JPH06508780A - graft material - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] graft material The present invention provides novel materials useful for repair and recovery of the skeletal system, methods for their preparation, and The present invention relates to a new method for skeletal repair using these methods.

A variety of surgical methods are employed to repair the skeleton in humans and animals. . These include the insertion of grafts to stabilize the fracture and promote healing; In areas of the body that have a naturally low tendency to heal, implantation of anterior beam material and support structures are recommended. healing of the fracture by insertion of a graft that acts as a membrane to regenerate the tissue. It will be done. Materials used as grafts should ideally be biocompatible and bone-compatible. It has the property of promoting growth.

Possible role of the enzyme alkaline phosphatase in promoting bone mineralization It has long been assumed that there is. Beersen and van den Hoos (Beer tsenJan　Den　Bos)　(l[atrix　Vol, 91989　 A recent notable inhydro study reported by (p. 159-171) The calcification process is stimulated by sphatase or perhaps by the action of increasing the local concentration of phosphate ions. The results showed a tendency to support the hypothesis that it may be involved in initiation.

However, the rather high concentrations of phosphate esters used in in vitro studies From this point of view, the rate of hydrolysis of phosphate ester at physiological pH level is considered to be too low to participate in the mineralization process. Therefore, whether such in vitro mineralization research is relevant to the in vivo situation is important. That (ヰis being questioned)

U.S. Pat. No. 4,394,370 describes regenerated collagen and demineralization ( dpim1neralization) bone particles or solubilized bony morphogenesis A bone graft material made of a sponge-like composite material with phase proteins has been described. It is. Complex these sponges with bovine intestinal alkaline phosphatase It can be formed. This implantation was achieved by dispersing collagen at a concentration of 15 mg/g. The inflammatory response to the material is eliminated and the formation of osteoid in the graft material is promoted. It has been stated that the graft is gradually resorbed to help it become more fully cortical. ing. There is no mention of mineralization occurring. U.S. Patent No. 4.40 No. 9.332 describes the use of drugs that reduce the inflammatory response that occurs when introduced into the body. Kolaken material that forms a complex with alkaline phosphatase for Porous membrane structures based on materials have been described.

The inventors have developed biocompatible carrier materials and minerals. By combining with an amount of phosphatase enzyme that promotes skeletal repair, We have now discovered that a useful new material can be obtained. The level of enzyme activity is preferably At least 0.5 milliunits (mU) of APa per 1.0 μg of hydroxyproline se (where IU is the same as that of Heelsen and Van den Bos). sen, Yan Den Bos) Matrix Yol, 9/1989 ．． p-nitrophenyl phosphate using the method reported in p16L. 1 μmol p-nitrophenol per minute at 37°C and pH 10,5 ).

The biocompatible carrier material itself has at least It is thought that it is mineralized to some extent and can remain in place for a long time. The material of the present invention is Promotes implant mineralization and skeletal repair to an extent that is easily detectable within 7 days. It has practicality.

These new materials are stable products that can be mineralized and regenerated when used for skeletal repair. It has the effect of promoting the growth of bones. According to one aspect of the invention, therefore, biocompatible an amount of a phosphatase enzyme combined with a carrier material to promote mineralization A novel material useful for skeletal repair is provided that has enzyme levels of .

The phosphatase enzyme is preferably an alkaline phosphatase enzyme (hereinafter simply referred to as “ APase”). APase is usually extracted from human or animal tissue. can be obtained. This enzyme is a cell surface sugar that can hydrolyze a variety of monophosphates. It is a protein. The major forms of APase in 3flj are distinguished. Liver II/Bone/ Kidneys: placenta, and intestines. All of these forms are useful in the present invention.

The carrier material is any biocompatible carrier material capable of binding the enzymes mentioned above. It may also consist of Preferably the conjugation involves the carrier and the enzyme described above. The carrier is incubated with the enzyme in the presence of a binding agent that can be covalently bonded to the enzyme. This is done by Appropriate binding agents significantly reduce the biological activity of the enzyme It binds to the enzymes mentioned above without any oxidation. Using a variety of polyfunctional compounds be able to. The binder used to form the preferred material of the present invention is It may be a material that binds to the carrier material. Examples of binders that may be used include is biotin-abine:glutaraldehyde and 1-ethyl-3(3-dimethyl). Contains thylaminopropyl) carbonimide/HCI. Preferred binders are In addition, it does not cause any untoward reactions when introduced into the body as part of the material according to the invention. It's a good thing. Highly preferred binders are carbodiimides, especially the compounds mentioned above. be.

A particularly preferred binder is that known as SATA-MHS, which Succinimidyl-8-acetylthioacetate (SATA) and maleimidohex Those using a combination of Sanoyl-N-hydroxysuccinimide (MHS) It is. Preferably the carrier is incubated with 5ATA and the enzyme with MHS. Bate. The products of these two types of incubation treatments are mixed and reacted. and prepare the graft material.

The carrier material may be of natural or synthetic origin. Diverse There is a possibility that synthetic polymers can be used. A particular group of synthetic materials that are useful are Polymers known to be absorbent It is. The use of synthetic materials makes them flexible and designed for specific uses It is also advantageous insofar as it can be formed into shaped pieces.

Various natural materials can also be used as carriers. especially humans or animals Tissues such as bones and teeth are useful. mineralized tissues, such as bones and Teeth must be demineralized before they can be used as carriers.

The material obtained by demineralization contains a substantial proportion of fibrinous male collagen. It consists of illar collagen. Collagen is a major fiber in many animals a fibrous protein that can be extracted from many parts of the human or animal body .

Primitive male kolaken is used as a material constituting a substantial proportion of the present invention. is the preferred carrier material for use. Examples of this material are human or animal hard It is a membrane. The carrier material is a material that is flexible enough to be formed into a shaped piece. That's preferable. Porous or fibrillar or fibrous material Materials made from polyesters also have relatively large surfaces that can bind enzymes. ) is advantageous. Useful carrier materials always cover the surface of the fiber or the exposed If the condition is to be maintained, the collagen fibers should be incorporated into or on a suitable support material. It can also be formed by letting it grow. An example of a support material is natural or synthetic phosphoric acid. Calcium, and these substances may be used to form part of the materials of the present invention. Ru.

The carrier material is sufficiently flexible to be useful as a graft material, and It is preferable that it be sufficiently strong. The degree of flexibility or strength is related to the material. will vary according to the particular intended use. Preferably the material is implantable It is a material that must be handled and manipulated before or during surgery. Preferred graft material The material contains at least 200 U/cm3 of phosphater. preferred carrier The material is capable of binding this amount of enzyme. Useful materials include carriers, or the pressure of materials produced by a combination of carrier and phosphatase enzymes. It can be produced by shrinking.

The preferred material is a relatively smooth surface and a densely organized fibrous structure. It is something that we have. These materials have osteoconductivity (osteoconductivity). ve properties). i.e. they promote osteoblast migration. This appears to induce bone movement, thereby promoting new bone growth.

The carrier has a tubular structure, i.e. a series of cracks or It is also preferable that the material has minute cracks. Enzymes bind within these tubules However, mineralization will occur within it. The strength of the material increases as mineralization progresses. As far as this is concerned, this is advantageous. Physiological concentrations of calcium and organic phosphates (e.g. β-glycerophosphate solutions) or solutions containing inorganic phosphates The carrier (or carrier-APase binding material) is pre-inked in Mineralization can be promoted by mining. preinquency Ili'! in the carrier. A nuclear center was formed and implanted into the body. It is expected that the minerals will grow rapidly and increase the rate of mineralization.

The carrier material must be free of substances that inhibit its mineralization. That's preferable. If the material is natural, it may be necessary to reduce the amount of inhibitors to an acceptable level. For this purpose, it will be necessary to sufficiently extract the material. calcified tissue minerals The removal process involves washing the clean tissue and placing it in an acid solution, for example acetic acid or hydrochloric acid. This is conveniently carried out by placing it in a liquid for a long period of time. Preferably, obtained The demineralized material is further treated with chaotropic substances ( chaotropic agent).

The demineralized material contains residual amounts of non-collagenous proteins and insoluble phosphoproteins. or dentin phosphophoryn (denjinal phosphophoryn) s). Bound phosphoprotein or dentin phosphophorin was found to be effective in promoting remineralization. preferred carrier - Materials that naturally contain these phosphoproteins or phosphophorins, or phosphoproteins or phosphophorins are introduced, preferably covalently linked to the carrier. It is a combination of Preferably the material of the invention contains hydroxyproline per μg. very preferably at least 0.03 μg of phosphate. contains in the form of phosphophorine.

The novel material of the present invention can be prepared by combining the carrier with APase in the presence of a binder. Prepared by incubation, optionally in the presence of an organophosphorus compound. Ru.

The binding reaction is carried out by introducing the carrier and APase into a solution of the binding agent. It would be convenient to implement it. The reaction is carried out under conditions that do not inactivate APase. Should. Conveniently at a temperature not higher than ambient temperature, preferably below 10°C, Generally at least 1 hour, conveniently longer, e.g. 24 hours, the reactants may be sufficient to prepare the products of the invention.

The amount of APase present in the novel material can vary widely. Preferably the purpose Materials sufficient to promote remineralization in applications such as hydroxyproline 1 ．． At least per 0 μg, indicating the level of OmU APase enzyme activity. Probably. The optimal amount of enzyme to incorporate depends on the properties of the constituent materials and the intended use of the materials. , the age of the human or animal to which it is to be transplanted, and the serum content of that human or animal. will vary depending on the concentration of inorganic phosphate present.

Materials containing lower amounts may be useful in animals undergoing relatively rapid growth. Probably. In adult animals, the graft preferably contains a higher concentration of the enzyme. very preferably in combination with phosphoproteins or phosphophorins. have

Alternatively, for example, organic or inorganic phosphates can be administered transdermally or intradermally. mineralization by increasing the phosphate concentration near the graft by administering can be promoted. The skeleton consists of the introduction of the graft material mentioned above for its repair. A method of treating cancer forms another aspect of the invention. From a favorable perspective, the treatment is It also consists of increasing phosphate levels near the graft.

The amount of binding agent used is simply the amount required to bind the desired amount of APase.

Generally, the binder is present in solution in large excess of the amount required by stoichiometry. Will. The presence of such an excess amount of binder cross-links the surface of the carrier. It is thought to have a bridging effect. Such cross-linking affects the mechanical properties of the carrier material. would be useful for improving. This is also useful for controlling the biodegradability of the graft Will. The graft must retain its mechanical integrity during mineralization or It may be useful to disassemble it afterwards.

The materials of the present invention can be used in a variety of surgical procedures. Carrier material is bound to APase It would be preferable to shape it into an appropriate shape before making it.

In particular, the material of the present invention stabilizes damaged or defective parts of the skeleton and promotes their healing. It is used as an internal wound dressing for After transfer it mineralizes and hardens . The new material can also be used as a bone substitute, fixing the material in place and It can also be used to repair fractures by mineralizing and hardening in situ. Ru. Treatment of bone fractures by these methods provides another aspect of the invention.

The material of the present invention is a 'guided tissue regenerator'. ation). These methods helps regenerate supportive connective tissue in areas of the body damaged by disease This is due to the insertion of a membrane for this purpose. The membranes currently used for this application are They are often insoluble and must be removed during a two-day surgical procedure. do not have. The material of the invention is biocompatible and may remain in place and therefore further It is advantageous in this application in that the need for surgical intervention is avoided.

The invention will be illustrated by the following examples.

(1-3 years old) and frozen at -80°C until use. Gums and teeth after thawing The circumferential ligament is removed and the roots are flushed with tap water parallel to their longitudinal axis, from the apex to the neck of the tooth. It was cut with a diamond disk and disassembled with a chisel while being constantly watered. then The tooth root was cleaned and the pulp and cementum were removed. inhibit them from proteinase Wash with water-cooled PBS in the presence of a harmful agent, and remove the outer dentin (mantle) while cooling with tap water. (including the soge through layer) was taken out with a diamond disk.

Minerals were removed by soaking in 0.5M acetic acid or 0.6M hydrochloric acid for 4 weeks. 6. Cryotome set at 30 μm to prepare dentin slices (DDS) Sections were cut using (cryotome). Then add them further 4°C with 4M guanidino HCI and 064M EDTA (pH 7,5) It was extracted for 3 days. Before use, wash the DDS in double-distilled water for 1 hour and remove antibiotics. The seeds were placed in supplemented twice-steamed seedling water overnight at 4°C. DDS is μg of hydroxyproline It contained 0.04 Mg of PO+/.

Binding of APase Bovine intestine APase is It was covalently bonded to dentin collagen sheets by the use of diimide.

Glutaraldehyde granular food 0. In PBS containing 1% glutaraldehyde, DDS was incubated in the presence of APase (700 U/ml) at 25°C for 2 hours. I bet. This material was then thoroughly washed with PBS and incubated at 4°C with 1mM Mg''. and O,LM glycine buffer M containing 0.1mM Zn” (pH 10,5) I saved it inside.

Calphonimide bond 0.13M i-ethyl-3(3-dimethylaminopropylene) ) DDS in carbodiimide/HCI (pH 4, 5) at 4°C for 2 days. During this period, the cells were incubated in the presence of APase (700 U/ml). Then that Distilled water, 0. IM LM NaCl in sodium acetate (pH 4,0), 0.1M NaHCOx Lp) (8,3), and finally washed thoroughly with distilled water. . This material was stored in Grinno's buffer (see section above) at 4°C. . The enzyme retained its activity for at least 10 months under these storage conditions.

In order to find out as a function of and their controls (treated with cross-linker alone or heat-inactivated enzyme) at 10% heat Iscob ([5c ove) Incubated for various periods at 37 °C in modified Darchenoco medium (IMDM). Cubated. Radiolabeled calcium (1 μCi per well [45Ca] C12) was added and the collagen sheet was monitored for label uptake. phosph A source of β-glycerophosphate (β-GP) was added at a concentration of 10 mM. Ta. At the end of the experiment, the specimens were incubated in 0.5 ml of LM HCI for 1 hour at 37°C. , decalcified. A 300aL sample was treated with obtifluor (Optif 1uor). Scintillation Cocktail (Z1 Kart Instruments) The coefficients were measured using a Rackard Tricard 4530 nontyrene 3-meter coefficient meter.

In vivo experiments APase-treated DDS was tested for its ability to mineralize under in vivo conditions. The force was tested as follows. Female Wistar rat (approximately 200 g each) were anesthetized with Hypnorm, including the sagittal suture. An incision was made in the skin covering the skull following the . Pull this skin backwards to the right and left sides. The neck muscles were exposed. Next, by making an incision at the exposed muscle point, Pockets were formed on each side. D with a thickness of 30 μm, a width of 1 cm, and a length of 2 cm. DS slices were inserted into the pocket. The APase treated one is on the right, the control on the left. side. Only the narrow edges of the graft make contact with the muscle, and the majority (90%) are true I tried to make contact with the skin. The skin wound was closed with nylon thread and kept for one week. The patient was cured for various periods from 1 to 4 days. The animal is then re-anesthetized and the wound area The incision was made again and the graft was excised. The 71-valent portion of each graft was excised along with the surrounding connective tissue. and prepared for histological examination. The remaining part was cleaned of surrounding tissue and subjected to chemical separation. When used for analysis, it becomes radioactive &! Calcium was stored in a tank. monophosphate In the absence of Stell, only a small amount of [+5Ca] was found in the snow. Ta. Tracking the uptake of 1fi3it in carboimide-treated samples as a function of time. When traced, a rapid influx of [Cal] was observed within one day. A chronic increase was observed. The control sheet treated with carbodiimide without enzyme , remained largely radioactive-free. APase-containing slices harden rapidly. and was positively stained by the Yon Kossa method. child The minerals are deposited in the form of needle-like microcrystals in tight association with the carrier collagen fibrils. , (according to Xi1 analysis) exhibited hydroxyapatite-like properties.

In vivo cutaneous wound healing was not significant. Transplanted during re-invasion treatment Inspection of the site revealed that the surrounding tissue was intact, especially for carbodiimide-treated grafts. It was observed that it was rich in collagen and that there was no inflammation. However, glutaraldehyde The treated graft is surrounded by highly vascularized, slightly edematous connective tissue. There were many cases. For both types of grafts, DDS (APase-conjugated) It hardened within and took on an opaque appearance. The control specimen was soft snow, and upon visual inspection It was transparent.

Chemical analysis showed that mineral uptake by APase-treated DDS occurred early after transplantation. This proved to be extremely rapid. About carbosimide treated samples further increased or occurred over time. According to linear regression analysis, this increase is due to phosph It was statistically significant for ate (p<0.05). Carbo in 4 weeks For diimide-bound samples, the calcium content per μg of hydroxyproline is The amount is about 80% of that found in normal hair/growth, whereas phosphate The amount was approximately 60%.

Expressed in molar a degree, the C a / P ratio in the remineralized Zougeyoi is Approximately 2.00 for diimide-treated samples, and approximately 2.00 for glutaraldehyde-treated samples. was found to be approximately 1.80. In normal bovine dentin, this ratio is 17 A series of graft materials were prepared using the materials and methods described in Example 1. .

The binder was 1-ethyl-3(3-dimethylaminopropyl) carbonimide. Ta.

Forty female Wistar rats (approximately 200 g each) were anesthetized with Vibnorm.

An incision was made in the skin parallel to the cranial suture. Invert the skin covering the right and left parietal bones. , the skull from the frontal to the occipital crest was exposed. along the frontal and occipital sutures After the incision was made, the periosteal flap was lifted to either side. attached to the dental handpiece. Using a low-speed rotating trephine bar (diameter 1.8 mm) attached to each parietal bone, Two complete fractures were created at a distance of approximately 1.0-15 mm. surgical procedure The tissue was kept moist with saline to prevent overheating during the procedure. beneath Special care was taken not to physically compromise the dura mater.

One wound on each side of the skull was made with a width of 5 cm and a length of 0.8 cm. Covered with sized grafts. On the right is the one containing APase, on the left is the control. (containing no enzyme) was transplanted. The periosteum and skin were closed. After surgery on the animal Animals were killed by decapitation at 3-week, 6-week, 9-week, and 12-week intervals. the cranial vault Extracted, 4% baraho in O, 1 mol/L sodium cacodylate (pH 7,4) Soaked in a solution of lumaldehyde and -% glutaraldehyde for 24 hours. inspection Bodies were subsequently fixed in 1% OsOn and embedded in epoxy resin.

Cut each half of the skull at right angles to the skull so that the two wounds are cut in the same plane. The stomach was placed on a microdome. Section with methylene blue or phono cossa It was stained according to the method. From each specimen, one methylene was extracted from the central region of two fractures. Blue-stained sections were taken and used for histomorphometric analysis.

Ultrathin sections were cut with a diamond knife and treated with uranyl acetate and citric acid. Stained with acid lead and examined with a Zeiss EMIOC electron microscope.

Near the Wtliff-3 Ma' cry, for each selected section, a magnification of 173 A trace test was conducted using a sine prism (Cs1gr, prism). X- Measure the mineralized area of each graft using the Y device and occupy in the cut plane Expressed as % of total area. Assess the thickness of the graft as well and compare it to the newly formed bone. The length of direct contact was measured. both on the inner surface of the graft (the side facing the skull) and and along the outer surface (mq facing the skin).

Refined barley The APase activity of enzyme-treated grafts, as shown by histochemical examination, was It was attached to the walls of the rodent tubules and to the outer surface of the sheet. Same dyeing along the entire length of the sheet The distribution of activity was uniform in that a color pattern was seen. APase failure Sections of control sheets incubated with crosslinker in the presence of histochemical staining did not show. Based on the conversion rate of p-nitrophenyl phosphate, the enzymatic treatment The collagen sheet contains 0.95 ± 0.24 mU per μg of hydroxyproline. It contained the enzyme.

Healing of the surgical site is assessed by microscopic examination and ignores the inflammatory response of the tissue surrounding the graft. It was not serious in that it could have been done. More specifically, edema, redness and no signs of swelling were observed. For microscopic examination and bin setting of grafts after fixation. Careful palpation revealed that the APase-containing grafts were hardened, whereas the controls were soft. It became clear that it remained.

Light and electron microscopy revealed that the test implants accumulated minerals over the course of the experiment. This has been proven. Within the first 3 weeks after transplantation, approximately 55% of their amputations occupied by minerals. After that, the amount of minerals increased slightly to about 70-80%. added. Areas that remained mineral-free are covered by bone that the graft covers. It was often the edge of the membrane that was in contact with the connective tissue. Graft to cover the fracture There was no significant difference in mineral density between the graft and the graft covering the intact bone. It was.

No substantial mineralization was detected in control grafts up to 9 weeks. However, the surgical department After 12 weeks of implantation, some control specimens were distributed more or less randomly along the graft. It contained mineralized areas. On average, these areas account for approximately 20% of the graft material. Ta.

In most cases, the minerals are small, tightly bound kolaken fibrils that make up the implant. It was deposited as crystals. The dentin tubules remain open and the implantation In two areas at the edge of the fragment, no layer of mineralized material was deposited in the adjacent periosteum.

There were also no signs of cartilage formation.

As the graft becomes mineralized, osteoblasts containing large amounts of Golthy bodies and rough endoplasmic reticulum A cyst appeared. Osteoid and bone are deposited between the osteoblast layer and the mineralized part of the graft. there was. Although no determination was made regarding the bonding strength between the graft and bone, the mineralized base The orientation of cracks caused by cutting artifacts in the material gives some indication. I got it. They were rarely seen along the border between the two. Usually they are It had passed through the bone-graft interface.

These phenomena occur along the inner surface of the enzyme-treated Kolaken sheet facing the calvarial bone. This was particularly noticeable. Bone is formed along the outer surface facing the extracranial periosteal layer. Even if it did, it was very little. According to correlation analysis, mineralization of the graft increases It is shown that the length of direct contact with the bone along the inner surface tends to increase as the (Correlation coefficient r=0.53.n=37; p<0.005).

Also for control implants, their degree of mineralization and length in direct contact with newly formed bone A positive correlation was found between (r=0.45.n=37;l)<0. OL>. death The degree of integration of the graft with bone is much lower than that on the experimental side of the skull. won. These two-sided differences proved to be statistically significant (pg0 01).

Little inflammatory reaction was observed in the tissue surrounding the implanted material. In more detail, 0 buys There were virtually no cells present, and polymorphonuclear leukocytes and lymphocytes were rare. deer The surface of the graft, except where it is in direct contact with the graft or newly formed bone. The presence of macrophages and multinucleated giant cells along the The graft showed evidence of mild foreign body reaction. This applies to both the control side and the experimental side. But it was. Number of giant cells along the graft for experimental and control sides Almost the same (Wilcoxon sign rank test) igned-ranks test), p>Q, 05), they are the mineralization of the graft. It was not associated specifically with either the section or the non-mineralized section.

Areas of non-mineralization or mineralization despite the presence of multinucleated giant cells associated with the graft No significant resorption of the graft occurred in any of the sections. evidence of this The grafts retained their full width during the experiment (32 μm) and the absorbing cell force width was affected. This result was obtained from the finding that there was no However, it penetrates deeply into the canal. Cells with cytoplasmic projections were seen in various places. None of the mineralized grafts contain No osteoclasts were observed indicating a rim, and no Haunpp concavity was observed. .

Harukoku Punishment Examining the mineralization of transplanted materials as a function of age, sex, transplant site, and phosphoprotein snow content. Betaou Male and female Wistarlands (5 or 20 weeks old) were anesthetized and their weight It was measured. Next, sheets of the material were implanted subcutaneously at the following sites: On the skull (parietal bone) and frontal bone), inferodorsal region, and abdominal wall. Control slices do not contain APase or heat-inactivated APase. Reweigh the animals 2 weeks later and After decapitation (under advanced anesthesia), the sheets were removed and analyzed for phosphorus content.

Serum samples were assayed for inorganic phosphate.

Two types of materials were tested: one with minerals removed and one with guanidine extraction. It was created from the excreted bovine carcass (which still contains some covalently bound carmine phosphorus). Contained phoholine: 0.035 = 0.019 μg phosphate/μg bi The other is demineralized and extracted bovine cortex (cartic). al bone) was created (0, O05 + 0.0025ag boss fate/ μg Hybron. Slices were prepared as described in Example 1.

APase was converted into a collagen carrier (mainly) according to the SATA-MH5M method. It was combined with type ■collagen).

A: 5ATA (succinimidyl-8-acetylthioacetate, /<-su(P 175 mg of collagen bound to a carrier was added to 5 ml of collagen. 0.05M phosphate (pH 7.5), 1mM EDTA and 500μl S Incubated in ATA (15 mg/ml dimethyl sulfoxide) for 30 min at room temperature. Cubated. The collagen sheet was then washed four times with phosphate buffer. Cola -Ken-5 ATA slice to APase-VHS (see below) Immediately before washing the slices, add 5 ml of 005M Phospheh (pH 7.5), 1mM EDTA and 500μl of deacetylated solution i'll [(1.75g of Hydroxylamine HCI and 0.475g EDTA, 50ml area 0 Incubate at room temperature in 5M Phospheh (pH 7,5). It was deacetylated by Decant the solution after 2 hours of incubation. Ta.

B: VHS (maleimidohexanoyl-N-hydroxysuccinimide or N - Succinimisyl-6-malate caproate, full strength (Fluka)) Peeters et al. (Immunol, i[ethods 12 0.133-143.1 conjugated to APase by the method described in 1989). 1.5 to 0 mg APase/ml 0.05M phosphate (pH 8.0) 4 mg of MH8/40 μm trimethylformamide was added. 5 minutes at room temperature Add 1 ml of 0.05M phosphate (pH 6.0) after the main application. , maleimidated APase was treated with Sephadex G-2. It was separated from smaller organic molecules by a 5 column (loxlcm). column Elute with 0.05M phosphate (pH 6.0) and collect 1.5ml of each aliquot. I took it. Fraction containing APase activity (Hoyt volume, vo id vo fu me) were pooled (approximately 4 ml).

Reaction deacetylation of C collagen-3ATA slice and AP as e-VHS collagen-5ATA slices in 0.05M phosphate (pH 6.0) Add 4 ml of APase-VHS and incubate the slices at room temperature for 4 hours. I did it. The solution was then decanted and the slices were washed as described in Example 1. did. Slices were stored in glycine buffer at 4°C. Elephant Collagen Sura The chair contains 1,36 ± 0.28 mU APase/μg, bone collagen slurry. The chair contained 2.39 ± 0.41 mU APase/μg for this experiment. The results are summarized in Figure 1. The numbers used were as follows: Skull OF Elderly female-- Elephant material/belly OM Elderly male -△-Bone/Ill YF Young female -+- Bone/Skull YM Young male The results are based on mineralization (Y-axis), sex, age, and phosphoprotein (P P] content. Especially bone-derived slices (PP-containing). compared to slices derived from Elephant Hyaku (which has a relatively high PP content). It was proven that the degree of mineralization was extremely low. Male rats have higher mineral uptake than females (measured by phosphate content). Young animals produce more minerals than older animals. showed uptake of substances. Graft mineralization and serum inorganic phosphate concentration (X axis) A high correlation was observed between them.

Only slight differences were seen between the various implantation sites, with dentin-derived The slices showed a slightly higher degree of mineralization than dorsal and ventral cases. lower back area No difference was found between the abdominal wall and the abdominal wall.

All control slices remained mineral-free within the two-week experimental period. .

IG 7 P;, = 4? (17111018) Submission of translation of written amendment (Article 184-8 of the Patent Law) January 7, 1994 1. Display of patent application PCT/GB92101247 2. Name of the invention graft material 3. Patent applicant Address: Nised 6B, London, UK.

Newington Causeway 101 Name British Technology ・Group Limited 4, Agent Address: Shin-Otemachi Building, 206-ku, 2-2-1 Otemachi, Chiyoda-ku, Tokyo Phone: 3270-6641~6646 Name (2770) Patent attorney Kyozo Yuasa 5. Date of submission of written amendment Article 34 amendment billing savings 1. Combined with a sufficient amount of phosphatase enzyme to promote mineralization of the material , consisting of a biocompatible carrier material having a dense W4-structured fibrous W4 structure; Material useful for skeletal repair.

2. Hydroxyproline 1. At least 0.5 mU of phosphatate per Oμg The material according to claim 1, characterized in that it exhibits enzymatic activity.

3. At least 1.0 mU of phosphater per 1.0 μg of hydroxyproline 3. The material according to claim 2, characterized in that it exhibits enzyme activity.

4 Claim characterized in that the phosphatase enzyme is alkaline phosphatase The material according to item 1, item 2 or item 3 of the range.

5 In addition, bound organic or inorganic phosphate, phosphoprotein and/or or Elephant Lightning phosphophorine. Material according to any of paragraph 4.

6. The carrier material is at least 0.03 μg per μg of hydroxyproline. Any one of claims 1 to 5, characterized in that it contains a phosphate of g. Materials listed in

7. The biocompatible carrier material demineralizes calcified human or animal tissue. Claims 1 to 6 characterized in that the product is prepared by Materials listed in any of the above.

8. characterized in that the carrier consists of at least a substantial proportion of proto-male collagen; 7. The material according to claim 7.

9. If the carrier is made from demineralized elephant lightning or demineralized bone. The material according to any one of claims 1 to 8, characterized in that:

10. A claim characterized in that it contains at least 200 U of phosphatase/cm'. The material according to any one of claims 1 to 9.

11 A claim characterized in that the phosphatase enzyme is covalently bonded to the carrier. The material according to any one of items 1 to 10.

12. The biocompatible carrier and enzyme are optionally combined in a solution of the multifunctional binding agent. in the presence of organophosphorus compounds and/or calcium and inorganic phosphates. Incubate in the presence of ions, in each case present in the carrier material The material according to claim 11, characterized in that it is added to any of the Production method.

13. combined with sufficient amounts of phosphatase enzymes to promote mineralization of the material , consisting of a biocompatible carrier material with a densely structured fibrous W4 structure. Shaped grafting material used for case repair.

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Claims (27)

[Claims] 1. raw materials in combination with sufficient amounts of phosphatase enzymes to promote mineralization of the material. A material useful for skeletal repair comprising a biocompatible carrier material. 2. At least 0.5 mU of phosphater per 1.0 μg of hydroxyproline 2. The material according to claim 1, characterized in that it exhibits a level of enzymatic activity. 3. At least 1.0 mU of phosphater per 1.0 μg of hydroxyproline 3. The material according to claim 2, characterized in that it exhibits a level of enzymatic activity. 4. A claimed invention, characterized in that the phosphatase enzyme is alkaline phosphatase. The material according to any one of items 1 to 4 of the requested range. 5. characterized in that the phosphatase enzyme is covalently bound to the carrier. The material according to any one of items 1 to 4 of the requested range. 6. Claim 5, characterized in that the binding is carried out using a polyfunctional binding agent. Materials listed in Section. 7. The binder is glutaraldehyde, 1-ethyl-3(3-dimethylaminopropylene) ) carbodiimide and SATA-MHS (succinimidyl-S-acetyl thioacetate and maleimidohexanoyl-N-hydroxysuccinide). 7. Material according to claim 6, characterized in that it is selected from the group consisting of: 8. According to claim 7, characterized in that the binder is SATA-MHS. Materials included. 9. In addition, bound organic or inorganic phosphates, phosphoproteins and/or or dentin phosphophorin. Material according to any of Item 8. 10. At least 0.03 μg phosphate per μg of hydroxyproline 9. Material according to claim 8, characterized in that it comprises. 11. The carrier material is at least 0.03 μg per μg of hydroxyproline. Material according to claim 9, characterized in that it inherently contains a phosphate of g. . 12. Additional amounts of organophosphate, phosphoprotein or dentin phosphophorine may be Material according to claim 9, characterized in that it is combined with a carrier material. . 13. Additional amounts of calcium ions and inorganic phosphate are bound to the carrier material. Claims 9 to 11, characterized in that Materials included. 14. The biocompatible carrier material demineralizes calcified human or animal tissue. Claims 1 to 1 are characterized in that they are prepared by The material described in any of Section 3. 15. that the carrier consists of at least a substantial proportion of fibrillar collagen; 15. A material according to claim 14, characterized in that: 16. Claimed characterized in that the carrier consists of demineralized dentin. The material according to any one of the ranges 1 to 15. 17. Claim 1, characterized in that the carrier consists of demineralized bone. The material according to any one of Items 1 to 15. 18. characterized in that it contains at least 200 U of phosphatase/cm3, A material according to any one of claims 1 to 17. 19. Incubate the biocompatible carrier and enzyme in a solution of the multifunctional binding agent. according to any one of claims 1 to 18, characterized in that: manufacturing method of the material. 20. Claims characterized in that the polyfunctional binder is glutaraldehyde. The method according to paragraph 19. 21. The polyfunctional binder is 1-ethyl-3(3-dimethylaminopropyl)carboxylate. 20. Process according to claim 19, characterized in that it is a diimide. 22. The biocompatible carrier is succinimidyl-S-acetylthioacetate. and the enzyme was incubated in a solution of maleimidohexanoyl-N-hydrochloride. Incubate with xysuccinimide and react the enzyme with this carrier. 20. The method according to claim 19, characterized in that the method comprises: 23. Incubation is carried out in the presence of an additional amount of organophosphate compound. The method according to any one of claims 8 to 22, characterized in that Law. 24. The organophosphate compound is a phosphoprotein or dentin phosphophorin 23. A method according to claim 22, characterized in that: 25. Incubation in the presence of calcium and inorganic phosphate ions Any one of claims 8 to 23, characterized in that the invention is implemented in Method described. 26. A graft made of the material according to any one of claims 1 to 17. processing the animal skeleton to promote bone mineralization and growth, consisting of inserting How to fix it. 27. Replenish organophosphorus compound concentrations near the implant site from an extracorporeal source 27. A method according to claim 26, characterized in that: