JP5046511B2 - Hard tissue substitute carrier material - Google Patents

Description

  The present invention relates to a hard tissue substitute carrier material (artificial bone material). More specifically, the present invention relates to a hard tissue substituting carrier material composed of eighth calcium phosphate and collagen and excellent in shape imparting property.

Currently, for patients with bone defects with functional impairment (after bone malignant tumor surgery, trauma such as cleft lip and palate, fractures, etc.) aiming at improving quality of life (QOL) Various surgical therapies have been applied. It is preferable to use autologous bone for bone grafting, but autologous bone has quantitative limitations, and there is a problem of remaining damage in the removed site, so there is no quantitative limitation instead of autologous bone transplantation. Development of artificial bone materials is expected.
As such artificial bone materials, hydroxyapatite (hereinafter referred to as HA) ceramics, β-tricalcium phosphate (β-TCP), and the like have been proposed.
The inventors of the present invention have studied synthetic octocalcium phosphate (Octacalcium
phosphate, Ca ₈ H ₂ (PO ₄ ) ₆ · 5H ₂ O (hereinafter referred to as OCP) has a larger amount of bone tissue formation compared to HA ceramics, which are bioceramics conventionally used as artificial bones. Moreover, it discovered that it was a material which shows higher material absorbency than (beta) -tricalcium phosphate ((beta) -TCP) which is bioceramics known as in-vivo absorbability (for example, nonpatent literature 1, patent document 1). reference).
J Biomed Mater Res 59: 29-34,2002 Japanese Patent No. 3115642

More specifically, the present inventors have experimentally embedded OCP, which is a precursor of artificially synthesized biological apatite, to elucidate the following.
(1) OCP implanted in the living body is converted into HA (Non-patent Document 2) and has a good bone forming ability (Patent Document 1).
(2) OCP acts on osteoblasts and their progenitor cells (Non-Patent Document 3) and promotes bone repair by becoming the nucleus of bone formation (Non-Patent Document 4) (Non-Patent Document 5) ).
(3) OCP is absorbed in vivo by osteoclast-like multinucleated giant cells (Non-patent Document 6) and is replaced by new bone to promote bone repair, and HA and β-tricalcium phosphate (β-TCP Compared with existing calcium phosphate materials such as), the bone is more easily replaced by new bone and easily absorbed (Non-patent Document 1).
(4) OCP implanted in the living body is known to specifically adsorb glycoprotein in the living body on its surface (Non-patent Document 7), but on the other hand, it has a bone-forming cytokine (bone) When morphogenetic protein-2, transforming growth factor β ₁ ) and OCP are combined, OCP becomes a carrier of these cytokines, and bone regeneration in vivo is further accelerated (Non-patent Documents 8 and 9).
Tohoku J Exp Med 164: 37-50, 1991 AnatRec 256: 1-6, 1999 Oral Dis 7: 259-265, 2001 J Dent Res 78: 1682-7, 1999 J Electron Microsc 46: 397-403,1997 Bone Miner 20: 151-166, 1993 J Biomed Mater Res 57: 175-182,2001 J Biomed Mater Res 71A: 299-307,2004

In this way, OCP has extremely excellent properties as an artificial bone material, and it is formed during bone defects surrounded by bone tissue (for example, jaw cleft, extraction fossa, craniotomy of cleft lip and palate children). For example, bone formation with OCP alone can be expected. However, OCP as a hard tissue regenerative material is an inorganic substance and therefore has poor shape imparting properties. Therefore, there is a problem that there is a limit to application to an alveolar ridge augmentation operation which is intended to increase bone mass for a wide-range bone defect part or an atrophy alveolar ridge (see Non-Patent Document 10).
Arch Oral Biol 41: 1029-1038,1996

In bone regeneration, the three elements of cells, growth factors and carriers (artificial materials) play an important role, and it has become clear that their coordination effectively restores bone. Therefore, as a next generation hard tissue substitute carrier material (1) fill the space caused by bone defects; (2) have biocompatibility; (3) activate the function of osteogenic cells; (4) Bio-replacement that satisfies the requirements of effectively retaining and diffusing growth factors and fully exerting their bone regeneration function; (5) The transplanted artificial material itself is finally absorbed and replaced by new bone; Development of mold artificial materials is desired.
As a result of various studies on hard tissue substitute carrier materials (artificial aggregates) having such characteristics, the present inventors have only excellent shape imparting properties when OCP and collagen having excellent characteristics as artificial aggregates are combined. However, the present invention has been completed by finding that it can be a carrier material having operability, safety and biocompatibility, and can be a hard tissue substitute carrier material capable of achieving the intended purpose.

The hard tissue replaceable carrier material of the present invention is a hard tissue replaceable carrier material composed of OCP and collagen. The carrier material preferably has an OCP / collagen composition of 0.5 to 35 (weight ratio) with respect to collagen 1 and more preferably heat-treated. Note that the carrier material, cytokines with osteogenic potential (e.g., bone morphogenetic protein-2, such as transforming growth factor β ₁₎ may contain.

The hard tissue substitute carrier material of the present invention is a composite of OCP and collagen. By making OCP into a composite with collagen, shape impartability that has been difficult with OCP alone is provided. OCP can be used for bone defect and alveolar ridge augmentation. In particular, the heat-treated OCP-collagen complex significantly promotes bone regeneration. As described above, the OCP-collagen complex can be a biomaterial without a quantitative limitation in place of autologous bone transplantation without losing the excellent bone forming ability of OCP.
Therefore, due to the clinical application of OCP-collagen complex, not only is it adapted to bone defects with dysfunction, but also bone defects formed with surgery that are often overlooked without dysfunction manifesting It can overcome obstacles such as restoration of brain (brain surgery, orthopedic surgery, dental field, etc.), periodontal disease with bone resorption, or lowering of the jaw ridge that destabilizes denture retention, and can contribute to improvement of the patient's QOL.

As described above, the present invention is a hard tissue substitute carrier material comprising an OCP-collagen complex.
The OCP used in the present invention is a known substance. For example, the dropping method of LeGeros (LeGeros RZ, Calcif Tissue Int 37: 194-197, 1985) or the synthesizer disclosed in Patent Document 1 (three-stream tube) is used. It can be prepared by the method used.

Moreover, as collagen, the origin, property, etc. are not specifically limited, Various collagen can be used. Preferably, an enzyme-solubilized collagen obtained by solubilization with a proteolytic enzyme (for example, pepsin, pronase, etc.) from which telopeptide is removed is used. As the type of collagen, type I (or type I + type III) collagen derived from skins such as pigs and cows, bones and tendons is advantageous from the viewpoint of raw materials. Since collagen is a biological component, it has the feature of high safety. In particular, enzyme-solubilized collagen is preferable because it has low allergenicity.
A commercial product may be used as the collagen.

The method for preparing the hard tissue substitute carrier material of the present invention is not particularly limited, and various methods can be used as long as they are a method for preparing a complex containing OCP and collagen. Is mentioned.
a) Method of mixing and complexing OCP Adjust the pH of a collagen solution with an appropriate concentration to a range that can be gelled, add OCP here, and knead well to make a mixture of OCP and collagen, The composite powder is obtained by lyophilization. The composite powder is added to an appropriate mold and molded to produce a molded product, and further sterilized by a conventional sterilization method (for example, electron beam irradiation, high-pressure steam sterilization, etc.).

b) Method of mixing OCP suspension and complexing it Aseptic pH of an acidic collagen solution of appropriate concentration with an appropriate buffer (eg phosphate buffer, Tris buffer, sodium acetate buffer, etc.) Prepare a suspension of collagen and OCP by adjusting to 5.5-7.5 and adding OCP before the collagen gels. Then, after pouring into a mold where the pH is maintained from neutral to weakly alkaline, after giving the shape, it is gelled at an appropriate temperature (for example, 37 ° C.), and washing with water is repeated to remove salts of the buffer solution. And sterilize as above.
In addition, the gelled and water-washed material that has not been molded is lyophilized as it is, sterilized in the same manner as described above, and applied to the affected area as an amorphous filling material.

c) Method of precipitating OCP on collagen and complexing it With an appropriate concentration of collagen acidic solution, aseptically adjusting the pH with an appropriate buffer (eg, phosphate buffer, Tris buffer, sodium acetate buffer, etc.) It adjusts to 5.5-7.5, and before collagen gelatinizes, a calcium solution and a phosphoric acid solution are added and OCP is precipitated on collagen. Then, after pouring into a mold while maintaining the pH from neutral to weakly alkaline, after giving the shape and gelling at an appropriate temperature (for example, 37 ° C), washing with water is repeated to remove salts such as buffer solutions. The composite carrier is then sterilized as described above.
Alternatively, the gelled material that has not been molded is lyophilized as it is, sterilized in the same manner as described above, and applied to the affected area as an amorphous filling material.

The precipitation of OCP on collagen will be described. The OCP precipitation is based on the degree of supersaturation (ionic product / solubility product) determined by Ca ²⁺ , PO ₄ ³⁻ , pH, and the like. Therefore, a Ca ²⁺ solution and a PO ₄ ³⁻ solution are poured into a collagen solution adjusted in pH under the condition of supersaturation with respect to OCP, and precipitated. OCP either spontaneously deposits in the collagen gap or precipitates with the surface of the collagen fiber as the nucleus. It is known that OCP is converted to OCP via dicalcium phosphate (DCP or its dihydrate DCPD) or amorphous calcium phosphate (ACP), and finally matures into HA (Non-patent Document). 2). Therefore, instead of OCP, DCP, DCPD or ACP may be precipitated by adjusting the supersaturation degree of the calcium phosphate solution.

  The blending ratio of OCP and collagen in the OCP-collagen complex, which is the hard tissue substituting carrier material of the present invention, can be suitably adjusted according to the desired shape imparting property, operability, biocompatibility, etc., but is preferable. The blending ratio is 0.5 to 35 (weight ratio, the same unless otherwise specified), more preferably 1 to 20, more preferably 2 to 10, and most preferably 3 to 3 with respect to collagen 1. It is adjusted to about 5. If the OCP is less than 0.5 with respect to collagen 1, the bone regeneration function of the obtained composite may be inferior, and if it exceeds 35, the shape imparting property may be lowered.

It is more preferable that the above-mentioned OCP-collagen complex is heat-treated. By heat treatment, part of the OCP molecular structure is destroyed and invasion of bone-forming cells occurs, which promotes bone regeneration and crosslinks collagen to improve shape retention. The heat treatment of the OCP-collagen complex is more preferably performed under reduced pressure conditions.
Examples of the heat treatment conditions include a temperature of 50 to 200 ° C., preferably 60 to 180 ° C., more preferably 95 to 150 ° C., and a pressure of 0 to 3000 Pa, preferably 0 to 300 Pa. About 1 to 10 days, more preferably about 0.5 to 5 days.

The hard tissue substitute carrier material of the present invention, in addition to the OCP and collagen, cytokines with osteogenic potential (e.g., bone morphogenetic protein-2, such as transforming growth factor β ₁₎ may also contain, according By containing cytokines, the bone regeneration rate can be increased. The cytokine content can be appropriately adjusted depending on the desired bone regeneration rate and the like.
In addition, the hard tissue replacement carrier material of the present invention can include ingredients conventional in this field. Examples of such components include bioabsorbable polymers (eg, polylactic acid, polylactic acid-polyethylene glycol copolymer), bioabsorbable calcium phosphate (eg, β-TCP), and non-bioabsorbable materials (eg, HA, ceramics). Etc.).

EXAMPLES Hereinafter, although this invention is demonstrated in detail based on an Example, this invention is not limited to the example which concerns.
The OCP and collagen used are as follows.
(1) OCP
OCP is synthesized, dried and solidified based on the synthesizer disclosed in Patent Literature 1, dried and solidified, crushed, sieved in the range of 300-500μm, and sterilized by dry heat at 120 ° C for 2 hours. We used what we did. For details, the same reference can be referred to, but its production method will be briefly described.
_Three solutions of 0.16 mol calcium acetate (Ca (CH ₃ COO) ₂ .H ₂ O) in a total volume of 1500 ml, 0.16 mol sodium dihydrogen phosphate dihydrate in a total volume of 1500 ml, and pure water 1000 ml Introduced in 15 minutes from each supply port of the three flow tubes, using a device heated to 70 ° C. and having three supply ports (three flow tubes). The resulting precipitate was aged at 70 ° C. for 15 minutes and then filtered. The precipitate was again dispersed in pure water, filtered, sized and dried.
(2) Collagen Pepsin-solubilized collagen freeze-dried powder derived from pig skin (type I + type III, neutral, with crosslinking: NMP collagen PS; manufactured by Nippon Ham) was used.
Collagen tablets dissolve the same collagen, adjust to a final concentration of 3%, pH 7.4, freeze-dry the collagen solution, then mold to 9mm diameter and 1mm thickness and sterilize by electron beam irradiation (5kGy) did.

Example 1
Preparation of OCP- collagen complex Dissolve the above collagen, adjust to a final concentration of 3% and pH 7.4, and add 10% (w / w) OCP (300-500μm diameter) to the collagen solution. Kneaded thoroughly. The kneaded OCP-collagen mixture was freeze-dried, molded into a 9 mm diameter and 1 mm thickness, and sterilized by electron beam irradiation (5 kGy) (the obtained OCP-collagen complex is hereinafter referred to as OCP / Col). In addition, the moldability was extremely good, and it was possible to easily prepare a molded product.
The OCP contained in the complex was about 10.5 mg, and the collagen was about 3.5 mg.

Test example 1
The OCP / Col of the present invention was embedded in a bone defect portion and its function was tested. Specific methods and results are as follows.
(1) Materials and methods
(1) Experimental animals:
Male Wistar rats (12 weeks old) were used.
(2) Surgical operation After injection of pentobarbital (50mg / kg) into the abdominal cavity, a standardized full-thickness bone defect of approximately 9mm diameter that cannot be spontaneously cured was created in the rat calvaria, and OCP / Col (9mm diameter) 1mm thick). At that time, the periosteum exfoliated at one end was restored and sutured.
For comparison, (a) OCP group: only OCP granules (300-500 μm diameter: 15 mg) were embedded, and (b) Col group: only collagen tablets (9 mm diameter, 1 mm thickness: about 3.5 mg) were embedded. A thing was used.
(3) Experimental period Samples were placed for 2 and 4 weeks, and 5 experimental animals were used for each group and each period.
(4) Specimen preparation method After pentobarbital (50 mg / kg) was injected intraperitoneally, 0.1 M phosphate buffer (PBS) (pH 7.4) was preperfused from the left ventricle and 0.1 M phosphate buffer 4 After perfusion fixation with% paraformaldehyde (pH 7.4), the calvaria and surrounding tissues were collected and fixed by immersion in the same fixative. After taking a soft X-ray photograph (20 KV, 5 mA, 1 min.), The X-ray opacity (% opacity) in the defect was measured using image analysis software (NIH Image 1.63). Thereafter, the tissue sections were prepared by decalcification with 10% EDTA, and histologically searched by staining with hematoxylin and eosin.

(2) Results
(1) Soft X-ray findings
OCP / Col group: Cotton-like and granular opacity images were observed throughout the defect from 2 weeks after implantation, and the opacity increased at 4 weeks after implantation (see FIG. 1a). % opacity was 98.7 ± 0.70% at 4 weeks. Increased opacity means accelerated bone formation and mineralization.
Col group: Slightly cotton-like impermeable images were observed within the defect 2 weeks after implantation. At 4 weeks of implantation, the opacity increased and spread within the defect (see FIG. 1b). % opacity was 49.2 ± 14.6% at 4 weeks.
OCP group: At 2 and 4 weeks after implantation, granular and solitary opaque images were observed in the defect (see FIG. 1c). % opacity was 48.7 ± 16.7% at 4 weeks.

(2) Histological findings
OCP / Col group: 2 weeks after implantation, in addition to bone formation from the bone defect margin and cerebral dura mater, bone-forming cells infiltrated into OCP / Col at high density, and they were deep . Vigorous bone formation using a network structure of collagen sponge by osteogenic cells was observed. Multinucleated giant cells around OCP were not noticeable.
At 4 weeks after implantation, bone formation further progressed and was confirmed throughout the defect. New bone was formed so as to fill the network structure of the collagen sponge (see FIG. 1d), and invasion of blood vessels was also observed. Some of the bones that were formed were remodeled, making collagen less noticeable.
Col group: After 2 weeks of implantation, in addition to bone formation from the bone defect edge, cells invaded into the network structure of the collagen sponge, but the density was low. In addition, bone formation from the cerebral dura mater side was observed, but these were limited to a small area. Inflammatory cell infiltration was not noticeable.
At 4 weeks after implantation, osteoblasts are not noticeable. Although the invasion of cells was observed in the network structure of the collagen sponge, the absorption of collagen by some multinucleated giant cells was observed (see FIG. 1e). Inflammatory cell infiltration was not noticeable.
OCP group: At 2 weeks of implantation, new bone was localized along the margin of the defect. Inflammatory cell infiltration was observed and OCP was surrounded by multinucleated cells. At 4 weeks of implantation, new bone was localized around the defect margin and around the implanted OCP (see FIG. 1f). Inflammatory cell infiltration was reduced. Some OCPs were surrounded by multinucleated cells.

(3) Evaluation
When the bone forming ability was examined by X-ray photography and histochemistry, in the OCP / Col group, it could not be expressed with conventional materials.
1) Engraftment of cells in the deep layer inside the composite material,
2) Promotion of deposition of calcium phosphate, an inorganic component of bone, on the composite material (promotion of calcification),
3) Bone formation earlier than OCP alone,
It was found that the OCP-collagen complex of the present invention can be an excellent hard tissue substitute carrier material.
Thus, the hard tissue substitute carrier material of the present invention in which OCP and collagen are combined is a material that mimics the growth process of hard tissue, satisfies the requirements of the above-mentioned bio-replacement type artificial material, and OCP or collagen alone This makes it possible to repair a wide range of bone defect sites that could not be achieved, so it is an excellent hard tissue substitute carrier material.

Test example 2
The heat-treated OCP-collagen complex was tested by the following method.
(1) Materials and methods
(1) Test sample The OCP / Col molded and prepared in Example 1 was treated in a vacuum dryer at 150 ° C. for 24 hours and then sterilized by electron beam irradiation (5 kGy). / Col). For comparison, the same OCP / Col that had been sterilized by electron beam without heat treatment (referred to as uncrosslinked OCP / Col for convenience) was used.
(2) Experimental animals, experimental procedures and specimen preparation are the same as in Test Example 1. Regarding the experimental period, the sample embedding period was 4 and 12 weeks, and five experimental animals were used for each group and each period.

(2) Results
(1) Soft X-ray findings Cross-linked OCP / Col group: Cotton and granular opacity images coexisted throughout the defect from 4 weeks after implantation (Fig. 2a), and opacity of the defect at 12 weeks after implantation. Increased further and became denser (FIG. 2b).
Uncrosslinked OCP / Col group: Granules and isolated opaque images are scattered within the defect 4 weeks after implantation (Fig. 2c), and the impermeable image of the defect shows a fusion tendency at 12 weeks after implantation. Compared to the cross-linked OCP / Col group, the impermeable image in the defect is significantly less (FIG. 2d).

(2) Histological findings Cross-linked OCP / Col group: In addition to bone formation from the bone defect margin at the 4th week of implantation, bridging OCP / Col is used as a scaffold, and vigorous bone formation is performed so as to fill the network structure. Observed (FIG. 2e). Multinucleated giant cells around OCP were not noticeable. At 12 weeks after implantation, bone formation was further enhanced throughout the defect, and vascular invasion and remodeling of the formed bone were also observed (Fig. 2f). Moreover, the embedded cross-linked OCP / Col was in an absorption tendency and was not noticeable.
Uncrosslinked OCP / Col group: At 4 weeks of implantation, new bone was confined to the defect margin and around the implanted OCP (FIG. 2g), and some OCP was surrounded by multinucleated cells. The structure corresponding to Collagen in the cross-linked OCP / Col was unclear, suggesting their absorption tendency. Although bone formation increased at 12 weeks after implantation, the new bone was confined to the defect margin and around the implanted OCP (FIG. 2h), and some OCP was surrounded by multinucleated cells.

(3) Histological Quantitative Findings FIG. 3 shows the results of a statistical examination of the proportion of new bone (n-Bone%) within the defect between both groups. At 4 weeks of implantation, the cross-linked OCP / Col group was 22.0 ± 7.80%, and the non-cross-linked OCP / Col group was 4.56 ± 3.19%. A significant difference in bone formation was observed between the two groups. At 12 weeks of implantation, the cross-linked OCP / Col group was 52.5 ± 13.7%, and the non-cross-linked OCP / Col group was 15.4 ± 13.7%. These also showed a significant difference in bone formation between the two groups.

Performance of cross-linked OCP / Col The actions of the cross-linked OCP / Col group are summarized below.
1) The cross-linked OCP / Col group significantly promotes bone regeneration than the non-cross-linked OCP / Col group.
2) The cross-linked OCP / Col group tends to replace the regenerated bone.
3) The good bone regeneration by the cross-linked OCP / Col group is considered to be due to the synergistic effect of OCP and collagen.

It is a figure which shows the soft X-ray findings (a-c) and histological findings (d-f) of OCP / Col group (a, d), Col group (b, e), and OCP group (c, f) in 4 weeks. In the drawing, a-c bars indicate 4 mm, and df bars indicate 100 μm. Crosslinked OCP / Col group at 4 weeks (a, e), Crosslinked OCP / Col group at 12 weeks (b, f), Uncrosslinked OCP / Col group at 4 weeks (c, g), Uncrosslinked OCP / at 12 weeks It is a figure which shows soft X-ray findings (ad) and histological findings (eh) of Col group (d, h). In the figure, the a-d bar represents 4 mm, and the e-h bar represents 200 μm. It is a figure which shows the ratio (n-Bone%) of the new bone in the defect | deletion by histological quantitative evaluation.

Claims (3)

A complex composed of eighth calcium phosphate and collagen, wherein the complex is subjected to heat treatment at a temperature of 150 ° C., a pressure of 0 to 3000 Pa, and a treatment time of 0.1 to 10 days. Tissue replacement carrier material.
  The hard tissue-replaceable carrier material according to claim 1, wherein the compounding ratio of the eighth calcium phosphate and collagen is 0.5 to 35 (weight ratio) of the eighth calcium phosphate with respect to collagen 1. The hard tissue-substituting carrier material according to claim 1 or 2, comprising a cytokine having bone forming ability .

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