suspended by trypsinization. On the other hand, examples of calcium phosphate ceramics include hydroxyapatite, tricalcium phosphate, octacalcium phosphate, and calcium hydrogenphosphate. These are used as a sintered body obtained by uniaxial pressing, then isostatic pressing, sintered in an electric furnace, and a cured body cured by a hydration curing reaction by kneading a buffer solution with calcium phosphate powder. . As an artificial bone material, it is also possible to appropriately use a metal such as titanium, a titanium alloy, and a stainless alloy, such as soda glass, phosphate glass, lactic acid resin, and acrylic resin, in the calcium phosphate-based ceramic sintered body. However, it is not particularly limited to other materials constituting the artificial bone.

The above artificial bone is polished to a mirror surface with, for example, a diamond paste having a particle diameter of 0.3 μm, but the method and means are not particularly limited. Next, the above-mentioned cell solution was impregnated with these materials in the above-mentioned cell solution at a constant temperature of about 30 ° C. for 1 to 4 days, and osteoblasts were engrafted and proliferated on the material surface.
Let it differentiate. Thus, an artificial bone material having osteoblasts adhered to the surface is obtained.

[0015]

EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to the following examples. Example 1 According to the test method shown in FIG. 1, bone tissue (cancellous bone) collected from the intertrochanter of a human femur, which is not necessary for treatment in total hip arthroplasty, was cultured in a flask for 5 to 7 weeks. ,
2.5 × 10 5 osteoblasts detached from the flask and suspended
^The cells were cultured until they reached ⁴ cells / cm ² to prepare a cell solution.
On the other hand, there are three typical calcium phosphates:
Hydroxyapatite (HA): Ca ₁₀ (OH) ₂ (PO ₄ )
₆ , tricalcium phosphate (β-TCP): Ca ₃ (PO
₄ ) ₂ , and octacalcium phosphate (OCP): Ca _{8
(HPO 4) 4 5H 2 O} : Pellets were produced in the sintered body and the cured body of the. That is, for hydroxyapatite, calcium nitrate is mixed with H ₃ PO ₄ and NH ₄ OH
To pH 9 to synthesize a precipitate, and then 20MP
a After molding by uniaxial pressing, sintering was performed at 1150 ° C. for 4 hours by CIP to produce pellets. For the tricalcium phosphate, a commercially available β-TCP powder was used, and
After molding under uniaxial pressure of 1 MPa, 1150 ° C, 4
After sintering for a time, pellets were prepared. Furthermore, for octacalcium phosphate, a commercially available α-TCP powder was used,
The mixture was kneaded with a pH 5 sodium acetate buffer solution, trained, and then hydrated and hardened at 60 ° C. for 3 days to produce pellets. The obtained sintered body and cured body (diameter 6 mm, thickness about 2
mm pellets) were buff-polished with a # 200 abrasive paper and buffed with a 0.3 μm-diameter diamond slurry to be mirror-polished. FIG. 2 shows the X-ray diffraction pattern of the pellet sample used. The above-mentioned sample was impregnated with the above-mentioned cell solution for 1 to 4 days in a stationary state, and the cells were fixed. Thus, an artificial bone material having osteoblasts adhered to the surface was prepared. Table 1 shows the synthesis conditions of the samples used in the test of this example.

[0016]

[Table 1]

Example 2 In Example 1, the surfaces of the pellet samples before the test and the pellet samples impregnated with the osteoblast culture solution for 4 days were observed with a scanning electron microscope. The electron micrographs are shown in FIGS. In the case of the hydroxyapatite sintered body paste-polished on the smooth surface shown in FIG. 3, spindle-shaped and polygonal osteoblasts were engrafted after culturing for 4 days, and further covered the surface densely. On the other hand, in the hydroxyapatite sintered body roughly polished on the rough surface in FIG. 4, osteoblast engraftment was observed, but the number of cells and its networking were small. A similar tendency was observed in other materials due to the difference in the polishing state of the material surface (FIGS. 5 to 8). Further, among the comparative calcium phosphate sintered bodies and the cured bodies, the hydroxyapatite sintered bodies had the best affinity for osteoblasts.

Example 3 In Example 2 above, the SEM photographs of FIGS.
Cell engraftment / proliferation was compared by 100 point counting methods prepared at equal intervals in the vertical and horizontal directions. FIG. 9 shows the result. From these results, it was also observed that the adherence of osteoblasts to the mirror-polished surface of hydroxyapatite was good, and that the survival rate of cells decreased in the order of tricalcium phosphate and octacalcium phosphate. Was.

[0019]

As described above in detail, the present invention provides a highly biocompatible artificial bone material having osteoblasts adhered to the surface thereof, wherein the osteoblast is obtained by culturing bone tissue. The artificial bone material is characterized in that the liquid is impregnated in a material mainly composed of a calcium phosphate ceramic polished to a mirror surface by a surface polishing treatment, and osteoblasts are attached to the surface of the material. According to the present invention, 1) an artificial bone material having autologous bone cells adhered to the surface can be obtained. 2) An artificial bone material having improved biocompatibility can be provided.
The artificial bone material can be produced by a simple manufacturing process, and 4) a new artificial bone grafting technique can be provided.

[Brief description of the drawings]

FIG. 1 is an explanatory diagram showing a test method of an embodiment in a flowchart.

FIG. 2 shows an X-ray diffraction (XR) of a sample used in a test of an example.
D) It is explanatory drawing which showed the pattern.

FIG. 3 shows an electron on the surface of a test sample before and after impregnation with an osteoblast culture solution for a hydroxyapatite sintered body mirror-polished by diamond paste polishing used in the test of the example. It is a microscope picture.

FIG. 4 shows the surface of the test sample before and after the impregnation of the rough hydroxyapatite sintered body prepared by polishing with a No. 200 abrasive paper used in the test of the example and in the osteoblast culture solution for 4 days. Electron micrograph.

FIG. 5 shows the surface of a test sample before and after impregnation with an osteoblast culture solution for 4 days before the test of the tricalcium phosphate sintered body mirror-polished by the diamond paste polishing used in the test of the example. It is an electron micrograph.

FIG. 6 shows a test sample of a roughened tricalcium phosphate sintered body prepared by polishing with a No. 200 abrasive paper used in the test of the example before the test and after impregnation with the osteoblast culture solution for 4 days. It is an electron micrograph of the surface.

FIG. 7 shows the surface of a test sample before and after impregnation with an osteoblast culture medium for 4 days before the test of the sintered body of octacalcium phosphate mirror-polished by mirror polishing with diamond paste used in the test of the example. It is an electron micrograph.

FIG. 8 is a test sample of a rough octacalcium phosphate sintered body prepared by polishing with a No. 200 polishing paper used in the test of the example before the test and after impregnation with the osteoblast culture solution for 4 days. It is an electron micrograph of the surface.

FIG. 9 is an explanatory diagram showing a comparison of the amount of osteoblasts engrafted / proliferated on the surface of each sample by the point counting method from electron micrographs used for sample observation in the tests of Examples.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI theme coat ゛ (Reference) A61F 2/32 A61F 2/32 (72) Inventor Kaji Koji 16-11 Yagiyama Midoricho, Taishiro-ku, Sendai City, Miyagi Prefecture −24 F term (reference) 4C081 AB03 AB05 BA12 CD34 CF131 CG02 CG03 CG05 DA16 DC03 EA02 EA06 4C097 AA04 BB01 DD05 DD06 DD07 DD09 DD10 DD15 FF17 MM02 MM04 SC01

Claims (4)

[Claims] 1. A highly biocompatible artificial bone material having osteoblasts adhered to its surface, wherein a culture solution of osteoblasts obtained by culturing bone tissue is mirror-polished by surface polishing treatment. An artificial bone material characterized by impregnating a polished calcium phosphate-based ceramic-based material as a main component and allowing osteoblasts to adhere to the surface of the material. 2. The method according to claim 1, wherein the calcium phosphate-based ceramic is α
—Ca ₃ (PO ₄ ) ₂ , β-Ca ₃ (PO ₄ ) ₂ , Ca
₈ (HPO ₄ ) ₄ 5H ₂ O, Ca ₁₀ (OH) ₂ (P
O _{4) 6} artificial bone material according to claim 1, further comprising one or more. 3. A calcium phosphate-based ceramic material.
Α-Ca_Three(PO_Four)_Two, Β-Ca_Three
(PO_Four)_Two , Ca₈(HPO_Four)_Four5H_Two O, Ca_Ten
(OH)_Two (PO_Four )₆ Containing at least one of
A calcium phosphate-based ceramics sintered body or cured body;
At least one of biopolymers such as soda glass and lactic acid resin, and metals
The artificial bone material according to claim 1, comprising: 4. A calcium-phosphate-based calcium phosphate solution obtained by culturing bone tissue collected between the trochanters of the femur during total hip arthroplasty for 5 to 7 weeks, and subjecting the resulting culture solution containing human osteoblasts to surface polishing treatment. The artificial bone material according to claim 1, wherein the ceramics material is immersed for a predetermined time to allow osteoblasts to adhere to the surface.