JPH10194987A - Composition for ossification and chondrogenesis - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject composition useful for treating diseases which need the restoration of bone and catilage tissues and the induction by including a bone-including factor and hedgehog protein. SOLUTION: This medicinal composition contains both of a bone-inducing factor (BMP) having bone-including effect and hedgehog protein. The BMP is the protein which acts on undifferentiated mesenchymal cells, differentiates them to cartilage cells or osteablasts and possesses the activity allowing them to form cartilage or bone. As the BMP, the human BMP produced by gene recombination technique is preferable. The hedgehog protein is preferably produced analogously by gene recombination technique. The BMP is added in an amount of more than 1μg/ml, suitably 1-300μg/ml, when a recombinant human BMP is utilized and the hedgehog protein is added in an amount of more than 2ng/ml, suitably 10ng-10μg/ml, when Shh-N is utilized. By using the BMP and the hedgehog protein in combination, the ability of forming bone and cartilage is drastically increased and defects of bone and cartilage caused by injury, disease and malformation are rapidly repaired.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a pharmaceutical composition containing an osteoinductive factor, and particularly to a pharmaceutical composition for forming bone and cartilage.

[0002]

2. Description of the Related Art Bone morphogen.
Enetic protein (BMP) is an active protein that acts on undifferentiated mesenchymal cells in subcutaneous or muscular tissue to differentiate them into chondroblasts or osteoblasts and form cartilage or bone. BMP was discovered as a substance exhibiting ectopic osteoinductive activity present in bovine demineralized bone matrix, but was not isolated purely and the specific structure remained unclear. However, the gene encoding human BMP was cloned by genetic engineering techniques, and the amino acid sequence was revealed. It has also been found that human BMP constitutes a group of families consisting of a plurality of closely related proteins having homology in amino acid sequence, and many types of recombinant human osteoinductive factors (rhBMP) 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);
No. 00241, Tokiohei 3-503649, Tokiohei 3-
No. 505098, WO91 / 18098, WO92 / 0
5199, WO93 / 09229 publications: Biochemica
l and Biophysical Research Communications Vol.204,
No.2,646-652, (1994): THE BONE, Vol.9, No.3,51-63, (19
95)]. Production by transformants has also been performed.
Methods for treating bone, cartilage damage, defect or dysplasia using the above-mentioned BMP have been studied, but an ideal clinical application method has not yet been established.

[0003] On the other hand, the hedgehog (hh) family is a group of protein factors that have attracted attention as a key to morphogenesis. In vertebrates, at least soni
c hedgehog (Shh), desert hedgehog (Dhh), indian
hedgehog (Ihh), tiggy-winkle hedgehog (twhh)
There are several types of hedgehog, especially Shh is attracting attention in vertebrates as a ventralizing factor of neural tube and somites and anterior-posterior axis determining factor of limb bud, and its functional analysis is being advanced
(Yodosha, Experimental Medicine Separate Volume BioScience Term Library Development and Neurology, Nakamura S., Ed., Pp. 38-39). The involvement of Shh and Ihh has also been reported in the control of skeletal formation (De
v. Biol. 1995, 172, 126-138; Nature 1996, 383, 407
-413). However, the bone / chondrogenic action of hedgehog protein is not known.

[0004]

[0007] Bone and cartilage tissue is involved in the invasion of bone and cartilage progenitor cells or bone and cartilage into a site to be formed, the proliferation and differentiation of bone and cartilage precursor cells or bone and cartilage cells, -It is formed through a series of processes such as synthesis of cartilage matrix. This process takes a long period of time, generally several weeks to several months, depending on the size of the site to be formed. Therefore, in order to overcome a disease requiring repair or induction of bone / cartilage tissue, creation of an excellent bone / cartilage-forming drug capable of promptly inducing bone / cartilage tissue has been desired.

[0005]

In studying the action of hedgehog protein, the present inventors have introduced an N-terminal domain (Shh-N), which is an active site of Shh, having a bone / cartilage-forming activity by itself. It has been found that transplantation of isolated fibroblasts into nude mice induces ectopic ossification. This finding indicated that hedgehog protein could be applied clinically as a novel hard tissue induction / repair factor.

[0006] Further, when the bone and cartilage forming effect of hedgehog protein was studied, it was surprisingly found that the combined use of BMP having an osteoinductive effect and hedgehog protein dramatically improved the bone and cartilage forming ability. And completed the present invention. That is, the present invention is a pharmaceutical composition characterized by containing BMP and hedgehog protein, particularly a pharmaceutical composition for bone and cartilage formation, which is significantly different from conventional BMP-only bone and cartilage formation pharmaceuticals. It has excellent bone and cartilage formation ability.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.
Osteoinductive factor (BMP) that can be used in the present invention
Is not particularly limited as long as it acts on undifferentiated mesenchymal cells to differentiate it into chondrocytes and osteoblasts and has the activity of forming cartilage or bone, and its preparation method is not limited. . However, human BMP produced by a genetic recombination technique is preferred in that a large amount of materials having stable clinical safety such as immunity and the like and quality can be obtained. 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 the transformant is isolated and purified. This is a recombinant human osteoinductive factor (rhBMP) prepared as follows. These human osteoinductive factors (rhBMP) include, for example, rhBMP
-2, rhBMP-3, rhBMP-4 (rhBMP-
2B), rhBMP-5, rhBMP-6, r
hBMP-7 (rhOP-1), rhBMP-8, rhB
MP-9, rhBMP-12, rhBMP-13, rh
Heterodimers of BMP-15, rhGDF-5, rhBMP, or modified or partially deficient variants thereof can be mentioned. These proteins can be used alone or as a mixture of two or more. Preferably rhBMP
-2.

[0008] These rhBMPs can be expressed in mammalian cells (for example, CHO cells), microorganisms (for example, Escherichia coli) or yeast cells. As rhBMP, for which mass production and purification methods have already been established,
Although there is hBMP-2, other rhBMPs can be similarly produced, purified and used [Progress
in Growth Factor Research
h, Vol. 1, pp. 267-280 (198
9)].

[0009] Purified rhBMP-2 already known is
It is a dimeric protein with a molecular weight of about 30,000. Each monomer has a high-mannose-type sugar chain at Asn56 residue [Abstract Sixth
InteractionSymposiumof
the Protein Society, SanDi
ego, CA (1992)].

The hedgehog proteins that can be used in the present invention have so far been such that Ihh and Shh have equivalent biological activities such as the ability to induce overlapping limbs (Science, 1996, 273, 613-62).
2) In addition, it has been reported that all activities of Shh are carried by the N-terminal domain (Shh-N) (Curr. Biol. 1995, 5, 791-796;
Cell 1995, 81, 313-316), Shh, Ihh, and Shh-N of the N-terminal domain which is the active site of Shh.

[0011] Hedgehog proteins produced by genetic recombination techniques are preferred in that large quantities of stable quality materials can be obtained. That is, a recombinant hedgehog protein prepared by culturing a transformant containing a recombinant DNA containing a nucleotide sequence encoding a hedgehog protein, isolating and purifying the recombinant hedgehog protein produced by the transformant, Hog protein. A number of researchers have already reported methods for producing large amounts of recombinant hedgehog proteins that retain bioactivity using Escherichia coli (C
urr. Biol. 1995, 5, 791-796; Nature 1995, 375, 322
-325).

The Shh and Ihh genes are highly conserved across species from chicken to human (Cell, 1993, 7
5, 1401-1416; Science, 1996, 273, 613-622; Cell 19
93,75,1417-1430; GeneBank Accession Number L38517
and L38518), the animal species of the hedgehog protein used is not limited. Furthermore, Shh-N of an N-terminal domain which is an active site of Shh having a smaller molecular weight than Shh and Ihh and having the same physiological activity is preferable.

In the bone / cartilage-forming composition according to the present invention, the amount of BMP to be added may be any concentration as long as the bone / cartilage-inducing action is exhibited. When rhBMP-2 is used, it is usually 0.1 μg / ml. ml or more, preferably 1 to
1,000 μg / ml, more preferably 1 to 300 μg
/ Ml.

[0014] The amount of hedgehog protein added may be any concentration as long as it exerts an osteo- and cartilage-inducing action. When Shh-N is used, it is usually 2 ng / ml or more.
Preferably it is 5 ng / ml to 500 μg / ml, more preferably 10 ng / ml to 10 μg / ml.

[0015] The composition for bone and cartilage formation of the present invention comprises BMP
These can be administered to a site where bone and cartilage formation is expected, by using a solution containing and Shh, or by supporting them on a suitable carrier. The cartilage-forming composition of the present invention may contain components other than those described above. As these desired components, specifically, a bioabsorbable carrier, a stabilizer, a preservative,
Solubilizing agents, pH adjusters, thickeners and the like can be mentioned. It can also contain additional components useful for bone and cartilage formation, such as fibronectin, osteonectin and the like. These desired components can be added by a suitable method at a suitable stage of formulation of the composition of the present invention.

The bone / cartilage-forming composition of the present invention can be used for repairing various bone / cartilage defects or adding bone / cartilage to any site where bone / cartilage formation is expected. Is administered. In the case of a liquid, it can be injected, and in the case of a paste or clay, it can be embedded in a desired shape by being supported by a suitable carrier and embedded as a solid. The composition for forming bone and cartilage of the present invention can be applied to a living body by a method known in the art, and can be appropriately applied depending on the purpose, use, site of application, condition of a patient, and the like. .

The bone and cartilage forming composition of the present invention may be used not only alone but also in combination with other known implants. For example, a biocompatible membrane for locally immobilizing the composition, such as a collagen membrane or GTR
A Gore-Tex film or a polylactic acid film used in the method may be combined.

The effectiveness of the pharmaceutical composition of the present invention for bone and cartilage formation can be confirmed as follows. The effects on bone formation are as follows: 1) C3H10T, an undifferentiated mesenchymal cell line that has been reported to have the ability to differentiate into osteoblasts
To examine the degree of differentiation promoting effect of 1/2 cells (Growth Factors 1993, 9, 57-71) to osteoblasts. 2) To determine the expression of differentiation function of primary cultured osteoblasts isolated from calvaria of chicken embryo. It can be evaluated by studying. In each case, alkaline phosphatase (Alkali) of cells was used as a differentiation marker.
A change in ne phosphatase (APase) activity can be used as an index.

The effect on chondrogenesis was determined by a rat undifferentiated mesenchymal cell line RMD cell (J.B.
One Miner. Res. 1996, 11, 544-553) can be evaluated by examining the differentiation into chondrocytes. The degree of differentiation into chondrocytes can be measured using the incorporation of 35S-sulfate into the extracellular matrix as an indicator of the synthesis of cartilage-type macromolecular proteoglycan (GAG) (J. Bone Miner. Res. 1996, 11, 544-553). . Also,
For the accumulation of cartilage-type macromolecular proteoglycan,
It can be easily determined by staining with Alcian blue at pH 1.0.

[0020]

Examples Examples showing the bone / cartilage-forming activity of the pharmaceutical composition of the present invention are shown below. However, the present invention is not limited at all by the following examples.

The hedgehog and BM used in the examples
The method for producing P is described below. <Preparation of chicken Shh-N recombinant virus and chicken Shh-N protein> total RNA of stage 25 chicken embryo
Then, chicken Shh cDNA was obtained by RT-PCR. Then N
The part encoding 200 amino acids on the terminal side was amplified by PCR. At that time, a stop codon was inserted at the 200th residue.
The obtained DNA fragment was blunt-ended, and Cla I adapter
gation and then chicken retrovirus vector RCAS (A) (J.
Virol. 1987, 61, 3004-3012). This viral DNA was transferred to 11-day-old virus-free eggs (Line-M
Chicken embryo fibroblasts prepared from Egg, Nisseiken, Yamanashi)
(CEF) was transfected by the calcium phosphate method (M
ol. Cell. Biol. 1987, 7, 2745-2752). CEF is a 10% fetal bovine serum (FBS; Cell culture Laboratories, Ohio, U.
Medim 199 (SA)) (GIBCO, Gaithersburg, MD, USA)
And cultured.

Since the culture supernatant of CEF transfected with viral DNA contains chicken Shh-N recombinant virus and chicken Shh-N protein, the virus in the collected culture supernatant was reported by Cepko et al. (Mol. Cell. Biol. . 1993,
13, 2604-2613) and used for infection experiments after concentration by ultracentrifugation. Culture supernatant containing chicken N-Shh protein
(Shh-N-CM) was used in a mouse or rat cell culture system without virus infection. CEF culture supernatant (RCAS-CM) into which only the virus vector was introduced was used as a subject.

<Mouse Recombinant N-Shh (rmSHH-N)
Preparation> Mouse Shh cDNA containing a substantially full-length coding sequence was obtained from the 10-day mouse total RNA by RT-PCR. Mouse Shh cD registered by MacMahon
NA sequence (GeneBank Accession Number X76290, Cell 199
3, 75, 1417-1430)
Amplification was performed using a primer pair containing a site. At that time, 65
A stop codon sequence was inserted after the first base sequence. After digesting the obtained DNA fragment with Sac I, QIA express
It was subcloned into the pQE30 plasmid attached to the kit (QIA express kit, QIAGEN GmbH, Germany). Then, E. coli is transformed with plasmid according to the usual method.
Mouse recombinant N- with a histidine tag at the end
Shh is synthesized. This was purified using a nickel chelating resin column (Proc. Natl. Acad. Sci. US
A, 1991, 88, 8972-8976). All the experimental methods were performed according to the protocol (The QIAexpressionist) attached to the kit.

<Preparation of human BMP-2 recombinant virus and human BMP-2 protein (hBMP-2)> Human osteosarcoma total RNA
The human BMP-2 cDNA containing the full-length coding sequence was cloned by RT-PCR. Using this DNA fragment, the RC of subgroups A and B was obtained in the same manner as in the construction of the chicken N-Shh recombinant virus described above.
AS vector (RCAS (A), RCAS (B)) (J. Virol. 1987, 61,
3004-3012). And the virus
The culture supernatant of CEF transfected with DNA was concentrated and used for infection experiments. The culture supernatant containing the hBMP-2 protein (hBMP-2-CM) was used in a mouse or rat cell culture system not infected with the virus.

<Preparation of mouse BMP-4 recombinant virus and mouse BMP-4 protein (mBMP-4)>
Mouse BMP-4 cDNA containing the full-length coding sequence was cloned from the otal RNA by RT-PCR. This DN
Using the A fragment, the RCAS vectors of subgroups A and B (RCAS (A), RCAS (B)) (J. Virol. 1) in the same manner as in the construction of the chicken N-Shh recombinant virus described above.
987, 61, 3004-3012). Then, the culture supernatant of CEF transfected with the viral DNA was concentrated and used for infection experiments. Further, a culture supernatant containing mBMP-4 protein (mBMP-4-CM) was used in a mouse or rat cell culture system not infected with virus.

<Cell culture method> Chicken embryo-derived fibroblasts
(CEF) was isolated from Virus-free 11-day embryos and 10%
Fetal bovine serum (FBS; Cell culture)
Laboratories, Ohio, USA)
Were cultured in Medim 199 (GIBCO, Gaithersburg, MD) containing The mouse fibroblast cell line C3H10T1 / 2 clone 8 was donated by RIKEN cell bank and contains Eagle's containing 10% FBS.
The cells were subcultured in Minimal Essential Medium (MEM, Nissui, Tokyo). Rat undifferentiated mesenchymal cell line RMD-1 is 10% FB
The cells were subcultured in Ham's F-12 medium containing S. However,
Experiments on cartilage differentiation of RMD-1 cells were performed using Ham F-1 with 5% FBS.
Performed on two media.

Example 1 The Shh-N and BMP genes were introduced into osteoblasts using a recombinant retrovirus, and the effect of the transgene product on osteoblast differentiation was examined. Gerste from virus-free chicken 18-day embryo calvaria
Primary cultured osteoblasts were isolated by the method of nfeld et al. (Dev. Biol. 1987, 122, 49-60). Medium contains 10% FBS
phaMEM was used. Two hours after the cells were seeded on a culture dish, chicken Shh-N recombinant virus (Shh-N
N-RCAS (A)), human BMP-2 recombinant virus (BMP-2-
RCAS (B)) and mouse BMP-4 recombinant virus (BM
P-4-RCAS (B)). The virus alone or double subinfected cells with different subgroups of Shh and BMP viruses. As a control, a group without any infection (None) and a group with only vector infection (RCAS (A), RCAS
(B)) was prepared. Six days later, the cells were harvested using trypsin. Some cells use the antiviral antibody AMV-3C2 (Develpo
Immunostaining was performed with mental Studies Hybridoma Bank, Baltimore, MD) to confirm that more than 95% of the cells were infected with the virus. The remaining cells are in a 48-well plate (Cornin
g, NY, USA) and seeded at 30,000 cells / well,
Cultured for days. The culture medium and CM were changed every three days.

After completion of the culture, the APase activity was measured by
(Exp.CellRes. 1991, 195, 3
8-46). That is, cells were grown in 0.9% NaCl, 0.2% Triton-X 100
Ultrasonic crushing was performed in the solution, and the supernatant was added to a reaction solution using paranitrophenol diphosphate as a substrate, and the generated paranitrophenol was colorimetrically determined. The DNA content of the cells was measured by the fluorescent dye method (Analyte Biochem. 1982, 122,
338-344; Exp. Cell Res. 1991, 195, 38-46).

The APase activity was 1 minute per minute at 1 μg DNA equivalent.
n moles of para-nitrophenol is produced 1
The unit is shown in the figure. FIG. 1 shows the results. None, APAS activity of 1 unit / ug DNA or less was detected in none, RCAS (A), and RCAS (B) groups. On the other hand, Shh-N-RCA
In the cells infected with S (A), BMP-2-RCAS (B), and BMP-4-RCAS (B), the APase activity was observed to be 2-3 times higher than in the control group. In addition, cells that were double infected with Shh-N-RCAS (A) and BMP-2-RCAS (B) or BMP-4-RCAS (B) detected an 8-11-fold APase activity compared to the control group. Was done. This result suggests that Shh-N and BMP-2 / -4 synergistically promote osteoblast differentiation and have good osteogenic effects.

Example 2 Effects of Shh-N-CM and BM on osteoblast induction (differentiation of undifferentiated mesenchymal cells into osteoblasts)
The effect of P-2 / -4-CM was studied. Transfer C3H10T1 / 2 cells to 48-well plate (Corning, NY, USA)
The seeds were seeded at 30,000 cells / well, and after 24 hours, Shh-N-CM, hBMP-2-CM, and mBMP-4-CM were added to a concentration of 25% and cultured for 7 days. The culture medium and CM were changed every three days. After completion of the culture, the APase activity was measured in the same manner as in Example 1.

FIG. 2 shows the results. C3H10T1 / 2 cells are osteoblast marker APase even when CEF culture supernatant (RCAS-CM) containing only control virus vector is added.
Was not expressed. However, Shh-N-CM, hBMP-2-CM, mB
MP-4-CM alone induced APase activity in C3H10T1 / 2 cells, albeit weakly. Furthermore, Shh-N-CM and hBMP-2-
Simultaneous addition of CM or mBMP-4-CM results in synergistically high APase
Activity was induced in C3H10T1 / 2 cells. The results of this experiment show that Shh-N and BMP-2 / -4 contained in the culture supernatant synergistically promote the differentiation of undifferentiated mesenchymal cells into osteoblasts and have a good osteogenic effect Suggest that.

Example 3 Effects of Shh-N-CM and rhBM on osteoblast differentiation of C3H10T1 / 2 cells
The effect of P-2 over time and its concentration dependence were examined. Transfer C3H10T1 / 2 cells to 48-well plate (Corning, NY, USA)
Seed at 30,000 cells / well and 24 hours later, 25% concentration of Sh
hN-CM, 1 μg / ml recombinant human BMP-2 (rhBMP-
2; provided by Yamanouchi Pharmaceutical Co., Ltd.)
Cultured for days. After 2, 4, and 7 days of the culture, the APase activity was measured by the method of Example 1. The results are shown in FIG. 3A. Also, 1 μ
Various concentrations of S in the presence and absence of g / ml rhBMP-2
APase activity was measured 7 days after hh-N-CM was added to the medium. The results are shown in FIG. 3B. In addition, 25% concentration of Shh-N
Various concentrations of rhBMP-2 were added to the medium in the presence and absence of -CM, and the APase activity was measured 7 days later. Fig. 3 shows the results.
C.

C3H10T1 / 2 with Shh-N-CM and rhBMP-2
The APase activity promoting action of the cells was clarified on day 2 and day 4 of culture, respectively. Its effect increased over time. In addition, the simultaneous addition of both resulted in a synergistic increase in APase activity expression (FIG. 3A). The effects of Shh-N-CM and rhBMP-2 and their synergistic effects increased in a concentration-dependent manner (FIG. 3).
B, 3C). The optimal concentration of Shh-N-CM was 25% (Fig. 3
B). The optimal concentration of rhBMP-2 was 1 μg / ml (FIG. 3C).

Example 4 The concentration dependence of rmSHH-N and rhBMP-2 on osteoblast differentiation of C3H10T1 / 2 cells was examined. Transfer C3H10T1 / 2 cells to 48-well plate (Corning, NY, USA)
24 hours after seeding at 30,000 cells / well, 1 μg / ml rhBM
In the presence and absence of P-2, rmSHH-N at a concentration of 10 ng / ml to 10 μg / ml was added and cultured for 7 days. After culture
APase activity was measured by the method of Example 1. Fig. 4 shows SHH-N
Shows the effect of is on the APase activity of 10T1 / 2 cells. SHH-N
Promoted APase activity in a concentration-dependent manner, similar to Shh-N-CM, and showed a marked synergistic effect in the presence of BMP-2.

[Example 5] Effect of Shh-N-CM and BMP- on the differentiation of RMD-1 cells into chondrocytes
The effect of 2 / -4-CM was studied. Transfer RMD-1 cells to type I collagen-coated 24-well plate
(Iwaki Glass, Chiba) Seeding at 400,000 / well and 5%
Ham F-12 medium containing FBS was added. After 24 hours, 25%
, And Shh-N-CM, hBMP-2-CM, and mBMP-4-CM were added alone or in combination to culture. After 48 hours, the medium was changed, and after that, the cells were further cultured without adding CM. Six days later, the amount of proteoglycan synthesis, an indicator of cartilage differentiation, was increased by 35S-
It was measured by the incorporation of sulfuric acid into the cetylpyrimidine chloride precipitate fraction (J. Bone Miner. Res. 1996, 11,544-55
3). That is, 1.5 μCi of 35S-sulfuric acid was added per well, and the cells were cultured for 6 hours and labeled. Thereafter, the amount of 35S-sulfuric acid incorporated into the proteoglycan of the culture supernatant and the cell layer was measured.

FIG. 5 shows the results. RMD-1 cells hardly showed differentiation into chondrocytes even when the culture supernatant (RCAS-CM) of CEF into which only the control virus vector was introduced was added. However, Shh-N-CM, hBMP-2-CM, mBMP-4-CM
Alone promoted cartilage differentiation of RMD-1 cells.
Furthermore, simultaneous addition of Shh-N-CM and hBMP-2-CM or mBMP-4-CM showed a synergistically high cartilage differentiation promoting activity. This experiment revealed that Shh-N and BMP synergistically promote cartilage differentiation of RMD-1 cells, suggesting that they have a good chondrogenic effect.

Example 6 The effect of the treatment time of Shh-N-CM on the differentiation of RMD-1 cells into chondrocytes was examined. Transfer RMD-1 cells to type I collagen-coated 24-well plate
(Iwaki Glass, Chiba) Seeding at 400,000 / well and 5%
Ham F-12 medium containing FBS was added. After 24 hours, 200
The cells were treated with 25% Shh-N-CM for the first 2, 4 or 6 days in the presence or absence of ng / ml rhBMP-2. The synthesis amount of proteoglycan, which is an index of cartilage differentiation, was measured by incorporating 35S-sulfuric acid into the cetylpyrimidine chloride precipitated fraction. From this experiment, it was found that the presence of Shh-N for the first two days promoted maximal induction of differentiation of RMD-1 cells into cartilage, and a good synergistic effect with BMP-2 was obtained.

Example 7 The effect of rmSHH-N concentration on the differentiation of RMD-1 cells into chondrocytes was examined. Transfer RMD-1 cells to type I collagen-coated 24-well plate
(Iwaki Glass, Chiba) Seeding at 400,000 / well and 5%
Ham F-12 medium containing FBS was added. After 24 hours, 200
10 ng / ml to 10 in the presence or absence of ng / ml rhBMP-2
rmSHH-N at a concentration of μg / ml was added and cultured. After 48 hours, the medium was changed, and after that, the cells were further cultured for 7 days without adding the recombinant protein. The amount of proteoglycan synthesis, which is an indicator of cartilage differentiation, was measured by incorporating 35S-sulfuric acid into the cetylpyrimidine chloride precipitated fraction. rmSHH-N increases proteoglycan synthesis in a concentration-dependent manner,
In the presence of P-2, a marked increase in proteoglycan synthesis was observed. (Fig. 5)

From the above results, the pharmaceutical composition of the present invention
It was found that bone and cartilage progenitor cells markedly promoted differentiation into bone and chondrocytes and osteoblasts themselves and had good bone and cartilage-forming effects.

[0040]

EFFECT OF THE INVENTION The implant of the present invention can be used in vivo
Induces cartilage formation and forms good bone and cartilage tissue (recovery)
can do. Therefore, in order to repair various bone / cartilage defects caused by trauma, disease, congenital abnormality, etc., the method can be applied to an affected area by a method known in the art. The implant of the present invention can be applied to various fields, for example, repair of bone or cartilage defect sites accompanying accidents, diseases, birth defects or various operations, promotion of healing of various fractures, artificial joints, artificial bones Or bone formation around artificial implants such as artificial roots, promotion of fixation when using artificial implants, promotion of spinal fixation, regeneration or supplementation of bone or cartilage in orthopedic fields such as leg extension, joint reconstruction, plastic surgery It is suitable for the replacement of bone or cartilage, or the restoration of jaw bone in the dental area, the regeneration of alveolar bone, the restoration of cementum, and the increase of bone for use of implants.

[Brief description of the drawings]

FIG. 1 shows that Shh-N was added to osteoblasts in Example 1.
FIG. 3 is a diagram showing the effect of a transgene product on osteoblast differentiation by introducing the BMP-2 / -4 gene and Apase activity.

FIG. 2 shows Shh-N-CM and BMP- in Example 2.
FIG. 2 is a diagram showing the effect of 2 / -4-CM on osteoblast induction by Apase activity.

FIG. 3 shows Shh-N-CM and rhBM in Example 3.
FIG. 2 is a diagram showing the effect of P-2 on osteoblast differentiation by Apase activity. A shows the change with time, B shows the concentration dependency of Shh-N, and C shows the concentration dependency of rhBMP-2.

FIG. 4 shows rmSHH-N and rhBMP in Example 4.
Effect of Apase-2 on Osteoblast Differentiation (Concentration Dependency)
It is the figure shown by activity.

FIG. 5 shows Shh-N-CM and BMP-2 in Example 5.
/ -4-CM affects the differentiation of RMD-1 cells into chondrocytes,
FIG. 3 is a diagram showing the amount of 35S-sulfuric acid incorporated into proteoglycans as an index.

FIG. 6 shows the effect of the treatment time of Shh-N-CM on the differentiation of RMD-1 cells into chondrocytes in Example 6,
FIG. 3 is a diagram showing the amount of 35S-sulfuric acid incorporated into proteoglycans as an index.

FIG. 7 shows rmSHH-N and rhBMP-2 in Example 7.
Effect of RMD-1 cells on chondrocyte differentiation (rmSHH-
FIG. 3 is a diagram showing the concentration dependence of N) using 35S-sulfuric acid incorporated into proteoglycan as an index.

Claims (2)

[Claims] 1. A pharmaceutical composition comprising an osteoinductive factor and a hedgehog protein. 2. The pharmaceutical composition according to claim 1, which is used for bone and cartilage formation.