JP3695511B2 - Bone regeneration material - Google Patents

Description

【００２２】
培養時間を増やしていった際におけるポリリン酸ナトリウム塩のアルカリ性フオスフアタ−ゼ活性の経時変化を図９に示す。
【００２３】
【比較例１】
実施例４で使用したポリリン酸ナトリウム塩（平均鎖長75）の代わりに、リン酸ナトリウム緩衝液を、リン酸濃度に換算してポリリン酸と同濃度培養液に加えて所定時間培養し、アルカリ性フオスフアタ−ゼ活性を測定した。
リン酸ナトリウム緩衝液のアルカリ性フオスフアタ−ゼ活性の経時変化を図９に示す。
図９から明らかなように、ポリリン酸処理した実施例４の細胞は、１週間後から顕著な酵素活性の上昇がみられたが、リン酸緩衝液で処理した比較例１の細胞は顕著な酵素活性の上昇はみられなかった。すなわち、細胞にポリリン酸を添加することによって骨芽細胞の分化（骨形成）が促進されたことを示している。
【００２４】
【発明の効果】
本発明の骨再生材料を、物理的衝撃による骨折や損傷、外科手術によって損傷した骨の治療に、新生骨の形成を促進し、治癒、回復にいたるまでの時間を短縮することができる。
【図面の簡単な説明】
【図１】 滅菌水をしみこませたコラ−ゲンテ−プで処理した１週間後の大腿骨の穴の顕微鏡の実体を示す図面に変わる写真である。
【図２】 ポリリン酸水溶液をしみこませたコラ−ゲンテ−プで処理した１週間後の大腿骨の穴の顕微鏡の実体を示す図面に変わる写真である。
【図３】 滅菌水をしみこませたコラ−ゲンテ−プで処理した１週間後の大腿骨の穴における新生骨再生の様子を組織学的に顕微鏡で観察した図面に変わる写真である。
【図４】 ポリリン酸水溶液をしみこませたコラ−ゲンテ−プで処理した１週間後の大腿骨の穴における新生骨再生の様子を組織学的に顕微鏡で観察した図面に変わる写真である。
【図５】 滅菌水をしみこませたコラ−ゲンテ−プで処理した２週間後の大腿骨の穴における新生骨再生の様子を組織学的に顕微鏡で観察した図面に変わる写真である。
【図６】 ポリリン酸水溶液をしみこませたコラ−ゲンテ−プで処理した２週間後の大腿骨の穴における新生骨再生の様子を組織学的に顕微鏡で観察した図面に変わる写真である。
【図７】 滅菌水をしみこませた骨粉砕物で処理した２週間後の大腿骨の穴における新生骨再生の様子を組織学的に顕微鏡で観察した図面に変わる写真である。
【図８】 ポリリン酸水溶液をしみこませた骨粉砕物で処理した２週間後の大腿骨の穴における新生骨再生の様子を組織学的に顕微鏡で観察した図面に変わる写真である。
【図９】 ポリリン酸ナトリウム塩およびリン酸ナトリウム緩衝液のアルカリ性フオスフアタ−ゼ活性の経時変化。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a bone regeneration material for promoting the formation of new bone tissue, and particularly relates to a bone regeneration material using polyphosphoric acid as the material.
[0002]
[Prior art]
Bone is a specific, hardened bone derived from connective tissue consisting of cells and extracellular matrix, and differentiated from other connective tissue in the bone matrix. Its main constituent is calcium phosphate composed of hydroxyapatite crystals (Ca ₁₀ (PO ₄ ) ₆ (OH) ₂ ). Since bone is a very hard tissue that supports physical stress and protects bone from stress, bone loss or damage due to fractures or pathological changes leads to lack of human ability and wasted time. Currently, when a bone is removed for some reason, it is connected with an artificial bone, or another bone of the body is used for resubstitution.
[0003]
In addition, for the treatment of bone damage (fractures) caused by physical impact and bone damage caused by surgery, various types of prosthetic devices, including artificial bones, artificial joints for bones, immobilization and fixation of fractures It takes a long time for the bone to regain its original shape and function, and the physical and mental stress of the patient is quite large. Also, the longer the course to cure, the greater the chances of being exposed to the risk of bacterial infection and the risk of not being completely cured. Various materials that can function as artificial fillers for bone repair, such as ceramics, composite materials, bone derivatives and natural and synthetic polymers, have been studied as current selection materials for skeletal augmentation and reconstruction. ing.
[0004]
In the case of teeth, the alveolar bone that supports the teeth is the basis for tooth replacement in the treatment of bone fractures or loss in the maxillofacial region or pathological / physical wounds. Alveolar bone is susceptible to bacterial infection and is difficult to regenerate to its original level once infected or killed. In general, transplantation in which a titanium-based metal implant is inserted into the jawbone to construct an artificial tooth is beneficial for the loss of the original tooth, but this transplantation is used to support the body surrounding the implant. Is inadequate and causes excessive occlusal forces in adjacent bone structures and has not always been successful. As a treatment method to solve these problems, there is a method of promoting tissue / bone regeneration, and demineralized bone, hydroxyapatite, and other transplant substitutes to promote bone regeneration in the defective bone region. Although it has been used, sufficient results have not been achieved.
[0005]
The ideal filler or graft material is biocompatible, sterilizable, osteoinductive activity (stimulation that phenotypes mesenchymal cells into osteoblasts for bone formation) and osteoconductive activity (lattice for new bone formation) As a function). The filler is preferably biodegradable, has no immunogenicity, and is not toxic to the body tissue. However, in reality, there are no materials that satisfy all of these requirements, but only partially.
[0006]
[Problems to be solved by the invention]
The object of the present invention is to overcome the disadvantages of these conventional bone treatment means or alternative bone materials, and promotes the formation of new bone and shortens the time until healing and recovery. It is to provide a bone regeneration material that can be used.
[0007]
[Means for Solving the Problems]
That is, the present invention is a bone regeneration material for promoting new bone tissue formation containing a linear condensed polyphosphate or / and a polyphosphate.
In the bone regeneration material according to the present invention, the material for promoting regeneration of damaged bone is a bone regeneration material in which a polyphosphate is contained in a base material made of a biocompatible material. .
Furthermore, the present invention is the bone regeneration material according to the above-mentioned bone regeneration material, wherein the material for promoting the formation of new bone tissue is an orthopedic filler containing polyphosphoric acid.
Furthermore, the present invention provides a bone regeneration material obtained by mixing a natural substance containing an osteoinductive protein or / and an osteoinductive factor in polyphosphoric acid.
[0008]
DETAILED DESCRIPTION OF THE INVENTION
Examples of polyphosphoric acid include linear condensed polyphosphoric acid obtained by dehydration condensation of orthophosphoric acid. Particularly, the general formula is represented by (P _n O _{3n + 1} ) ^{(n + 2)} H, and two or more PO ₄ tetrahedra are apexes. linear condensed polyphosphate sharing of oxygen atoms to the continuous structure to a linear thereof. Polyphosphate has a molecular structure in which hydrogen of hydroxyl group of polyphosphoric acid is substituted with metal. Examples of the metal include sodium and potassium. n is 15-2000.
[0009]
Examples of the substrate made of a biocompatible material include sheets, films, fibers, porous bodies, and the like made of biocompatible synthetic polymers, natural materials, and the like. Polyphosphoric acid is mixed with these materials, coated on the surface of a substrate, or impregnated into fibers and porous materials. Synthetic polymers include non-bioabsorbable polymers such as polypropylene, polyethylene, polyvinyl chloride, polyester, polycarbonate, cellulose, polyamide, polyfluorinated ethylene, polyurethane, etc., or polyglycolic acid, polylactide And bioabsorbable polymers such as collagen, polyvinyl alcohol, polyvinylpyrrolidone, polyamino acid, polycaprolactone, polydioxanone, pinyl acetate and unsaturated carboxylic acid copolymer, and the like. Polyphosphoric acid is contained in these biocompatible materials. The content of polyphosphoric acid contained in the biocompatible material is 0.001 to 20% by weight, preferably 0.005 to 10% by weight, and more preferably. Is 1 to 5% by weight. If the polyphosphoric acid content exceeds 20 % by weight, the cells tend to be necrotic, and if it is less than 0.001% by weight, the bone regeneration effect is small.
[0010]
The orthopedic filler is an artificial bone component used as a bone filler in the orthopedic field, and examples thereof include hydroxyapatite, dicalcium phosphate, tricalcium phosphate, and tetracalcium phosphate. In addition, examples of osteoinductive proteins include Bone Morphogenetic Proteins such as BMP-1, BMP-2, and BMP-3, Transforming Growth Factors such as TGF-β, Osteopontin, and Osteocalcin. Etc. Furthermore, examples of natural substances containing osteoinductive factors include animal bone pulverized products and mineral-depleted bone matrix. Natural substances containing osteoinductive factors can be mixed with polyphosphoric acid in combination with osteoinductive proteins and administered locally as implants or devices. In this case, the administration substance is encapsulated or injected in a viscous form suitable for delivery to the bone injury site in a physiologically acceptable form, free from pyrogens. Thereby, a bone and cartilage structure is formed at the site of bone damage, and a matrix that can be resorbed into the body in an optimal state is formed.
[0011]
The bone regeneration material of the present invention is used as an osteogenic preparation containing polyphosphoric acid for preventive uses such as closed fractures, reduction of complicated fractures, and improvement of fixation of artificial joints. In addition, new osteogenesis is induced by osteogenic preparations, which are used for congenital, traumatic, tumor repair, and for restoration of defects, for cosmetic plastic surgery, treatment of periodontal disease, other dental restoration processes, etc. Or used. Furthermore, the bone regeneration material of the present invention is used by coating a material containing polyphosphoric acid on the surface of a base material made of a biocompatible material such as a sheet, a film, a fiber, or a porous material. The
[0012]
【Example】
Hereinafter, an example of the present invention will be described with reference to examples.
[Example 1]
A white rabbit (New Zealand, 3 kg body weight) was injected with anesthesia to expose the femur. Using a sterilized drill (3 mm in diameter), two holes were drilled near the femoral head and the condyle at the end of the femur until the tip of the drill reached the soft tissue in the femur. A 1 cm × 1 cm collagen tape (CollaTape, manufactured by Calcitec) was impregnated with 30 μl of a 2% aqueous solution of sodium polyphosphate (average chain length 75) and dried aseptically. The collagen tape was packed into a hole in the femur of the right leg of the white rabbit. In addition, a collagen tape soaked with sterilized water as a control product was packed in a hole formed in the femur of the left leg. In this state, the incised portion was sutured, and the state of new bone regeneration in the femoral hole after 1 week and 2 weeks was observed. The femur removed for observation was fixed with 10% formalin.
[0013]
FIG. 1 and FIG. 2 are the ones after one week, in which the hole is cut in the vertical direction and observed with a stereomicroscope. FIG. 1 shows a treatment with a collagen tape soaked with sterilized water, and FIG. 2 shows a treatment with a collagen tape soaked with a polyphosphoric acid aqueous solution. No formation of new bone was observed in the collagen tape soaked with sterilized water in FIG. 1, but the collagen tape soaked with the polyphosphate aqueous solution in FIG. 2 corresponds to the periphery of the bottom edge of the hole. It was observed that an amount of new bone was formed and extended to soft tissue.
[0014]
Using the collagen tape soaked with the same sterile water and the collagen tape soaked with the polyphosphate aqueous solution as described above, regeneration of new bone in the femoral hole after 1 and 2 weeks is necessary. For histological observation, a part of the tissue sample of the femoral hole was taken out and treated with 10% EDTA for 2 months for decalcification. The decalcified samples were dehydrated with various concentrations of ethanol and xylene as a final step and embedded in paraffin. The embedded sample was cut at a thickness of 5 μm, stained with Azan, and observed under a microscope. The tissue state after 1 week was treated with a collagen tape soaked with sterilized water, and FIG. 4 shows the tissue treated with a collagen tape soaked with a polyphosphoric acid aqueous solution.
[0015]
In the collagen tape soaked with sterilized water, the collagen tape (C) is covered with the fibrous tissue (F) as shown in FIG. 3, and the dense bone (B) The trabecular bone (TB) from the endosteum was approaching its fibrous tissue. In contrast, in the case of FIG. 4 treated with a collagen tape soaked with an aqueous polyphosphoric acid solution, the collagen tape was almost absorbed and replaced with a fibrous structure (F). In addition, immature fibrous bone trabecular clumps (*) derived not from the dense bone (B) but derived from the new fibrous tissue in the hole were already confirmed at six locations in the fibrous tissue.
[0016]
Next, with regard to the state after 2 weeks, FIG. 5 shows that treated with a collagen tape soaked with sterilized water, and FIG. 6 shows that treated with a collagen tape soaked with a polyphosphoric acid aqueous solution. It was shown to. As shown in Fig. 5, the collagen tape treated with sterilized water is connected to both ends of the hole by the trabecular bone (TB), but the collagen tape (C) is completely The bone trabeculae (TB) derived from the endosteum surrounded the collagen tape (C) and formed a callus bone. On the other hand, the collagen tape treated with the collagen tape soaked with the polyphosphoric acid aqueous solution was almost completely absorbed as shown in FIG. 6 and could not be confirmed. In addition, bone trabeculae, which are newly formed primary bones, adhered to each other and replaced with collagen tape. Bone trabeculae (*) derived from neoplastic tissue replaced with collagen tape are combined with bone trabeculae derived from endosteum, and callus is formed by bone trabeculae of two different origins, They repaired the hole by connecting the ends of the hole.
[0017]
[Example 2]
In the same manner as in Example 1, a femur of white birch was punctured and demineralized human bone crushed material (particle size 250-300 μm) 10 mg 2% sodium polyphosphate (average chain length 75) aqueous solution 30 μl And aseptically dried. The bone crush was pinched in a hole in the femur of the right leg of the white rabbit. Further, as a control, a pulverized bone soaked with sterilized water was packed in a hole formed in the femur of the left leg of the white rabbit. In this state, the incised portion was sutured, and the appearance of new bone regeneration in the femoral hole two weeks later was observed histologically in the same manner as in Example 1. Fig. 7 shows the result of a micrograph processed with a crushed bone soaked in sterile water. The holes were mainly filled with bone powder (DB) and fibrous tissue (F), and new bone trabeculae were observed sparsely. It was about. FIG. 8 is a result of a micrograph treated with a bone pulverized product impregnated with an aqueous polyphosphoric acid solution. The holes were filled with new bone trabeculae (*) and bone powder (DB).
[0018]
[Example 3]
In the same manner as in Example 1, a hole was made in the femur of a white rabbit, and a collagen tape was soaked with 30 μl of a 2% aqueous solution of polyphosphate sodium salt (Polyphosphate glass, manufactured by Sigma) with various chain lengths and aseptic Dried. The collagen tape was packed into a hole in the femur of the right leg of the white rabbit. Further, a collagen tape soaked with sterilized water as a control product was packed in a hole formed in the femur of the left leg of the white rabbit. In this state, the incised portion was sutured, and the appearance of new bone regeneration in the femoral hole 2 weeks later was observed histologically. The chain length of the polyphosphate sodium salt used was calculated as (1) average chain length 15 (Na ₁₇ P ₁₅ O ₄₆ ) (2) average chain length 25 (Na ₂₇ P ₂₅ O ₇₆ ) (3 ) Average chain length 35 (Na ₃₇ P ₃₅ O ₁₀₆ ) (4) Average chain length 65 (Na ₆₇ P ₆₅ O ₁₉₆ ).
In experiments conducted using any of the polyphosphates, new bone formation was promoted, and new bone formation was promoted without being affected by the chain length of the polyphosphate.
[0019]
[Example 4]
In order to confirm the bone differentiation-inducing effect of polyphosphate, normal human osteoblasts (manufactured by Bio Whittaker) are cultured, and the degree of bone differentiation of the cells when polyphosphate is added is measured using alkaline phosphatase activity as an index. Therefore, it was evaluated in an in vitro system.
It is well known that the alkaline phosphatase activity of osteoblasts increases when cells form new bone, and this increase in activity is an indicator of new bone formation. Normal human osteoblasts were seeded on a 35 mm culture dish at 10,000 cells / cm ² and cultured in osteoblast basic medium (Bio Whittaker) containing 10% calf serum for 48 hours. Thereafter, the medium was replaced with a culture solution containing 1 mM sodium polyphosphate (average chain length 75), and the alkaline phosphatase activity of the cells was measured every day. The culture solution containing polyphosphoric acid was changed every 3 days after the start of measurement.
[0020]
The alkaline phosphatase activity was measured by the following method.
The cells were washed with PBS [mixture of 20 mM Na ₃ PO ₄ buffer (pH 7.0) and 150 mM NaCl], detached from the dish with trypsin EDTA, and suspended in PBS. Cells were separated by centrifugation and the supernatant was removed. The cell-containing solution was suspended in 1 ml of TBS [mixture of 20 mM Tris-HCl (pH 7.5) and 150 mM NaCl]. After disrupting the cells using an ultrasonic cell disrupter, the suspension was centrifuged again, and the supernatant was used as a crude enzyme solution. The protein concentration (A) in the crude enzyme solution was measured using a Bio-Rad protein assay kit (BIORAD). Next, an appropriate amount of the crude enzyme solution is added to a substrate solution in which 1.2 M Tris-HCl (pH 8.2) and 20 mM p-Nitrophenyl phosphate disodium are mixed at a ratio of 1: 1, and reacted at 28 ° C. for a predetermined time (Δt). Then, 2M K ₂ HPO ₄ was added to stop the reaction, the absorbance (B) at 410 nm was measured, and the alkaline phosphatase activity was calculated according to the following formula.
[0021]
[Expression 1]

[0022]
FIG. 9 shows the time course of the alkaline phosphatase activity of sodium polyphosphate when the culture time was increased.
[0023]
[Comparative Example 1]
Instead of the polyphosphate sodium salt (average chain length 75) used in Example 4, the sodium phosphate buffer was converted to the phosphate concentration and added to the culture solution having the same concentration as the polyphosphate, and cultured for a predetermined time. The phosphatase activity was measured.
FIG. 9 shows the time course of the alkaline phosphatase activity of the sodium phosphate buffer.
As is clear from FIG. 9, the cells of Example 4 treated with polyphosphate showed a marked increase in enzyme activity after 1 week, but the cells of Comparative Example 1 treated with phosphate buffer were marked. There was no increase in enzyme activity. That is, it is shown that osteoblast differentiation (bone formation) was promoted by adding polyphosphoric acid to the cells.
[0024]
【The invention's effect】
The bone regenerating material of the present invention can promote the formation of new bone and shorten the time required for healing and recovery in the treatment of bone fractures and injuries caused by physical impact and bones damaged by surgery.
[Brief description of the drawings]
FIG. 1 is a photograph instead of a drawing showing the microscopic substance of a femoral hole one week after treatment with a collagen tape soaked with sterilized water.
FIG. 2 is a photograph instead of a drawing showing the substance of a microscopic view of a femoral hole one week after treatment with a collagen tape soaked with an aqueous polyphosphoric acid solution.
FIG. 3 is a photograph changed to a drawing obtained by histologically observing a state of new bone regeneration in a hole of a femur one week after treatment with a collagen tape soaked with sterilized water.
FIG. 4 is a photograph changed to a drawing in which a state of new bone regeneration in a femoral hole one week after treatment with a collagen tape soaked with an aqueous polyphosphoric acid solution is observed histologically under a microscope.
FIG. 5 is a photograph in place of a drawing in which the appearance of new bone regeneration in the femoral hole two weeks after treatment with collagen tape soaked with sterilized water was observed histologically under a microscope.
FIG. 6 is a photograph changed to a drawing in which the appearance of new bone regeneration in a hole in a femur two weeks after treatment with a collagen tape soaked with an aqueous solution of polyphosphoric acid is observed histologically under a microscope.
FIG. 7 is a photograph in place of a drawing obtained by histologically observing a state of regenerating new bone in a hole of a femur two weeks after treatment with a bone pulverized product soaked with sterilized water.
FIG. 8 is a photograph in place of a drawing obtained by histologically observing a state of new bone regeneration in a hole of a femur two weeks after treatment with a bone pulverized product soaked with an aqueous polyphosphate solution.
FIG. 9: Time course of alkaline phosphatase activity of polyphosphate sodium salt and sodium phosphate buffer.

Claims (9)

General formula
(P _n O _{3n + 1} ) ^{(n + 2)} H ^(where n represents 15 to 2000), and a new bone tissue formation containing a linear condensed polyphosphoric acid obtained by dehydration condensation of orthophosphoric acid To promote bone regeneration material. The bone regeneration material according to claim 1, wherein the polyphosphoric acid is a polyphosphate. The bone regeneration material according to claim 2, wherein the polyphosphate is sodium or potassium polyphosphate. The bone regeneration material according to any one of claims 1 to 3 , wherein polyphosphoric acid is contained in a base material made of a biocompatible material. The bone regeneration material according to claim 4, wherein the polyphosphoric acid is contained in a biocompatible material in an amount of 0.001 to 20% by weight. The bone regeneration material according to any one of claims 1 to 5 , wherein the base material made of a biocompatible material is formed into a sheet shape. The bone regeneration material according to claim 1 or 2 , wherein the orthopedic filling contains polyphosphoric acid. The bone regeneration material according to any one of claims 1 to 7 , wherein a bone induction protein selected from BMP-1, BMP-2, BMP-3, TGF-β, osteopontin and osteocalcin is mixed with polyphosphoric acid. Bone regeneration material of animal bones pulverized and natural materials containing osteoinductive factor selected from mineral loss bone disposition is formed by mixing claims 1-8, wherein the polyphosphoric acid.

Images