JPH0788174A - Implant for osteogenesis - Google Patents

Abstract

PURPOSE:To provide an implant for osteogenesis contg. a recombination human bone induction factor (rhBMP). CONSTITUTION:This implant is composed of the rhBMP and a carrier consisting of at least one kind among atherocollagen, a polymer or copolymer of a lactic acid and/or glycolic acid or a block copolymer of the polymer or copolymer of the lactic acid and/or glycolic acid and a polyethylene glycol. As a result, the implant has an excellent osteogenetic ability even with Primates with which osteogenesis is considered to hardly take place.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to an osteogenic implant. In particular, it relates to an osteogenic implant comprising a recombinant human osteoinductive factor (rhBMP) and a biodegradable carrier.

[0002]

2. Description of the Related Art Bone morphogen
Enetic protein (BMP) is an active protein that acts on undifferentiated mesenchymal cells in subcutaneous tissue or muscle tissue to differentiate them into chondroblasts or osteoblasts to form cartilage or bone. BMP was discovered as a substance showing ectopic osteoinductive activity present in bovine demineralized bone matrix, but it was not isolated purely and its specific structure remained unclear. However, due to the technology of genetic engineering, human B
The gene encoding MP was cloned and the amino acid sequence was revealed. It was also found that human BMPs constitute a family of closely related proteins having amino acid sequence homology, and many kinds of recombinant human osteoinductive factors (rhBMPs) have been created [[ Science Vol. 242, pp. 1528-
1534 (1988); Proc. Natl. Aca
d. Sci. USA Vol. 87, pp. 2220-
2224 (1990); Progress in Gr
owth Factor Research, Vol.
1, pp. 267-280 (1989); Tokuyohei 2-5
No. 00241, Tokuyo Hyodai 3-503649, Tokuyo Hyodai 3-
No. 505098, WO91 / 18098, WO92 / 0
5199, WO93 / 09229]. In addition, production by transformants is also performed.

Various methods have been proposed for the treatment of damages, defects or hypoplasia of bone or cartilage using the BMP since the structure of the BMP was unclear. For example, a transplant body in which a ceramic material support is impregnated with BMP and a collagen carrier (JP-A-60-253).
455) or a polymer or copolymer of lactic acid and / or glycolic acid, which is a biodegradable polymer, or a block copolymer of the polymer or copolymer and polyethylene glycol, as a support for BMP. Body (JP-A-2-203861 or JP-A-3-45265)
Each of the publications) has been proposed as a technique for adding or transplanting it to the treatment site.

However, the BMPs described in these patent publications are crude BMPs obtained by extracting and purifying bone sarcoma cells or bone tissues of animals other than humans (for example, bovine).
And contains multiple types of BMPs. Similarly, as an extracted component from bone tissue, various bone formation-related proteins other than BMP, such as transforming growth factor (TGF-
β: transforming growfac
tor β), osteogenic factor (OIF)
ND, insulin-like growth factor (IGF)
owth factor) or platelet-derived growth factor (P
DGF: platet driven growt
h factor) and the like, and it is also suggested that the conventionally known crude extraction BMP may be a mixture containing substantially these proteins [Progre.
ss in Growth Factor Resea
rch, Vol. 1, pp. 267-280 (198
9); The Journal of Biologi
cal Chemistry Vol. 264, No. 264.
32, pp. 20805-20810 (1989); Japanese Society for Bone and Metabolism 7 (3) p196 (1989)].

Therefore, the above-mentioned JP-A-60-253455 is used.
When the techniques described in JP-A-2-203861 and JP-A-2-203861 are applied to humans, not only there is a problem of immunity but also interaction between a plurality of proteins and a carrier, and a support which may be further used in some cases. It is not known whether there are side effects due to the interaction with or the combination thereof, and there were many problems in clinical use in humans. Further, since the crude BMP itself also contains many impurities other than the above, which are derived from the raw material thereof, it was not satisfactory in terms of immunity and safety when used in humans. Also,
Since it is a trace amount extract, it is difficult to mass-produce components having a certain standard, industrial production is difficult, and there are many problems in practical use.

[0006] Actually, the above-mentioned JP-A-60-253455.
In Japanese Patent Laid-Open No. 2-203861 and Japanese Patent Laid-Open No. 2-203861, crude BM is used.
The osteogenic effect of P has only been confirmed in mice. It has also been widely known that it is more difficult to form bone in primates such as humans when forming bones with crude BMP than in rodents such as mice. For example, it has been reported that crude BMP does not induce osteoinduction in primates (P. Aspenberg, et al, J. of B.
one and Joint Surgery, 70-
B: 625, 1988), a report that the osteoinductive reaction is poor (S. Hosny, et al, J. Oral. Ma).
xillofac Surg. , 43: 837, 198.
5), and further reports that the osteoinductive response is delayed (U.
Ripamonti, et al, Clin. orth
op. , 269: 284, 1991). As described above, even in the above-mentioned JP-A-60-253455 and JP-A-2-203861, even if a carrier in which bone formation was observed in a mouse using crude BMP, it is clinically useful in humans. Sex has been questioned.

On the other hand, recombinant human osteoinductive factor (rhBM
Unlike the above-mentioned crude BMP, P) has no problem in immunity when applied to humans, and can be expected to exhibit clinically stable pharmacological action and safety, and is highly practical. However, conventionally, the osteoinductive activity of rhBMP in primates has not been clarified, and therefore, no carrier or support effective for osteoinduction of rhBMP in primates has been known at all.

[0008]

Therefore, the object of the present invention is to ensure that rhBMP is safe in terms of immunity and the like even when applied to humans.
Can be applied to, and has sufficient bone formation ability even in primates, which have been considered to be difficult to form bone,
It is intended to provide a clinically useful bone forming implant.

[0009]

The above objects are at least (1) a human osteoinductive factor substantially free of other human-derived proteins, and (2) atelocollagen; lactic acid and / or glycolic acid. Bone of the present invention, characterized in that it contains a carrier or a copolymer or a polymer of lactic acid and / or glycolic acid and at least one block copolymer of polyethylene glycol. This can be accomplished with a plastic implant.

The present invention will be described in detail below. The transplant of the present invention comprises at least (1) rhBMP and (2) a biodegradable carrier. The transplant of the present invention is mainly a bone (including cartilage) in vivo, similar to the conventional allograft.
To be used by transplanting to the site to be formed. When the transplant of the present invention is transplanted in vivo, rhBMP acts locally at the implanted site to induce bone formation. Delivery that locally retains this rhBMP and forms the desired bone
The biodegradable carrier plays a role as a system. The carrier of the present invention is a pure carrier that does not cause foreign body reaction or immune reaction, and as shown in Examples below, rhBMP
It has been confirmed to induce periosteal bone formation or ectopic bone formation in primates in combination with.

The human osteoinductive factor (human BMP) that can be used in the present invention is a human osteoinductive factor that does not substantially contain other human-derived proteins, and is a human BMP produced by gene recombination technology. Any of them can be used. That is, a transformant (cell or microorganism) containing a recombinant DNA containing a nucleotide sequence encoding a human osteoinductive factor is cultured, and the recombinant human osteoinductive factor produced by these transformants is isolated and purified. It is a recombinant human osteoinductive factor (rhBMP) prepared by. Examples of these human osteoinductive factors (rhBMP) include rhBMP-
2, rhBMP-3, rhBMP-4 (rhBMP-2
B), rhBMP-5, rhBMP-6, rh
BMP-7, rhBMP-8, rhBMP heterodimer or recombinant human BMP-2-like protein other than these can be used alone or as a mixture of two or more kinds.

These rhBMPs can be expressed in mammalian cells (eg CHO cells), microorganisms (eg E. coli) or yeast cells. As rhBMP for which mass production and purification methods have already been established,
There is hBMP-2, but other rhBMPs can be similarly produced, purified and used [Progress.
in Growth Factor Research
h, Vol. 1, pp. 267-280 (198
9)]. The already-known purified rhBMP-2 is a mature peptide composed of a plurality of monomers having different N-terminal amino acid sequences, and is a dimeric protein having a molecular weight of about 30,000. Each of the monomers, high to Asn ⁵⁶ residue
It has a mannose-type sugar chain [Abstract
Sixth Interaction Symposs
ium of the ProteinSociet
y, San Diego, CA (1992)].

The transplant of the present invention is substantially the same as the rhBM.
It can be composed of P and a carrier. The carrier in this case holds the rhBMP locally for a certain period of time in which the implant is implanted, and also provides a field in the early stage of the bone (or cartilage) formation induced by rhBMP. Therefore, it must not easily move from the implanted site by flow or the like, and it must be one that does not dissolve or disperse within the period required for bone formation in the living body. Further, the carrier is required to have a certain shape as a transplant and to be transplantable to a predetermined site in a living body when the transplant is formed together with rhBMP. Implants are, for example, solids, plastic semi-solids (eg clay-like or pasty), or highly viscous fluids (eg highly viscous gel).
Can be Further, it preferably has a property of adhering to in-vivo tissue.

During the bone formation process by rhBMP, rhB
The carrier containing MP is decomposed in vivo and replaced with it, and bone formation proceeds. Therefore, the carrier used in the present invention must be biodegradable.
At this time, the decomposition rate affects the size and properties of the formed bone. That is, if the decomposition rate is too fast, the bone formation becomes insufficient, so that the bone formed becomes small and the bone strength becomes insufficient. On the contrary, if the decomposition rate is slow, even if a part of the bone is formed, the carrier remains for a long period of time, which may cause the delay of the bone formation, and further, the mechanical properties or the bone structure may be defective. Therefore, those having an optimum biodegradability are preferable. The optimal biodegradation rate varies depending on the site of bone formation, purpose, etc., but is generally several days to several weeks, preferably about 2-3 weeks. In addition, the carrier must have biocompatibility, cytotoxicity, antigenicity, carcinogenicity, and the like, and should not cause foreign body reaction, and can be supplied industrially at low cost, and transplant surgery At this time, it is preferable that the handling is easy.

In the present invention, as biodegradable carriers satisfying the above conditions, atelocollagen and polylactic acid (PL) are used.
A), polyglycolic acid (PLG), lactic acid-glycolic acid copolymer (PLGA), block copolymer of polylactic acid and polyethylene glycol (PLA-PEG), block copolymer of polyglycolic acid and polyethylene glycol (PLG-PEG) and / or a block copolymer of a lactic acid-glycolic acid copolymer and polyethylene glycol (PLGA-PEG) is used.

Atelocollagen is a non-antigenic collagen obtained by removing an antigenic telopeptide from collagen derived from an animal tissue other than human such as bovine or porcine (for example, Japanese Patent Laid-Open No. 62-89629; (2) S43 (1
988)], for example, a commercially available product (eg, Cellmatrix LA, manufactured by Nitta Gelatin Co., Ltd.) can be used. Further, polymers or copolymers of lactic acid and / or glycolic acid, and block copolymers thereof with polyethylene glycol have also been conventionally used as biocompatible materials (see, for example, Japanese Patent Application Laid-Open No. HEI-2).
-203861 and JP-A-3-45265), these synthetic polymer materials can also be used as carriers in the present invention.

The preferred carrier for the implant of the present invention is
Atelocollagen or number average molecular weight of about 300-10
It is a polymer or copolymer of 50,000, especially about 400 to 50,000 lactic acid and / or glycolic acid. Further, a polymer or copolymer of lactic acid and / or glycolic acid having a number average molecular weight of about 300 to 10,000, particularly about 400 to 5,000, and a number average molecular weight of about 150 to 10,
000, especially about 150 to 2,000 polyethylene glycol, and the equivalent ratio of the latter to the former is 0.3 to
A block copolymer prepared by polymerizing at a ratio in the range of 5.0 is also preferable. Among these carriers, particularly preferable ones are those which are biodegradable in vivo within a few days to a few weeks (especially about 2 to 3 weeks) and have a biodegradation rate suitable for bone formation. Those having such a biodegradation rate include, for example, atelocollagen or a number average molecular weight of about 55.
0-750 polylactic acid and number average molecular weight of about 150-25
No. 0 block copolymer with polyethylene glycol, particularly preferably polylactic acid (number average molecular weight about 650) -polyethylene glycol (number average molecular weight about 200) block copolymer and the like.

The implant of the present invention consisting essentially of rhBMP and a carrier can be produced by supporting rhBMP on a carrier. The method for supporting rhBMP on a carrier is not particularly limited as long as it is a method known to those skilled in the art, and a rhBMP solution (or suspension) in a suitable liquid is used.
A solution or suspension containing the above and a carrier is mixed, dried (preferably freeze-dried), and molded into an appropriate shape by compression or the like, a method of molding the fine powder rhBMP and the carrier as it is by compression, or the like. After the carrier is molded into the shape of (for example, granules, microspheres, films, porous solids, etc.), it is impregnated with a rhBMP solution (or suspension) and dried (preferably freeze-dried), the carrier itself. When is a liquid or gel, there is a method of directly dissolving or suspending rhBMP. These carriers can be appropriately used by mixing or combining carriers of any type and / or any form.

The transplant of the present invention is easy to handle,
Maintaining a certain form as an implant, improving mechanical strength,
Alternatively, for the purpose of providing a microenvironment more suitable for osteogenesis of rhBMP, in addition to the completely same carrier as described above, a secondary carrier, that is, a support can be contained.
The support is a molded body (hereinafter,
Sometimes referred to as a molded support), or rhB
Before being combined with the MP and the carrier, it may have an amorphous shape (for example, powder or granules) and be molded into a predetermined shape together with them (hereinafter, also referred to as an amorphous support). Good. These supports are appropriately selected according to the site to which the transplant is applied, the size and shape of the bone defect requiring repair, the required mechanical strength, and the like.

Like the above-mentioned carrier, the support used in the present invention is required to have biocompatibility, have no cytotoxicity, antigenicity, carcinogenicity, etc. and not cause foreign body reaction. Further, it is required that the carrier can be physically and easily combined with the carrier. Further, it is preferable that it is supplied industrially at low cost and is easy to handle during transplant surgery. The support used in the present invention may be non-degradable or biodegradable in vivo, but when a non-biodegradable support is used, it should be stable without changing its properties in vivo for a long period of time. Is preferred. Further, it is advantageous that the physical properties thereof are as equal as possible to the bone tissue, because the bone-like tissue composed of the formed bone and the residual support has properties close to those of natural bone.
Furthermore, when the affinity with bone tissue is excellent, more preferable continuity with autologous bone in a living body is expected.

In the present invention, as the biocompatible support satisfying the above conditions, for example, a support made of a polymer or copolymer of lactic acid and / or glycolic acid, a ceramic material or a metal material is used. it can. The polymer or copolymer composed of lactic acid and / or glycolic acid used as a support has a number average molecular weight of about 5,000.
~ 1,000,000, especially about 10,000-50
Mention may be made of polylactic acid (PLA), polyglycolic acid (PLG) or lactic acid-glycolic acid copolymer (PLGA) which is solid at room temperature and has a temperature of 10,000. These biodegradable polymers or copolymers have good moldability and strength, and have a function as a support. The polymer or copolymer is required to support rhBMP and a carrier for a certain period of time to impart plasticity or shape retention to the implant,
Degradability in vivo is slower than that of the carrier. That is, it is desirable that the rhBMP and the carrier function as a support during the initial bone formation and then gradually decomposed and absorbed. The decomposition rate varies depending on the size, shape and application site of the support, but is generally several weeks to 2 years, preferably about 1 to 10 months.

Examples of the ceramic material used as the support include bio-related ceramics (implant ceramics) which are preferably bioceramics. Specifically, hydroxyapatite [Ca
₁₀ (PO ₄ ) ₆ (OH) ₂ ], tricalcium phosphate (T
CP) [Ca ₃ (PO ₄ ) ₂ ], bioglass [SiO
_{2 -CaO-Na 2 O-P} 2 O 5 ], Serabitaru [S
_{iO 2 -CaO-Na 2 O-} P 2 O 5 -K 2 O-Mg
O], CPSA glass fiber composites [CaO-P ₂ O ₅ -
SiO ₂ -Al ₂ O _3], apatite - wollastonite (A-W) glass ceramic [SiO ₂ -CaO-
MgO-P ₂ O _{5], β-Ca 3 (PO 3} ) 2 glass ceramic [CaO-P ₂ O _5], alumina [Al ₂ O
₃ ], zirconia [ZrO ₂ ], carbon [C], titania [TiO ₂ ], sapphire [Al ₂ O ₃ ], silicon carbide [SiC], silicon nitride [Si ₃ N ₄ ] or calcium phosphate [Ca ₃ (PO _{3 2} )] and the like. Of these, particularly preferable are hydroxyapatite, TCP, bioglass, cerabital, CPSA glass fiber composite material, or AW glass ceramics, which have excellent affinity to bone, or alumina or zirconia, which has excellent strength. Hydroxyapatite is most preferred.

Examples of the metal material include titanium alloy, Co-Cr alloy, stainless steel, nickel-titanium type shape memory alloy and the like. It is preferably a titanium alloy. These materials can be used alone or in combination to prepare the support.

As the non-degradable support in vivo, when a support having excellent affinity with bone tissue and having the same physical properties as bone tissue as much as possible is used, a new bone to be formed and the remaining support are used. And are present in good condition, and the overall properties thereof are close to those of natural bone, which is advantageous. Examples of such a support include bioceramics. Especially hydroxyapatite, TCP, bioglass, cerabital, CPSA, which has excellent affinity with bones
Glass fiber composites and AW glass ceramics are preferred.

When the transplant of the present invention contains the above-mentioned support and the support or the rhBMP-support complex is supported on the support, the support itself is a liquid (solution or suspension, etc.). ) Or a low-viscosity fluid (low-viscosity gel, etc.). The amorphous support that can be used in the present invention is, for example, in the form of powder or granules, solid, plastic semi-solid,
It can be mixed with rhBMP and / or carrier in any state such as high-viscosity fluid state or low-viscosity fluid state, or liquid state. In particular, when the rhBMP-carrier complex is a liquid or a low-viscosity fluid, a powdery or granular amorphous support is added to impart plasticity to them, and a plastic semi-solid (for example, clay-like or paste-like) is added. ) Or a highly viscous fluid (eg highly viscous gel) implant is preferably prepared. That is, by adding these supports to the rhBMP-carrier complex, it is possible to impart a certain morphological maintenance ability to the transplant, facilitate the handling of the transplant during surgery, and, in vivo, to improve the transplantability of the transplant. Fixing and maintaining the shape to give more strength.

As the powdery or granular amorphous support, the above-mentioned materials which are non-degradable or degradable in vivo can be used. The size of the granule is not particularly limited, but from the viewpoint of operability and adaptability, the diameter is preferably several mm.
Hereafter, it is more preferably 1 mm or less. Furthermore, it is preferable to use porous granules. The powder or granule used in the present invention is required to have biocompatibility, cytotoxicity, antigenicity or carcinogenicity and not cause any foreign body reaction, and is supplied industrially at low cost. Those are preferable.

The implant of the present invention using an amorphous support is, for example, powdered with rhBMP and a gel or liquid carrier [eg PLA (average molecular weight 550 to 750) -PEG (average molecular weight 150 to 250)]. Paste-like or clay-like transplant in which body-shaped or granular hydroxyapatite and / or PLGA is combined, powdered rhBMP and atelocollagen are mixed with powdered or granular hydroxyapatite and / or PLGA, and compressed, etc. The transplant body etc. which were shape | molded by these can be mentioned.

The molded support of the present invention is one which has been molded into a predetermined shape in advance and is capable of supporting rhBMP and a carrier on the surface and / or inside thereof. In particular, having a porous portion on the surface of the molded support,
This is advantageous because rhBMP and a carrier can be easily loaded in the pores, and cells involved in bone formation in a living body can easily enter. Therefore, porous molded supports are most preferred. That is, the above-mentioned porous molded support has at least a porous portion on its surface, and the entire support is porous, or is composed of a porous surface layer and a non-porous core portion. It may be one. Further, a support comprising at least a porous portion having a porous surface and a non-porous portion having a non-porous surface is also included. The porous portion has a large number of pores, and those pores preferably have an average pore diameter of 50 to 1,000 μm, particularly preferably 100 to 300 μm. When the average pore diameter is within the above range, particularly, the bone formation of rhBMP inside the pores of the support proceeds well.

The porosity of the porous portion is preferably 30%.
The above is more preferably 70 to 95%. When the porosity is high, new bone enters the pores due to bone formation in the pores, and finally, the proportion of the new bone tissue formed is high, and the proportion of the residual support is high. Since it becomes low, bone formation close to that of natural bone can be expected. However, on the other hand, if the porosity is high, the mechanical strength of the support may decrease. Therefore, it is preferable to appropriately adjust the porosity in accordance with the conditions such as the adaptation site, the size of the transplant, and the properties of the support.

Furthermore, the biocompatible porous support formed into a predetermined shape by using a material which is not degradable or hardly degradable in the living body, maintains its shape stably in the living body for a long period of time. As described above, the rhBMP-carrier complex is provided with a field of initial bone formation, and the three-dimensional morphology of new bone finally formed by further progress of osteogenesis induced by the rhBMP-carrier complex. Is also advantageous because it also has the effect of controlling mechanical strength and imparting mechanical strength.

When the implant of the present invention having a support is transplanted in vivo, the osteogenic process proceeds due to the action of rhBMP. The undifferentiated mesenchymal cells and capillaries invade the surface and / or the inside of the transplant, and particularly inside the pores of the porous part when the support has a porous structure, and the bone (cartilage) by the action of rhBMP is introduced. ) Inducing action occurs. After that, as the bone formation process progresses, the biodegradable carrier gradually decomposes and disappears, and gradually replaces the newly formed bone (or cartilage). With a porous support, new bone (or cartilage) is gradually formed in the internal pores. Bone formation progresses even after the biodegradable carrier has decomposed and disappeared, and finally,
At the implant site, new bone tissue is formed along the shape of the implant. That is, a bone (or cartilage) -like tissue consisting of a non-degradable or undegraded residual support and new bone formed around the support or new bone that has entered the porous portion of the porous support is It is formed.

The implant of the present invention, including the support, is a rhBM
It can be produced by supporting P on a carrier and then adding or supporting P on a support, for example, a porous molded support or a powdery or granular support. rhBM
The method for supporting P on the carrier is not particularly limited as long as it is a method known to those skilled in the art, and a rhBMP solution (or suspension) in a suitable liquid and a solution or suspension containing the carrier are mixed,
Drying (preferably freeze-drying) and molding into a suitable shape by compression, molding of fine powder rhBMP and carrier as it is, compression of a certain shape (eg granules, microspheres, membranes) , A porous solid form, etc., and then impregnated with a rhBMP solution (or suspension) and dried (preferably freeze-dried). If the carrier itself is liquid or gel, rhBMP is directly dissolved. Alternatively, it can be carried out by a suspension method or the like.

The implant of the present invention containing a powdery or granular support that imparts plasticity can be prepared by adding these powders or granules at an appropriate stage of the step of supporting rhBMP on a carrier, or rhBMP-. It can be manufactured by adding the carrier complex after it has been prepared. For example, when the carrier is a solid, a method of molding by adding a support when molding the rhBMP and the carrier by compression or the like, and when the carrier is a gel or liquid, after mixing the rhBMP and the carrier It can be added to a support to prepare a paste or clay.

Furthermore, the implant of the present invention containing a molded support (particularly a porous support) can be produced by allowing rhBMP and a carrier to be carried on a porous support molded into a predetermined shape. . The shape of the porous support, the proportion of the porous portion in the support, and the like are arbitrarily determined depending on the implantation site, the shape of bone to be formed, and the like. The method of supporting rhBMP and the carrier on the support is not particularly limited, and for example, a rhBMP solution (or suspension) in a suitable liquid may be used.
To the support by a method such as dipping, impregnation, spraying, coating or dropping with a solution (or suspension) containing a carrier.
MP and carrier can be added, followed by drying (preferably vacuum or lyophilization) to obtain the desired implant.
When the carrier itself is in liquid or gel form, it may be treated in the same manner as above, but rhBMP is directly dissolved or suspended in the liquid carrier and added to the support (preferably impregnation under reduced pressure, etc. It can also be used as a transplant. In any case, a method is preferred in which the rhBMP and the carrier can be dispersed almost uniformly throughout the porous portion of the support without losing the activity of the rhBMP and the carrier.

The amount of rhBMP carried by the transplant, whether it contains a support or not, may be higher than the minimum rhBMP concentration required to induce bone formation (minimum bone formation expression concentration). The amount of bone formation increases with an increase in rhBMP concentration, but above a certain value, the amount of bone formation becomes almost constant. In general, this amount of bone formation roughly matches the size of the implant. That is, even if the concentration is excessively increased, bone larger than the size of the implant is not formed. Further, the concentration of rhBMP in the transplant at that time varies depending on the carrier and the support.

The size and shape of the bone formed by implanting an implant containing an appropriate concentration of rhBMP depends in principle on the size and shape of the implant. Therefore, the specific mixing ratio and rhBMP concentration are such that the type of carrier, the type and shape of the support, the porosity of the porous portion, the average pore diameter, and the powder or granular support when the support is used. Is
It can be appropriately determined according to its type, particle size, application site of the implant, preferable bone formation rate, and the like. For example, when atelocollagen or a block copolymer of PLA (number average molecular weight = about 650) and PEG (number average molecular weight = about 200) is used as a carrier, and rhBMP-2 is used to induce osteoinduction in primates It is preferable that rhBMP-2 is loaded in an amount of 30 μg / volume of bone or cartilage to be formed of 1 ml or more, preferably 50 to 1,000 μg / 1 ml, more preferably 70 to 500 μg / 1 ml. When a support is used, for example, the above rh
When BMP is supported on a carrier as hydroxyapatite (porosity = about 90%; average pore size = 200 μm) as a support, rh per volume of bone or cartilage to be formed
The concentration of BMP is the same as the above or lower.

The amount of the carrier in the case of having a porous support as the support varies depending on the type of the carrier, the type and shape of the support, the degree of porosity, etc., but the porosity of the support is preferred. The amount is such that a preferable amount of rhBMP can be carried on the entire quality portion for a certain period. However, it is not preferable to use a large amount of the carrier so as to block the porous portion of the support, because the invasion of cells in the living body into the porous pores is hindered. For example, atelocollagen or PLA-PEG
In the case of, it is preferable that the amount of rhBMP is 10 to 10,0.
It may be about 100 times by weight, and may be in the range of the above-mentioned preferable rhBMP concentration per volume of bone or cartilage to be formed. When a powdery or granular amorphous support is used, the support may be added in any amount as long as it can impart appropriate plasticity to the transplant, and rhBMP
The concentration may be in the range of the above-mentioned preferable rhBMP concentration per volume of bone or cartilage to be formed.

The implant of the present invention can carry components other than those mentioned above with or without a support. As those desired components, specifically, stabilizers,
Preservatives, solubilizers, pH adjusters, thickeners and the like can be mentioned. Also, additional components useful for bone or cartilage formation,
For example, mineral components such as calcium phosphate and hydroxyapatite, action promoting components such as co-factors for bone or cartilage formation, fibronectin, osteonectin, non-antigenic IBM (insoluble bone matrix) and the like can be included. These desired components can be added by a suitable method at a suitable stage of producing the transplant of the present invention.

The implant of the present invention substantially defines the shape of the new bone formed as described above. That is, bone formation occurs corresponding to the shape of the implant. Therefore, it is desirable to mold the implant of the present invention along a shape that is expected to cause bone formation. Of course, a plurality of transplants may be used in combination. When the above-mentioned transplant is locally fixed, the shape is maintained or the strength is not sufficient, other known reinforcing materials can be used together. The reinforcing material is, for example, a biocompatible membrane for fixing an implant, such as a collagen membrane or G.
A GORE-TEX film or the like used in the TR method, or a fixture for the implant of the present invention and a tissue in the living body (particularly bone), such as a metal plate, a bone-coupling pin or a fixing nail, and these reinforcing materials are necessary. If so, it can be surgically removed after bone formation. Also, it may be used in combination with other known transplants.

The transplant of the present invention may be prepared at the time of use, or may be stored under suitable conditions after preparation until use. The transplant of the present invention can be transplanted to an affected area by a method known in the art in order to repair various bone or cartilage defects. Like a conventionally known transplant, it is applicable to a living body, its purpose, use,
It can be appropriately applied according to the usual method of those skilled in the art, depending on the indication site, patient's condition, and the like. The implant of the present invention containing no support is suitable as a relatively small implant for use in repairing a partial defect of bone. On the other hand, the implant of the present invention containing a support is capable of maintaining its shape in a living body for a long period of time and has sufficient strength, so that it can be made into a large implant and can be used for large bone defects. Can be used.

The implant of the present invention can effectively induce osteogenesis even when it is applied to primates, which generally have difficulty in osteogenesis. Therefore, the transplant of the present invention,
Although it is preferably used for primates such as humans, it can naturally induce bone formation when applied to other rodents.

[0042]

EXAMPLES The present invention will be described in more detail below with reference to examples, but these do not limit the scope of the present invention. Example 1 Pig skin-derived atelopeptide type I collagen (hereinafter,
An aqueous solution (collagen I concentration = 0.3%; pH 3.0) containing 20 mg of collagen I was added to rhBMP-
2 (manufactured by Yamanouchi Pharmaceutical Co., Ltd.) 0.5 including 100 μg
M arginine-10 mM histidine buffer (rhBMP
-2 concentration = 1.67 mg / ml) was added and mixed.
The mixture was freeze-dried and then compression molded to prepare an implant. A compression molded implant was prepared that differs from the implant only in the absence of rhBMP-2 and was used as a control.

Each of these transplants was implanted under the tibial periosteum of cynomolgus monkeys (12 cases each). It was collected 4 weeks after the implantation, and the presence or absence of osteoinduction was observed by X-ray and histology.
In the rhBMP-2 containing transplant, periosteal bone formation was observed in 10 out of 12 cases. A bony prominence was formed continuously from the periosteum, and histologically, it contained bone and cartilage and was callus-like. No bone formation was observed in the control implants without rhBMP-2. The soft X-ray photographs of both are shown in FIG. The upper side of FIG. 1 shows the case where the rhBMP-2 containing transplant was used, and bone formation was observed (in the upper photograph of FIG. 1, a shadow observed below the middle portion between the center and the right end is newly generated. Bone). In the lower side of FIG. 1, when the control transplant was used, bone formation was not observed in the transplant.

Example 2 Viscous polylactic acid (molecular weight 650) -polyethylene glycol (molecular weight 200) block copolymer (hereinafter referred to as P
LA-PEG) 100 mg and rhBMP-2 2
The mixture was mixed with 0 μg, and the viscous gel-like mixture obtained was implanted under the tibial periosteum of cynomolgus monkeys (8 cases). rhB
A gel-like implant different from the above-mentioned implant only in that it did not contain MP-2 was used as a control, and was similarly implanted (8 cases). It was collected 4 weeks after the implantation, and the presence or absence of osteoinduction was observed by X-ray and histology. In the rhBMP-2 containing transplant, periosteal bone formation was observed in 8 out of 8 cases. A bony prominence was formed continuously from the periosteum, and histologically, it contained bone and cartilage and was callus-like. No bone formation was observed in the control implants.

Example 3 An aqueous solution containing 5 mg of collagen I used in Example 1 (concentration of collagen I = 0.3%; pH 3.0) was added with r.
0.5M arginine-1 containing 50 μg of hBMP-2
0 mM histidine buffer (concentration of rhBMP-2 = 1.
67 mg / ml) was added and mixed. The resulting mixed solution was treated with a porous hydroxyapatite block (porosity 90
%; Average pore diameter 200 μm: hereinafter abbreviated as HA-B) 1
Implants were impregnated into 0 × 10 × 5 mm pores and freeze-dried to prepare transplants. This transplant was implanted under the dorsal peritoneum of cynomolgus monkeys (12 cases). As a control, rhBMP-2
Transplants similar to those described above were prepared except that the above was not included, and the same test was performed (12 cases). Each sample was collected 4 weeks after the implantation, and the presence or absence of osteoinduction was observed histologically. That is, each sample was decalcified with EDTA, embedded in paraffin to prepare thin slices, stained with hematoxylin and eosin (HE), and observed under an optical microscope.

In the rhBMP-2 containing transplant, bone formation was observed in the pores of HA-B in 10 out of 12 cases. The tissue image of one sample is shown in FIG. New bone is HA
It was formed in direct contact with -B, and formation of bone marrow-like tissue was observed in part. Cartilage formation was also observed in part. A histological image of a portion where cartilage formation is observed is shown in FIG.
In all cases, bone formation was not observed on the outside of HA-B. No bone formation was observed with the control implants without rhBMP-2. The tissue image is shown in FIG. In each figure, the stained part is new bone and the white part is hydroxyapatite.
In addition, FIG. 5 is a soft X-ray photograph of the rhBMP-2 containing implant 4 weeks after the implantation.

Example 4 100 m of the viscous PLA-PEG used in Example 2
g and 20 μg of rhBMP-2 were mixed, and the resulting mixed solution was impregnated into the pores of HA-B (5 × 5 × 3 mm) under reduced pressure to prepare a transplant. Using the transplants described above, they were implanted (12 cases) under the back fascia of cynomolgus monkeys in the same manner as in Example 3, and collected 4 weeks after the implantation, and the presence or absence of osteoinduction was observed histologically. In the rhBMP-2-containing transplant, bone formation was observed in the HA-B pores in 12 out of 12 cases. The tissue image of one of the samples is shown in FIG. New bone was formed in contact with HA-B. No bone formation was observed on the outside of HA-B. No bone formation was observed with the control implants without rhBMP-2.

Example 5 The following four types of transplants (a) to (d) were prepared. (A) 50 μg of rhBMP-2 and 5 m of collagen I
The solution containing g was lyophilized and further compression-molded to obtain a transplant. (B) 50 μg of rhBMP-2 and PLA-PEG10
0 mg was mixed to obtain a gel-like transplant. (C) 50 μg of rhBMP-2 and PLA-PEG20
Hydroxyapatite granules (hereinafter, abbreviated as HA-g) 20 having a particle diameter of 44 to 100 μm mixed with 0 mg 20
0 mg was mixed to obtain a paste-like transplant. (D) 50 μg of rhBMP-2 and PLA-PEG20
0 mg was mixed, and further 200 mg of granular lactic acid-glycolic acid copolymer (PLGA-g) was mixed to obtain a paste-like transplant.

Evaluation The constitutions of the transplants of the above-mentioned examples and the results of the bone formation test are summarized and shown in Tables 1 to 3 below, and the transplants of the present invention are evaluated from the results.

[0050]

[Table 1] Constitution of each transplant rhBMP amount Carrier Support Example 1 100 μg Collagen I (20 mg) None Example 2 20 μg PLA-PEG (100 mg) None Example 3 50 μg Collagen I (5 mg) HA-B (10 ×) 10 × 5 mm) Example 4 20 μg PLA-PEG (100 mg) HA-B (5 × 5 × 3 mm) Example 5 (a) 50 μg Collagen I (5 mg) None Example 5 (b) 50 μg PLA-PEG (100 mg) None Example 5 (c) 50 μg PLA-PEG (200 mg) HA-g (200 mg) Example 5 (d) 50 μg PLA-PEG (200 mg) PLGA-g (200 mg)

[0051]

[Table 2] Frequency of bone formation in cynomolgus monkeys Tibia Periosteum (Orthotopic) Sub- back fascia (ectopic) Example 1 10/12 (not implemented) Example 2 8/8 (not implemented) Example 3 (Not implemented) 10/12 Example 4 ( Not implemented) 12/12

As is clear from Tables 1 and 2 above,
When collagen I and PLA-PEG were used as a carrier for rhBMP, it was confirmed that bones (or cartilage) were formed in primates, which had previously been difficult to form bone, and it was confirmed to be an effective carrier. .

In the above-mentioned Examples 1 to 4, the bone formation ability of each transplant was orthotopically (subtibial periosteum: Examples 1 and 2) or ectopic (back fascia subterior: in primates). Evaluation was carried out by the bone formation test of Examples 3 and 4). The orthotopic bone formation test evaluates the bone formation ability at the site where bone tissue originally exists, and is a model close to clinical application. This is to evaluate the bone forming ability in a region where bone tissue does not originally exist, such as subcutaneously. Therefore, clinical application is rare, and
It is known that bone formation is less likely to occur compared to orthotopic. However, since the bone formation ability of the transplant can be easily evaluated experimentally, the evaluation by an ectopic bone formation test is usually performed. The inventors of the present invention also described the above Examples 3-5.
By the ectopic bone formation test according to the method described in Example 3, the bone formation ability of the transplant of the present invention was compared between the rodent mouse and the primate cynomolgus monkey. The results are shown in Table 3. In Table 3, + indicates the case in which bone formation was observed, and-indicates the case in which bone formation was not observed.

[0054]

[Table 3] Species difference in bone formation in ectopic bone formation test ( subfascial back) Example of composition of transplant Mouse cynomolgus monkey Example 5 (a) rhBMP / collagen I + -Example 5 (b) rhBMP / PLA -PEG + -Example 5 (c) rhBMP / PLA-PEG / HA-g + -Example 5 (d) rhBMP / PLA-PEG / PLGA-g + -Example 3 rhBMP / collagen I / HA-B + + Example 4 rhBMP / PLA-PEG / HA-B ++

As is clear from Table 3, in a rodent mouse, a transplant consisting only of rhBMP and a carrier,
Heterotopic bone formation occurred in all of the pasty implants with a granular support and the implants with a shaped porous support. On the other hand, in the primate cynomolgus monkey, unless it is a transplant using a molded porous support,
Heterotopic bone formation did not occur. This result is rhBMP
It has been shown that there are species differences in bone formation by the engrafted implants. The reason for the existence of this species difference is currently unknown, but the sensitivity of in vivo cells involved in osteogenesis to rhBMP, metabolism of rhBMP, distribution of in vivo cells involved in osteogenesis, osteogenesis to carriers or supports. It is possible that this is caused by species differences such as the sensitivity of in vivo cells involved in the, the metabolism of carriers, and the reactivity of tissues around the transplant to carriers and supports.

As described above, there is actually a species difference in the osteogenic ability of the transplant using rhBMP, and even in the case of the transplant confirmed to be effective in rodents, it is When applied to, it does not always show the same bone forming ability. Therefore, when examining the clinical utility in humans, it is necessary to examine the efficacy in primates.
Among the implants according to the invention, the implants with a particularly shaped porous support show that even in the ectopic bone formation test, which is a more difficult condition than the orthotopic bone formation test, primates (cynomolgus monkeys) Since a sufficient bone formation ability was confirmed in, it is suitable for clinical application to humans.

[0057]

INDUSTRIAL APPLICABILITY The transplant of the present invention has osteogenic ability even in primates, which are generally considered to be unlikely to undergo osteogenesis.
It is an effective means for clinical application of BMP. Therefore, trauma,
In order to repair various bone or cartilage defects caused by a disease or a congenital defect, it can be applied to the affected area by a method known in the art. The transplant of the present invention has low inflammation and excellent biocompatibility when transplanted into a living body. Further, the continuity between the new bone and the existing bone in the living body is good, and the bone or cartilage can be repaired in a state close to that of nature. INDUSTRIAL APPLICABILITY The transplant of the present invention can be applied to various fields, for example, trauma such as bone fracture, tumor or inflammatory disease, degenerative or necrotic bone disease and other diseases, and bone collection accompanying surgery such as brain surgery or orthopedic surgery. Repair of bone or cartilage defect site by, etc., promotion of healing of various fractures, formation of bone around artificial materials such as artificial joints, artificial bones or artificial roots,
Adhesion promotion in the use of artificial materials, promotion of spinal fixation, bone replacement in the orthopedic field such as leg extension, cartilage regeneration, joint reconstruction, bone or cartilage replacement in the field of plastic surgery, or bone in the dental field or It is suitable for cartilage repair.

Of the transplants of the present invention, substantially rhBMP
Implants composed of and a biodegradable carrier (and optionally a biodegradable powdery or granular support) will eventually replace all newly born natural bone or cartilage at the implantation site. Is advantageous. Orthotopic for easy fixation of transplants, such as repair of small defects of bone or cartilage, promotion of healing of various fractures, repair of bone or cartilage under the periosteum, or repair of bone or cartilage in the dental field Is highly useful for bone formation, while large-scale bone defects, bone defects in areas requiring constant strength, or in areas requiring specific shape maintenance, or ectopic bone formation Etc., it is advantageous to use a shaped porous support or an implant containing a non-biodegradable powdery or granular support.

[Brief description of drawings]

FIG. 1 shows osteogenesis by a rhBMP-2 containing implant carried out in Example 1 in comparison with a rhBMP-2 free implant.
It is a photograph replacing a drawing shown by a soft X-ray photograph.

FIG. 2 is a photograph instead of a drawing, which shows a bone tissue image produced by inducing osteogenesis of a rhBMP-2 containing graft obtained in Example 3.

FIG. 3 is a photograph instead of a drawing, which shows a cartilage histological image generated by osteogenic induction of a rhBMP-2 containing graft obtained in Example 3.

FIG. 4 is a photograph as a substitute for a drawing, which shows a histological image of a state in which bone formation did not progress in a transplant containing no rhBMP-2, which was carried out in Example 3.

FIG. 5: rhB performed in Example 3 4 weeks after implantation
It is a photograph replacing a drawing which shows the state of an MP-2 containing transplant by a soft X-ray photograph.

FIG. 6 is a photograph as a substitute for a drawing, which shows a bone tissue image produced by inducing osteogenesis of a rhBMP-2 containing graft obtained in Example 4.

Claims (27)

[Claims] 1. At least (1) a human osteoinductive factor substantially free of other human-derived proteins, and (2)
Atelocollagen; a polymer or copolymer of lactic acid and / or glycolic acid; or a carrier comprising at least one polymer or copolymer of lactic acid and / or glycolic acid and a block copolymer of polyethylene glycol An osteogenic implant characterized by the following. 2. The osteogenic implant according to claim 1, wherein the carrier is atelocollagen. 3. The carrier has a number average molecular weight of about 400 to 500.
The osteogenic implant according to claim 1 or 2, which is a block copolymer of a lactic acid and / or glycolic acid polymer or copolymer of 0 and polyethylene glycol having a number average molecular weight of about 150 to 2000. 4. The carrier has a number average molecular weight of about 550 to 750.
4. A block copolymer of polylactic acid according to claim 1 and polyethylene glycol having a number average molecular weight of about 150 to 250.
The bone-forming implant according to item 1. 5. Recombinant human produced by a transformant containing a recombinant DNA containing a human osteoinductive factor-containing human osteoinductive factor, which is substantially free of other human-derived proteins. Osteogenic factor (rhBMP)
The osteogenic implant according to any one of claims 1 to 4. 6. Recombinant human osteoinductive factor (rhBMP)
, RhBMP-2, rhBMP-3, rhBMP-
4, rhBMP-5, rhBMP-6, rhBMP-
The osteogenic implant according to claim 5, which is one or more rhBMPs selected from the group consisting of 7, rhBMP-8, a heterodimer of rhBMP, and a recombinant human BMP-2-like protein other than these. 7. The osteogenic implant according to claim 6, wherein rhBMP is rhBMP-2. 8. The osteogenic implant according to claim 1, which consists essentially of the human osteoinductive factor and the carrier. 9. The bone forming implant according to claim 8, which is a compression molded body. 10. The osteogenic implant according to claim 8, which is a highly viscous fluid. 11. The osteogenic implant according to any one of claims 1 to 7, further comprising a biocompatible support. 12. The osteogenic implant according to claim 11, wherein the biocompatible support is made of a polymer or copolymer of lactic acid and / or glycolic acid, a ceramic material, or a metal material. 13. The bone formation according to claim 12, wherein the biocompatible support is a polymer or copolymer of lactic acid and / or glycolic acid having a number average molecular weight of about 5,000 to 1,000,000. For transplant. 14. The osteogenic implant according to claim 13, which is produced by using a powdery or granular polymer or copolymer as a biocompatible support and is a plastic semi-solid. 15. The ceramic material is hydroxyapatite, tricalcium phosphate, bioglass, cerabital, CPSA glass fiber composite material, apatite-wollastonite glass ceramics, β-Ca ₃ (PO ₃ ).
13. The osteogenic implant according to claim 12, which is ₂ glass ceramics, alumina, zirconia, carbon, titania, sapphire, silicon carbide, silicon nitride, or calcium phosphate. 16. The osteogenic implant according to claim 15, wherein the ceramic material is hydroxyapatite. 17. The osteogenic implant according to claim 15 or 16, which is manufactured using a powdery or granular ceramic material as a biocompatible support and is a plastic semi-solid. 18. The metal material is a titanium alloy or Co—Cr.
The osteogenic implant according to claim 12, which is an alloy, stainless steel or a nickel-titanium based shape memory alloy. 19. The porous molded body as a biocompatible support, which carries the human osteoinductive factor and the carrier, according to any one of claims 11 to 13, 15, 16 or 18. The osteogenic implant as described. 20. The osteogenic implant according to claim 19, wherein the porous molded article has a porous portion having an average pore diameter of 50 to 1000 μm on at least the surface thereof. 21. The osteogenic implant according to claim 20, which has an average pore diameter of 100 to 300 μm. 22. The porous molded article has a porous portion having a porosity of 30% or more on at least the surface thereof.
The osteogenic implant according to any one of 9 to 21. 23. The osteogenic implant of claim 22, having a porous portion having a porosity of 70-95%. 24. The entire porous molded body is porous.
The osteogenic implant according to any one of claims 19 to 23. 25. The osteogenic implant according to any one of claims 19 to 23, wherein the porous molded body comprises a porous surface layer and a non-porous core. 26. The osteogenic implant according to any one of claims 1 to 25, which is for a primate. 27. The osteogenic implant according to claim 26, which is used for orthotopic bone or cartilage formation.