WO2010114319A2 - Composition for bone regeneration - Google Patents

Abstract

The present invention relates to a composition for bone regeneration including a hyaluronic acid and a mesenchymal stem cell into which a bone regeneration inducing factor is introduced, and a manufacturing method thereof. The composition for bone regeneration can include a stem cell into which a gene coding a protein with a bone formation function is introduced or cells differentiated from the stem cell and the hyaluronic acid. The protein with the bone formation function can be a bone morphogenic protein (BMP). The stem cell can be a mesenchymal stem cell, and it can be a stem cell into which a suicide gene is additionally introduced. An average molecular weight of the hyaluronic acid can be from 50,000 to 300,000 daltons.

Description

Bone Regeneration Composition

The present invention relates to a composition for bone regeneration and a method for producing the same.

Bone is a representative feature of vertebrates and is one of the living tissues in vivo. The bone not only protects the organs, but also hematopoiesis to produce blood cells that carry nutrients and waste products necessary for each tissue throughout the body, and plays a wide variety of roles such as accumulating calcium and minerals.

The cells constituting the bone include osteocytes maintaining bone homeostasis, osteoblasts forming bones, osteoclasts absorbing bones, and cartilage responsible for joint function at the ends of bones. Cells (chondrocyte) and the like. During the growth phase of bone, osteoblasts actively form bone, and after the growth phase, bone formation and bone absorption are remodeled.

If the balance between the bone formation and bone absorption is broken and biased to either side, it causes bone disease, which can lead to severe symptoms and severely intractable. Refractory bone disease includes osteoporosis and non-union fractures that are broken bones due to diabetes, osteopenia due to congenital bone defects, osteomalacia due to lack of mineral deposits in the bone matrix, and bone tumors and There is a bone defect from an accident.

The most common of these intractable bone diseases is osteoporosis. In the osteoporosis patients, fractures, in particular nonunion fractures, are frequently caused by low bone mass and degeneration of the microstructure of the bone structure.

Osteoporosis, characterized by the low bone mass and the degeneration of the microstructure of the bone structure that causes fractures, is the most common health problem for older people, but it is not simply progressed by aging. The osteoporosis or osteoporosis disease may be caused by various factors, such as menopause, calcium deficiency diet, ovariectomization, glucoticoid-induced osteoporosis, hyperthyroidism and immunosuppressive induction It is not limited. The osteoporosis fracture is a high risk of fracture even in the degree of impact that can occur in everyday life, and because it is not particularly noticeable before the fracture is more dangerous and may cause death.

Such osteoporosis can cause fractures in all parts of the body, of which spinal fractures and hip fractures are most representative. Once a fracture occurs due to osteoporosis, the risk of recurring fractures over and over again is about five times higher. In addition, the risk of death from hip fracture due to osteoporosis is very high. For example, the first year mortality rate due to hip fracture in osteoporosis patients is reported to be approximately 25%.

There is a need for an effective treatment method for various refractory bone diseases, such as fractures due to reduction of bone density according to the age, destruction and deficiency of bone tissues due to accidents and disasters, and poor growth due to congenital osteoplasia.

However, since the treatment of fractures that can be caused by various causes is a very complex process, little is known about the healing mechanisms associated with fracture healing.

In addition, problems have also been raised in the case of autografting, allograft and artificial bone grafting, which are proposed as traditional treatments. Autologous bone grafts have complications such as infection, pain or hematoma at the bone harvest site. Allogeneic bone graft has the problem of possible disease transmission from donors, and has the disadvantage of poor bone union and osteogenic effect. Artificial bone graft is not a fundamental bone production, it takes a long time to regenerate bone, there is a problem that it is not well fused with the existing bone tissue around.

Recently, a treatment method proposed as an alternative to the conventional treatment method is a method of implanting a biodegradable carrier injected with osteoinductive protein in a wound site. Since the osteoinductive protein, which is a protein capable of inducing bone regeneration or bone formation, has a short active period in vivo, administration of a large amount of recombinant protein is required to achieve a therapeutic effect. Therefore, the therapy not only raises safety problems such as side effects due to excessive administration of the recombinant protein, but also has a disadvantage in that the therapeutic effect cannot be guaranteed.

Several cytokines have been considered in the orthopedic field as candidates for the treatment of plastic diseases, of which bone forming proteins have been reported as effective stimulators of bone formation. In addition, growth factors known to stimulate proliferation of bone cells include bone morphogenic protein (BMP), transforming growth factor-β protein (TGF-β), and insulin type growth factor (IGF). ) And basic fibroblast growth factor (bFGF).

There are three factors involved in bone formation: osteoinduction by growth factors, osteoconduction by scaffolds, and osteoogenesis by stem cells. When these three elements are in harmony, they can maximize bone formation.

Among them, bone morphogenic proteins (BMPs), which are bone regeneration inducing factors related to bone induction, have been reported to induce bone formation. For example, bone morphogenetic proteins BMP-2 (Bone Morphogenic Protein-2) and BMP-4 (Bone Morphogenic Protein-4) have been reported to be involved in the healing of cartilage-resistant membrane fractures. In addition, BMP-2 is not only a protein that promotes bone growth, but it has also been reported that BMP-2 proteins are essential for natural regeneration reactions.

Recently, expectations for stem cell treatment that can differentiate into all organs are rising. However, embryonic stem cell transplantation with superior differentiation has side effects such as development of uncontrollable teratomas or developmental problems through imprinting, and is limited by ethical problems. Therefore, interest in adult stem cells has recently increased.

Hematopoietic stem cells are attracting attention as adult stem cells. Two kinds of stem cells are known to the bone marrow, the hematopoietic stem cell source. First is hematopoietic stem cell, and second is mesenchymal stem cell.

Hematopoietic stem cells of the bone marrow are rarely differentiated into cells of other organs, while mesenchymal stem cells are capable of differentiation into all three germ layers, ectoderm, mesoderm and endoderm, and almost all organs when injected into balstocyst. Excellent differentiation ability to differentiate into cells has been reported. In addition, it is expected that similar stellate stem cells will be isolated from human bone marrow and applied to the field of cell therapy against disease and injury.

However, the need for alternatives has been continuously raised as bone marrow is very disadvantageous in practical application utility due to the decrease in hematopoietic stem cell and mesenchymal stem cell zones as the age and the bone marrow harvesting itself cause pain.

Umbilical cord is the source of all nutrients and waste excretion for the fetus, and connects the mother's placenta with the fetus, and cord blood refers to blood in the umbilical cord.

Umbilical cord blood has been suggested as the most suitable alternative to bone marrow for harvesting hematopoietic stem cells because cord blood stem cells are more primitive and easier to harvest than bone marrow.

Suicide genes are genes that convert prodrugs, which are harmless to the human body, into cytotoxic anti-cancer substances. For example, cytosine deaminase is a 5-FC (5-fluorocytosine). It has the function of converting 5-FU (5-fluorouracil), a cytotoxic anticancer agent, where 5-FU has a bystander effect that is secreted out of cells and kills adjacent cells. 5-FU is highly cytotoxic when administered systemically, but there are many side effects. However, the use of suicide gene and 5-FC prodrug increases the concentration of 5-FU only in the vicinity of suicide gene, resulting in the anticancer effect of 5-FU. Has been reported to appear locally around cancer cells (Bourbeau et al ., The Journal of Gene Medicine, 6, 1320-1332 (2004)).

In addition, thymidine kinase, which is originally present in cells, is an enzyme used for salvage pathways in DNA synthesis. HSV-tk is a nucleoside analog and antiviral agent as well as thymidine. Acyclovir or GCV can also be phosphorylated. When tri-phosphorylated GCV enters instead of deoxy-guanosine tri-phosphate during DNA synthesis, chain elongation by DNA polymerase is interrupted, thereby inhibiting DNA synthesis, which results in cell killing ability. Chemotherapy that kills cancer cells by administration is being studied.

An important advantage of the cancer treatment strategy is that it exhibits a bystander effect that kills surrounding cancer cells to which the HSV-tk gene is not injected, and genes are applied to all cancer cells, which is the biggest problem of gene therapy for cancer treatment. This is important in that it can partially compensate for the drawback of not being able to inject.

The bystander effect is understood to be achieved by endocytosis of surrounding cancer cells through apoptotic vesicles of cancer cells where phosphorylated GCV is transmitted through gap junctions formed between adjacent cells or dying by HSV-tk and GCV treatment.

In order to solve the problems of the prior art related to the fracture treatment, an object of the present invention is to provide a composition for bone regeneration having a bone formation function to effectively regenerate or form bone.

The present invention also provides a method for producing the composition for bone regeneration.

In order to achieve the above object, the present invention provides a composition for bone regeneration and its manufacturing method. In detail, the bone regeneration composition may include stem cells and hyaluronic acid.

In addition, the present invention provides a composition for preventing, reducing or treating bone diseases comprising the composition for bone regeneration as an active ingredient, and a method for treating bone diseases using the composition.

The present invention also provides a vector comprising a protein gene having a suicide gene and a bone formation function.

In addition, the present invention, in order to achieve the above object, there is a binding region of the protein gene having the bone formation function, the protein expressed by the protein gene having the bone formation function, the protein gene having the bone formation function Provided is a gene expression cassette comprising a promoter and suicide gene whose transcription initiation is determined by a protein expressed by the present invention.

EMBODIMENT OF THE INVENTION Hereinafter, this invention is demonstrated in detail.

The present inventors have conducted studies to solve the problems of existing bone disease treatment methods, stem cells and the average molecular weight into which the gene encoding BMP-2 (Bone Morphogenic Protein-2), a protein having bone formation function, was introduced. When the composition for bone regeneration comprising hyaluronic acid having a range of 50,000 Da to 300,000 Da is treated at a site requiring bone regeneration or bone formation, when treated with another kind of biocompatible material or hyaluronic acid having a different molecular weight It was confirmed that it can exhibit significantly superior bone regeneration effect or bone formation effect. In addition, when the stem cells are used with the stem cells introduced with the BMP-2 and suicidal genes, while showing excellent bone formation effect, it is possible to eliminate the side effects due to the expression of proteins with excessive or persistent bone formation function It was confirmed.

In view of the above facts, the inventors of the present invention, when using the composition for bone regeneration of the bone disease group including osteoporosis, osteoporotic fracture, diabetic fracture and bone defect, osteopenia, osteomalacia and fractures thereof, Treatment and alleviation of various bone diseases such as bone reconstruction, fracture defects due to tumor removal, disorders caused by bone diseases such as bone growth disorders, nonunion fractures, diseases requiring bone growth due to genetic bone growth defects, or bone height And / or it was confirmed that it can prevent, to complete the present invention based on this.

As used herein, "bone precursor cells" or "bone precursor cells" refer to cells that have the potential to become bone cells and are present in the periosteum and bone marrow.

As used herein, the term "bone growth" relates to bone mass and includes an increase in the number and size of osteoblasts or an increase in the incidence of osteogenic lining bone surfaces after systemic administration. to be. The "bone mass" refers to the amount of bone per unit area and is often referred to as bone mineral concentration.

As used herein, "mature bone" refers to bone that has been mineralized as opposed to non-mineralizing bone, such as osteogenic.

As used herein, “osteolytically effective amount” means an amount that affects the formation and development of mature bone.

As used herein, the term "biological activity" with respect to nucleic acids, proteins, protein fragments or derivatives thereof is defined as the ability of a nucleic acid or amino acid to mimic a known biological function derived by a wild type nucleic acid or protein.

As used herein, an “individual” may be a vertebrate, preferably a mammal, more preferably a human. As used herein, "patient" includes members of the animal kingdom, including but not limited to humans.

As used herein, "treatment" means an approach for obtaining advantageous or desired clinical outcomes. For the purposes of the present invention, advantageous or desired clinical outcomes may include relief of symptoms, reduction of disease extent, stabilization (ie, not worsening) of disease, delayed or delayed disease progression, relief and temporary relief of disease state, And a detectable or undetectable loss (either in part or in whole). In addition, the term "treatment" may also mean prolonged survival compared to the expected survival if not treated.

In addition, in this specification, the term "treatment" means both therapeutic treatment and preventive measures. Those in need of treatment include those already suffering from the disease, as well as those to whom the disease is to be prevented. The term "temporarily relieving" a disease means that the extent of the disease state and / or undesirable clinical signs and / or delayed or lengthened the progression of the condition as compared to the untreated situation.

In the present specification, when the recipient animal is resistant to administration of the composition, the composition is "pharmaceutically or physiologically acceptable", otherwise suitable for administration to said animal. Such agents are meant to be administered in therapeutically effective amounts when the dosage is physiologically effective. The drug is physiologically significant if the presence of the drug causes a detectable change in the physiology of the receptor patient.

As used herein, "pharmaceutically acceptable carriers and / or diluents" includes any and all solvents, dispersion media, coatings, anti-bacterial and anti-fungal agents, isotonic and absorption delaying agents, and the like.

As used herein, administering "with" one or more additional therapeutic agents includes simultaneous (co) or continuous administration in any order.

In the present specification, the terms "vector", "polynucleotide vector", "construct" and "poly nucleotide construct" may be used interchangeably. The polynucleotide vectors of the invention may be RNA, DNA, RNA encapsulated in a retroviral coating, DNA encapsulated in an adenovirus coating, or other viral or viral type (e.g., herpes simplex). And DNA packed with adeno-associated virus (AAV), DNA encapsulated in liposomes, DNA complexed with polylysine, DNA complexed with synthetic polyvalent cationic molecules, and molecules DNA conjugated with non-viral proteins or DNA complexed with compounds such as polyethylene glycol (PEG) to mask and / or increase half-life. It may be anything. The polynucleotide may be DNA. As used herein, the term "DNA" includes the bases A, T, C and G, as well as analogs and modified forms of these bases, such as methylated nucleotides, uncharged bonds and thioates ( internucleotide modifications such as thioate, the use of sugar analogs, and modifications and / or alternative backbone structures such as polyamides.

As used herein, "promoter" means activity and refers to any sequence of DNA that regulates transcription in eukaryotic cells. The promoter may be active in each or both eukaryotic or prokaryotic cells. The promoter may be structurally expressed or inducible and may be induced by external stimulation. Also, "enhancer element" that regulates transcription is meant to be inserted into a DNA vector construct and used with the construct of the invention to enhance expression of the gene of interest.

As used herein, the term "host cell" includes individual cells or cell cultures which may or may be receptors of the vectors of the invention. Host cells include the progeny of a single host cell, and the progeny are initially initially (in morphological or total DNA) due to natural mutations and / or changes, accidental mutations and / or changes, and intentional mutations and / or changes. It may not be exactly the same as the parent cell. As used herein, "mammal host" includes members of the animal kingdom, including but not limited to humans.

One example of the present invention is a bone regeneration inducer derived from mammals, preferably for bone regeneration comprising stem cells into which a gene encoding a protein having a bone formation function or cells differentiated from the stem cells and hyaluronic acid as an active ingredient It relates to a composition.

Another embodiment of the present invention relates to a composition for preventing, alleviating or treating a bone disease comprising the composition for bone regeneration.

The present invention relates to a composition for bone regeneration comprising a stem cell into which a gene encoding a protein having a bone formation function is introduced or a cell differentiated from the stem cell and hyaluronic acid as an active ingredient.

The bone regeneration inducer (promoter) refers to a gene encoding all proteins that induce bone formation in mammals, preferably, may be a protein having a bone generation function (bone generation function).

The protein having a bone formation function means a protein that stimulates proliferation of bone cells, and is also referred to as a protein or bone regeneration protein having a bone formation function. The protein gene having a bone formation function means a gene encoding a protein having a bone formation function.

Examples of the protein having a bone forming function include bone morphogenic protein (BMP), transforming growth factor-β protein (TGF-β), insulin type growth factor (IGF), and fibroblast growth factor (fibroblast growth). factor, FGF), specifically, basic fibroblast growth factor (bFGF) and Vascular Endotherial Growth Factor (VEGF), but are not limited thereto.

The bone forming protein (BMP) is a protein that acts to induce differentiation of mesodermal cells into chondrocytes and osteoblasts before initiating bone formation. The bone morphogenetic protein may promote cartilage and differentiation of osteogenic cells at the periphery or ectopic location of the fracture site. In addition, some bone-forming proteins can induce the synthesis of alkaline phosphatase and collagen in osteoblasts or act directly on osteoblasts to enhance their maturation, while at the same time inhibiting myogenous differentiation. In addition, some bone morphogenetic proteins enhance the conversion of typical fibroblasts to chondrocytes and may induce the expression of osteoblast phenotypes in non-osteogenic cell types.

The bone morphogenetic protein consists of BMP-2, BMP-3, BMP-4, BMP-7, BMP-8, BMP-8b, BMP-9, BMP-10, BMP-11, BMP-12 and BMP-14 It may be at least one selected from the group, but is not limited thereto.

The BMP-2 (eg, GenBank Accession No. AAF21646; encoded by GenBank Accession No. M22489) may be BMP-2A or BMP-2-α (114 amino acids). The BMP-3 (eg, GenBank Accession No. NP — 001192; encoded by GenBank Accession No. NM 001201) is a glycoprotein and may be identical to Osteogenin. BMP-2B or BMP-2-β (116 amino acids) was renamed as BMP-4 (e.g., GenBank Accession No. ACB21039; encoded by GenBank Accession No. EU518936), 72% with drofila protein Homology. The BMP-7 (eg, GenBank Accession No. NP_001710; encoded by GenBank Accession No. NM_001719) is identical to OP-1 (osteogenic protein-1). Mouse and human proteins have 98% homology. The BMP-8 (139 amino acids) may be referred to as BMP-8a, and may have the same sequence as OP-2 (osteogenic protein-2). The BMP-8b (139 amino acids) OP-3 (osteogenic protein-3) may have the same sequence. The BMP-9 (110 amino acids) is also referred to as GDF-5. The BMP-11 (109 amino acids) has been isolated from bovine sources, also referred to as GDF-11. The BMP-12 (104 amino acids) may also be referred to as GDF-7 or CDMP-3. The BMP-14 (eg, GenBank Accession No. AAH32495; encrypted by GenBank Accession No. BC032495) may be identical to GDF-5 and CDMP-1.

The bone forming protein (BMP) may be BMP-2, BMP-3 or BMP-7.

The gene encoding the protein having the bone formation function may be used by cloning or synthesizing the cDNA obtained from the sequencing information encoding the protein having the bone formation function, and a part of these sequences if it can exhibit the same or similar activity Modified, deleted, substituted may also be used.

In the present invention, the stem cell is a concept including both the stem cells and osteoblasts differentiated and / or proliferated from the stem cells. The stem cells may be mesenchymal stem cells or mesenchymal stem cells (MSC).

The mesenchymal stem cells may be collected from the patient himself or used to separate human leukocyte antigen (HLA) type matched bone marrow using a blood bank database in order to minimize immune rejection.

The mesenchymal stem cells may be isolated from bone marrow, blood (peripheral blood), cord blood, amniotic membrane, amniotic fluid, adipocytes, etc. of all mammals including humans, rodents, etc., in particular, human bone marrow, cord blood Or mesenchymal stem cells isolated from adipocytes, wherein the age is irrelevant, and human leukocyte antigen (HLA) type is matched from a patient or using a blood bank database to minimize immune rejection. It can be used separately from the bone marrow.

Stem cells into which the gene encoding the protein having the bone formation function is introduced, stem cells proliferated from the stem cells and / or stem cells differentiated from the stem cells, for example, a gene encoding the protein having the bone formation function Osteoblasts were introduced into osteoporotic fractures, diabetic fractures, bone defects, osteopenia, bone fractures without major shock or special causes, osteomalacia and its resulting fractures, craniofacial restorations, and defects in bones (eg, tumor removal Due to bone defects), bone growth around graft correction (eg, bone growth around hip graft correction), bone growth disorders, diseases due to nonunion fractures, genetic bone growth, etc. Good for cell replacement and gene therapy to treat or prevent more than one disease An effect.

Stem cells into which the gene encoding the protein having the bone formation function is introduced may be prepared by transfecting the stem cells, specifically the mesenchymal stem cells, using a vector containing the gene encoding the protein having the bone formation function. Can be.

The vector containing the gene encoding the protein having a bone formation function means all substances that serve as a carrier used to introduce the gene encoding the protein having a bone formation function into the cell, for example adenovirus expression Vector, adeno-associated virus, retroviral vector or plasmid.

The vector containing the gene encoding the protein having the bone formation function may further include a suicide gene. More specifically, the vector has a binding site of a protein having a bone formation function, a protein expressed by the protein gene having a bone formation function, and is transcribed by a protein expressed by a protein gene having the bone formation function. It may be a vector comprising a gene expression cassette comprising a promoter and suicide gene to determine the initiation.

The gene expression cassette may be one in which the protein having the bone formation function, the promoter and the suicide gene are connected in the transcription direction.

The suicide gene is Herpes simples virus thymidine kinase (HSV-tk) gene, Cytosine deaminase (CD) gene, Varicella zoster virus thymidine kinase (VZV-tk) gene, Escherichia coli nitroreductase (NTR) gene, Cytochrome P450 B1 (CYP2B1) gene It may be one or more selected from the group consisting of Carboxypeptidase G2 (CPG2) gene, Escherichia coli gtp (XGPRT) gene and Escherichia coli Deo (PNP) gene.

The suicide gene is a gene that has a function of converting a prodrug that is harmless to the human body into an anticancer substance having cytotoxicity, for example, 5-FC (5-Fu, a prodrug of an anticancer agent 5-FU (5-fluorouracil) Phosphorylation of cytosine deaminase (CD) and thymidine, which can convert fluorocytosine to 5-FU, as well as acyclovir and GCV, both nucleoside analogs and antiviral agents Thymidine kinase (TK), which can inhibit DNA synthesis by stopping chain elongation by DNA polymerase by entering tri-phosphorylated GCV instead of deoxy-guanosine tri-phosphate during DNA synthesis May be a gene.

The HSV-tk gene may be a HSV-tk gene (GenBank Accesion No. EU814922) or a gene having a sequence of SEQ ID NO: 2, and the CD gene may be a CD gene (GenBank Accesion No. S56903) or a sequence of SEQ ID NO: 3 It may be a gene, the VZV-tk gene may be a VZV-tk gene (GenBank Accesion No. M36160) or a gene having a sequence of SEQ ID NO: 4, the CYP2B1 gene is a CYP2B1 gene (GenBank Accesion No. NM_001134844) It may be a gene having a sequence of SEQ ID NO: 5, the XGPRT gene may be an XGPRT gene (GenBank Accesion No. M15035) or a gene having a sequence of SEQ ID NO: 6, the PNP gene is a PNP gene (GenBank Accesion No. M60917 ) Or the gene having the sequence of SEQ ID NO.

A promoter where a binding site of a protein expressed by the protein gene having the bone formation function exists, and whether transcription initiation is determined by the protein expressed by the protein gene having the bone formation function is an example of osteocalcin. It may be at least one selected from the group consisting of a promoter of protein, a promoter of osteopontin, a promoter of osteonectin, a promoter of alkaline phosphatase, and a promoter of type I collagen.

The osteocalcin (OC) protein may be Bone Gla protein (BGP), the osteocalcin protein gene may be OC (eg, GenBank Accession No. X53698), and the promoter of the osteocalcin protein is GenBank Accession No. . AY147065, the osteopontin (Osteopontin, OP) protein may be Bone Sialoportein (BSP), the osteopontin protein gene may be OP (eg, GenBank Accession No. X13694), the osteo The nectin (Osteonecin, ON, SPARC) protein gene may be ON (eg, GenBank Accession No. J03040), and the promoter of the osteonectin protein is eg GenBank Accession No. It may be a promoter having a sequence of U65081, the alkaline phosphatase gene may be OC (eg, GenBank Accession AB011406), the promoter of the alkaline phosphatase is, for example, GenBank Accession No. It may be a promoter having a sequence of AB035417, wherein the type I collagen (COL1A1) protein gene may be OC (eg, GenBank Accession No. NM_000088).

For example, a stem cell into which a gene encoding the protein having a bone formation function is introduced may be a packaging cell transduced with an expression vector prepared by inserting the protein gene having the bone formation function into an adenovirus vector. After culturing, it can be prepared by infecting mesenchymal stem cells using the adenovirus solution obtained by filtration.

As another example, the stem cell is inserted into the adenovirus vector, the suicide gene and the protein gene having the bone formation function to produce an expression vector, and then transduce the vector into packaging cells, transduced packaging After culturing the cells, the cells are filtered to obtain an adenovirus solution, which can be prepared by infecting mesenchymal stem cells.

The stem cell may be a stem cell into which the gene encoding the protein having the bone formation function and the suicide gene are introduced. In addition, the stem cells can express the gene encoding the protein having the bone formation function and the suicide gene, the expression of the suicide gene is regulated by the protein expressed by the protein gene having the bone formation function It may be a stem cell.

When the bone regeneration composition is in a gel state, it is easy to in vivo transplantation of the stem cells, which is an essential component in order to exhibit a therapeutic effect in the bone regeneration composition, and it is easy to fix the position after implantation in vivo. have. On the other hand, the composition of the solution state has a disadvantage in that it is difficult to fix the position after implantation in vivo and sufficient bone regeneration effect at the desired position due to the fluidity and flow properties. Therefore, the bone regeneration composition is preferably in a gel state.

In this aspect, the present invention includes hyaluronic acid as an active ingredient. The hyaluronic acid is one of the mucopolysaccharides consisting of amino acids and uronic acid, in the present specification, hyaluronic acid is a concept including both hyaluronic acid and modified hyaluronic acid.

Bone regeneration composition comprising a stem cell and a hyaluronic acid, a gene encoding a protein having a bone-forming function of the present invention is 4 ℃ to 50 ℃, including room temperature and body temperature range by including the hyaluronic acid, preferably May be in a gel state at 20 ° C to 40 ° C. When the composition for bone regeneration is in the gel state can be facilitated in vivo transplantation, the position is almost fixed after the transplantation can be obtained a sufficient bone regeneration effect in the desired position.

The inventors of the present invention are natural biopolymers and synthetic biomaterials that can adjust the viscosity of the composition in order to maintain the gel state of the composition containing the stem cells into which the gene encoding the protein having the bone formation function is introduced at 4 ° C to 50 ° C. A study on compatible polymers was conducted.

Specifically, the inventors of the present invention, while conducting experiments on a polymer that can be clinically used for the treatment of bone disease, to study a substance that can adjust the viscosity of the composition, the most desirable effect is when using hyaluronic acid It was confirmed that there is. On the other hand, Puramatrix (3DM, Japan), a hydrogel obtained in clinical practice, and stem cells containing the gene encoding the protein having the bone formation function were used together, and thus, there was little bone regeneration effect. It was confirmed that. The Puramatrix is a synthetic peptide hydrogel consisting of 1% (w / v) synthetic peptide and 99% water. The synthetic peptide is a synthetic peptide composed of 16 amino acids (can-RADARADARADARADA-CNH2).

The hyaluronic acid-containing composition for bone regeneration can maintain the gel state, thereby facilitating the transplantation, and can maintain the gel state for at least one week after the implantation, and can be almost fixed after the transplantation. have. Therefore, the hyaluronic acid can double the bone regeneration effect by contributing to the stable expression and delivery of the genes introduced into the stem cells and increase the viability of the stem cells, there is an advantage that can exhibit a long-term and continuous therapeutic effect .

In addition, the inventors further confirmed the bone regeneration effect according to the molecular weight and content of the hyaluronic acid, when the average molecular weight of 110,000 daltons (Da) when adding 0.5% (w / v) of the hyaluronic acid based on the total composition The bone regeneration effect was confirmed to be the best.

Therefore, the hyaluronic acid may have an average molecular weight of 10,000 to 2,000,000 Daltons, preferably 50,000 to 300,000 Daltons, more preferably 80,000 to 200,000 Daltons. In addition, the content of the hyaluronic acid is 0.01 to 3% (w / v), preferably 0.05 to 2% (w / v), more preferably 0.25 to 1% (w / v) day based on the total composition Can be.

The bone regeneration composition comprising hyaluronic acid under the above conditions may not only have a gel state at 4 ℃ to 50 ℃, but also excellent cell proliferation rate, bone regeneration effect can be remarkably excellent.

The bone regeneration composition may further include an additive depending on the degree of bone disease. The additive serves as a scaffolding skeleton (support) or a scaffold, matrix, or bioadhesive, and the like, and after transplanting the bone regeneration composition, stem cells included in the bone regeneration composition may be implanted in the transplanted position for a longer time. It may be a material added to allow it to be immobilized.

The support may be any biocompatible material used for the purpose of the support, for example, bone components such as hydroxyapatite, beta-tricalcium phosphate (β-TCP), or a complex thereof. It may be a synthetic biocompatible polymer such as a polymer or copolymer made of a substitute or polyphosphazine, polyacrylate, and PLGA (poly (lactic-co-glycolic acid)), but is not limited thereto. no.

The additive may be transplanted in vivo with the transduced stem cells. The content of the additive included in the bone regeneration composition is not particularly limited and can be adjusted appropriately according to the intended role, for example, may be 50 to 95% by weight based on the total composition weight.

The present invention relates to a composition for the prevention, alleviation or treatment of bone diseases comprising the composition for bone regeneration as an active ingredient.

The bone diseases include osteoporosis, osteoporotic fractures, diabetic fractures, bone defects, bone dysplasias that easily break bones without major impacts or special circles, osteomalacia and resulting fractures, craniofacial restorations, fracture defects (e.g., , Defects due to tumor removal), bone growth around graft correction (eg, bone growth around hip implant correction), bone growth disorders, defects in bone tumors, nonunion fractures and genetic bone growth. And diseases caused by "bone defects" or "defective bones" including bone kidneys and the like, and include all diseases treatable by bone formation or bone regeneration.

As used herein, the term "bone defect" or "defective bone" may include fractures, fractures, and / or degeneration of bone, including such diseases caused by a wound or disease, and spinal column of the spinal column. And may further comprise a further degeneration of the disc region between the defect and the spine.

The bone diseases are specifically osteoporosis, osteoporotic fractures, diabetic fractures, bone defects, osteoplastic insufficiency, osteomalacia and resulting fractures, fracture defects, bone growth around graft correction, bone growth disorders, bone tumors, nonunion fractures and It may be at least one selected from the group consisting of defects in genetic bone growth.

The osteoporosis is a structural degeneration of the skeleton caused by loss of bone mass that causes imbalance in bone formation, bone resorption, or both, so that resorption governs the bone formation stage, thereby reducing the weight bearing capacity of the affected bone. Means.

In healthy adults, the rate at which bone is formed and resorbed is tightly adjusted to maintain regeneration of skeletal bone. However, in individuals with osteoporosis disease, imbalances in these bone remodeling cycles progress, resulting in loss of bone mass and the formation of microstructure defects in continuity of the skeleton. Accumulation of skeletal defects created by confusion in the bone remodeling cycle ultimately severely impairs structural preservation of the skeleton, leading to fractureable time points. This imbalance develops gradually with age in most individuals (senile osteoporosis) and can occur radically in postmenopausal women.

In another aspect, the present invention uses the composition for bone regeneration to produce bone of a patient suffering from bone fracture in an individual infected with a disease that reduces skeletal bone mass, particularly a disease causing imbalance in the bone remodeling. It is about how to.

The present invention also provides a composition for enhancing bone growth and the composition comprising the composition for bone regeneration in another aspect, to treat, alleviate and / or prevent fractures of children suffering from bone diseases, including metabolic bone diseases. A method of treating, alleviating and / or preventing pediatric metabolic bone disease by use thereof.

In another aspect, the present invention also includes the composition for bone regeneration, which treats, alleviates, and / or treats fractured bone in individuals at risk of reducing bone mass, including postmenopausal women, the elderly, and patients undergoing dialysis. Or a composition for preventing or a method of treating, alleviating and / or preventing the bone to be fractured using the composition.

The present invention also relates to a method for treating a defect in a microstructure of a structurally damaged bone, comprising treating the bone fracture using the bone regeneration composition in another aspect. Accordingly, the present invention aims to stimulate bone formation and, optionally, to increase bone mass over a long period of time, and in particular to reduce the occurrence of new fractures resulting from structural degeneration of the skeleton.

In another aspect, the present invention may also be directed to a method of strengthening a bone graft in a vertebrate, eg, a mammal, by administering the composition for bone regeneration at or near a fracture or osteoclast.

There is no particular limitation on the manner of administering the composition for bone regeneration according to the present invention and / or the composition for preventing, alleviating or treating the bone disease, and may be, for example, by dropping the bone disease site during injection or bone disease site surgery.

The composition for bone regeneration according to the present invention and / or the composition for preventing, alleviating or treating the bone disease is characterized in that it comprises the stem cells and hyaluronic acid, it can be formulated for topical administration. In the present invention, the composition for bone regeneration and / or the composition for the prevention, alleviation or treatment of the bone disease is generally a filler, thickener, binder, humectant, dispersant, surface active agent, corrosive polymer depending on the mode of administration and the characteristics of the dosage form. And diluents of excipients, which may include lubricants.

The composition for bone regeneration and / or the composition for the prevention, alleviation or treatment of the bone disease is advantageously formulated parenteral composition in dosage unit form for ease of administration and uniformity of dosage.

Pharmaceutical forms suitable for injection, which are examples of such formulated forms, include sterile distilled water (ie, soluble aqueous solutions) or dispersions, and sterile powders for immediate preparation of sterile injectable solutions or dispersions. In all cases, the pharmaceutical form should be sterile and fluid to the extent that it can be easily injected. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier may be, for example, a solvent or dispersion medium, including water, ethanol, polyols (eg, glycerol, propylene glycol and liquid polypropylene glycol, etc.), suitable mixtures thereof, or vegetable oils.

The dosage of the composition for bone regeneration and / or the composition for preventing, alleviating or treating bone diseases according to the present invention may vary depending on the age, weight, sex, dosage form, health condition, type of disease and symptoms of the patient. For example, in order to obtain sufficient bone regeneration and / or prevention, alleviation or treatment effect of bone disease, the dosage of the composition for bone regeneration according to the present invention and / or the composition for preventing, alleviating or treating bone disease may be 0.05 to 2 mL. However, it is not limited thereto.

Another example of the present invention relates to a method for transdifferentiating stem cells into osteoblasts and a method for preparing a composition for bone regeneration.

Another example of the present invention is a protein gene having a bone formation function, a binding site of the protein expressed by the protein gene having the bone formation function is present, and by the protein expressed by the protein gene having the bone formation function It relates to a gene expression cassette comprising a promoter and suicide gene is determined whether transcription initiation.

Another example of the invention relates to a vector comprising said gene expression cassette, preferably an adenovirus expression vector.

The method of differentiating stem cells comprises the steps of preparing a recombinant expression vector comprising a DNA sequence encoding a bone regeneration inducer, preferably a protein having a bone formation function; And transfecting the recombinant expression vector with stem cells to transdifferentiate the stem cells into osteoblasts.

The method for preparing a composition for bone regeneration includes a gene encoding a protein having a bone forming function or a vector containing a gene encoding a protein having a bone forming function, and a protein expressed by a gene encoding the protein having a bone forming function. Producing a vector comprising a gene expression cassette consisting of a promoter and a suicide gene which is determined whether to initiate transcription by connecting in the transcription direction; Introducing a gene encoding a protein that functions to form bone into stem cells using the vector; And mixing the stem cells or cells differentiated from the stem cells with hyaluronic acid to prepare a gel composition.

The bone-forming protein may be a bone morphogenetic protein (Bone Morphogenetic Protein, BMP), preferably a mammalian bone morphogenetic protein.

The protein gene having the bone formation function, the bone formation protein is BMP-2, BMP-3, BMP-4, BMP-7, BMP-8, BMP-8b, BMP-9, BMP-10, BMP-11, It may be one or more selected from the group consisting of BMP-12 and BMP-14, but is not limited thereto.

The protein gene having a bone formation function may be at least one selected from the group consisting of BMP-2 gene, BMP-3 gene, BMP-4 gene, BMP-7 gene, and BMP-14 gene, wherein the BMP-2 gene is May be a gene having a sequence of SEQ ID NO: 1

The stem cells may be mesenchymal stem cells. The stem cells may be differentiated into osteoblasts by the introduced gene, specifically, a protein having a bone formation function by expression of the protein gene having the bone formation function.

The suicide gene may be one or more selected from the group consisting of HSV-tk gene, CD gene, VZV-tk gene, NTR gene, CYP2B1 gene, CPG2 gene, XGPRT gene and PNP gene.

A promoter where a binding site of a protein expressed by the protein gene having a bone formation function exists, and whether to initiate transcription by a protein expressed by the protein gene having a bone formation function is an example, an osteocalcin protein. It may be one or more selected from the group consisting of a promoter of, a promoter of osteopontin, a promoter of osteonectin, an promoter of alkaline phosphatase, and a promoter of type I collagen.

The gene expression cassette may be one in which the bone morphogenetic protein, the promoter and the suicide gene are linked in the transcription direction.

The method of producing the expression vector or the method of introducing the gene is a method using an adenovirus, a method using an adeno-associated virus, a method using a retrovirus, a method using a plasmid, for example DNA-calcium precipitation or electrophoresis It may be a method of introducing a gene known in the art, such as electroporation, liposomes, and / or polyamines.

The method of introducing the gene is preferably a method using an adenovirus in consideration of the characteristics of stem cells. More specifically, the introduction of a gene encoding a protein having a bone formation function in the stem cells is inserted into the protein gene having a bone formation function into an adenovirus vector to produce an expression vector, using this to transfect the stem cell It can be carried out by the method.

For example, the protein gene having the bone formation function or the suicide gene and the protein gene having the bone formation function are inserted into an adenovirus vector to produce an expression vector, and then the vector is transduced into packaging cells. After culturing the transduced packaged cells, and filtered to obtain the adenovirus solution, by using it to infect the mesenchymal stem cells using the suicide gene or the suicide gene and the protein gene having the bone formation function introduced into the mesenchymal stem cells By doing so, the stem cells can be prepared.

According to the method, it is possible to significantly increase the concentration of bone regeneration inducing factors, such as proteins having bone formation function in the mesenchymal stem cells, thereby obtaining an excellent bone regeneration effect in vivo injection.

Compositions according to the invention can be stored at appropriate conditions prior to implantation, for example stem cells can be stored frozen in 10% DMSO in liquid nitrogen, and storage conditions and methods can be as commonly known in the art. .

The bone forming composition of the present invention is capable of differentiating stem cells into osteoblasts with excellent efficiency, easy in vivo transplantation, and being almost fixed at the transplanted place, thereby obtaining sufficient bone regeneration effect at a desired location. Since good bone forming effects can be obtained, it can be used to treat bone diseases such as osteoporotic fractures, osteomalacia, osteopenia and bone growth disorders. In addition, the side effects caused by stem cells moved to other parts of the body other than the treatment site through the expression of suicide genes, and side effects due to the expression of a protein having excessive bone formation function, etc. can be controlled, the composition for treating existing bone diseases In comparison, the treatment of side effects has a significantly elevated effect.

The composition for bone formation of the present invention can be used in cell replacement therapy or gene therapy for bone disease, or widely used as a material for verifying drug effects or various studies in developing new drugs.

1 is a photomicrograph of the morphology of mesenchymal stem cells (passage 6) isolated from umbilical cord blood and cultured in vitro.

Figure 2 is a graph confirming the immune phenotype expression pattern of cord blood-derived mesenchymal stem cells (passage 6).

3 is a cleavage map of an adenovirus vector expressing BMP-2, a bone morphogenic protein.

Figure 4 is a photograph of the green fluorescent protein expressed in mesenchymal stem cells introduced after transduction of adenovirus expressing the green fluorescent protein GFP into mesenchymal stem cells at 50 M.O.I and 500 M.O.I under a fluorescence microscope.

5 is a graph of bone mineral density (BMD) measured for 20 weeks by inducing osteoporosis from 6 week old Sprague-Dawley rat females. In the graph, OVX means an ovariectomized experimental group, Ca free means an experimental group fed with a calcium-free feed, and Low Ca refers to an experimental group fed with a calcium-containing feed in a low concentration. it means. In the graph, the horizontal axis represents breeding period, and the vertical axis represents bone mineral density.

FIG. 6 is a graph showing calcium concentration in bone blood for 20 weeks by inducing osteoporosis from 6 week old Sprague-Dawley rat females. In the graph, OVX means an ovariectomized experimental group, Ca free means an experimental group fed with a calcium-free feed, and Low Ca refers to an experimental group fed with a low-calcium feed. it means. In the graph, the horizontal axis represents breeding period, and the vertical axis represents blood calcium concentration.

Figure 7 is a radiographic picture of a defect site that induced bone defects in the tibia of the rabbit.

Figure 8 is a photograph of the tissue of the defect site induced bone defects in the skull of a nude mouse.

9 is a bone retaining composition containing bone marrow mesenchymal stem cells (MSC / BMP2) and 2% (w / v) hyaluronic acid for transplantation introduced BMP-2 of the present invention maintained at an angle of 10 degrees at room temperature The photograph shows the result maintained for 10 minutes after application to the site.

FIG. 10 is a photograph of bone regeneration of the osteoporotic fracture experimental animals after radiation, 7 days, 21 days, 35 days, 49 days, 63 days, 77 days and 91 days after the injection of PBS.

FIG. 11 is a 7-day, 21-day injection of a bone regeneration composition containing hyaluronic acid and transplanted mesenchymal stem cells (MSC / BMP2) into which a BMP-2 of the present invention was introduced to a fracture site of an osteoporotic fracture experimental animal. Photographs of bone regeneration observed by radiation after days 35, 49, 63, 77 and 91.

Figure 12 is a bone regeneration composition containing the mesenchymal stem cells (MSC / BMP2) and hyaluronic acid for transplantation BMP-2 introduced into the skull defect site, hyaluronic acid solution, mesenchymal stem cell culture and BMP-2 After treatment with the transplanted mesenchymal stem cells (MSC / BMP2) and the composition for bone regeneration including the furametrics, the skull defect is photographed again after 6 weeks.

FIG. 13 shows the skull defect at 6 weeks after treatment of the bone regeneration composition including transplanted mesenchymal stem cells (MSC / BMP2) into which a skull defect was introduced and hyaluronic acid having different molecular weights. This is a picture taken again.

14 is a bone regeneration mesenchymal stem cells (MSC / BMP2) and bone regeneration of the present invention to the bone defect site and the bone regeneration composition containing hyaluronic acid of different contents, after 6 weeks have elapsed It is a photograph of the skull defect site again.

15 shows the examination of the regenerated bone tissue. Paraffin cuts of regenerated bone tissue were prepared 28 and 91 days after cell injection, and stained with Masson's trichrome (MT staining) and with hematoxylin and eosin (H & E staining). A picture of the result of dyeing. 15A and 15B are MT staining results, FIG. 15C and D are H & E staining results, A and C are photographs taken at 28 days, and B and D are 91 days. The picture was taken when the elapsed time. The inverted triangle-shaped arrow of FIG. 15 indicates an initial fracture site.

FIG. 16 is a photograph of a paraffin-cutting portion of regenerated bone tissue, and confirmed by immunohistochemical staining using an antibody against osteocalin. FIG. A and C of FIG. 16 are photographs taken after A and C are reacted with only a secondary antibody (2 ″ ab), and B and D of FIG. 16 are reacted with an antibody and a secondary antibody against osteocalcin and then photographed. A and B are photographs taken when 28 days have elapsed, and C and D are photographs taken when 91 days have elapsed, and the inverted triangle-shaped arrow of FIG. Indicates.

Figure 17 is a bone regeneration composition containing the mesenchymal stem cells and hyaluronic acid for transplantation, hyaluronic acid solution and hyaluronic acid and mesenchymal stem cell culture medium containing BMP-2 of the present invention introduced into an ectopic bone formation induction experimental animal model After inserting the support treated with the solution, the photograph of the result of staining the bone tissue paraffin cleaved portion of the site where the support was inserted by staining with hematoxylin and eosin (H & E staining) at 4 weeks. .

Hereinafter, the present invention will be described in more detail in the Examples. However, the following examples are only illustrated to help understanding of the present invention, but the scope of the present invention is not limited by the following examples.

Example 1 Isolation, Culture and Passage Culture of Mesenchymal Stem Cells

Isolation, culture, and passage of mesenchymal stem cells were made from cord blood or bone marrow.

Example 1-1. Isolation of Mesenchymal Stem Cells

Umbilical cord blood obtained by consent from the donor was centrifuged with Ficoll-Hypaque gradient (density 1.077 g / cm ³ , Sigma) to obtain monocyte cells, followed by basal culture medium (10% FBS). After washing several times with the minimal medium α-MEM (Gibco Inc.) to which Gibco was added, the cells were inoculated and suspended in a basic culture medium at a concentration of ⁴ × 10 ⁶ cells / cm ² , which was maintained at a temperature of 37 ° C. and 5% CO _2. Incubated for 7-14 days with medium exchange twice a week under wet atmospheric conditions, including fibroblast-like adherent cells observed after treatment with 0.25% trypsin (invitrogen). After the cells were removed and collected, the cells were increased by repeating subcultures at a concentration of 5 × 10 ⁴ cells / cm ² in the minimal medium α-MEM medium to which the basic culture medium was added.

Example 1-2. Cultivation and passage of mesenchymal stem cells

Mesenchymal stem cells isolated in Example 1-1 using a CO ₂ incubator while maintaining 37 ℃ mesenchymal stem cell medium 89% (α-MEM (Thermo Co.) + 1X antibiotic-antimycotic (antibiotic- antimycotic, Gibco) + 10% FBS (Hyclone)). Incubate the cells while replacing the medium every two days. When the cell is about 80% full, the cells are dropped using 0.25% trypsin-EDTA (Gibco), and then the medium is added 1:20. Diluted with and then subcultured in fresh medium. Some of the cells were stored frozen using medium added with 10% DMSO (Sigma). 1 is a photograph taken on an optical microscope after culturing mesenchymal stem cells (passage 6) isolated from human bone marrow in vitro for 3 days, and FIG. 2 shows the immune phenotype expression of these mesenchymal stem cells. One result.

Example 1-3. Checking the Differentiation of Mesenchymal Stem Cells

The differentiation ability of the mesenchymal stem cells cultured in Example 1-2 and the mesenchymal stem cells to adipocytes, chondrocytes and bone cells in Example 1-1 was confirmed as follows.

1) Differentiation into adipocytes

After culturing the mesenchymal stem cells about 80% of the culture vessels using the mesenchymal stem cell medium, the adipocyte differentiation medium (1 μM dexamethasone (dexamethasone, Sigma), 0.5 mM methyl-isobutyl xanthine (Sigma), insulin (10 μg / ml, Gibco), 100 nM indomethacin (indomethacin, Sigma) and 10% FBS (DMEM medium containing Gibco) for 48 hours. After the incubation, the cells were cultured in adipocyte maintenance medium (DMEM medium containing 10 μg / ml insulin and 10% FBS) for 1 week and stained using oil red O. As a result of the staining, it was confirmed that oil droplets stained red by oil red O were generated in the cells, thereby confirming that the mesenchymal stem cells were differentiated into adipocytes.

2) Differentiation into chondrocytes

After the mesenchymal stem cells were cultured about 80% of the culture vessels using the mesenchymal stem cell medium, the cells were separated by trypsin. 0.5 ml of serum-free chondrocyte differentiation induction medium (50 ml high-glucose DMEM (Gibco)), 0.5 ml 100x ITS (0.5 mg / ml bovine pancreas derived insulin, 0.5 mg) into 2 x 10 ⁵ cells obtained by centrifugation. / Ml human transferrin, 0.5 μg / ml sodium selenate; Sigma), 50 μl linoleic acid-albumin (Sigma), 100 nM dexamethasone (Sigma) and 10 ng / ml TGF-β1 (Sigma) Incubated. After culturing for 3 weeks while replacing the chondrocyte differentiation induction medium at 3 days intervals, the cells were fixed with 4% paraformaldehyde solution, sections were made with microtome, and alkian blue staining was performed. As a result of the staining, it can be confirmed that the mesenchymal stem cells were differentiated into chondrocytes from the fact that the cartilage matrix components outside the chondrocytes were stained blue in the Alcian blue staining solution and the presence of the chondrocytes in the cartilage cavity (lacuna).

3) Differentiation into Bone Cells

After mesenchymal stem cells were cultured in the culture vessel by using mesenchymal stem cell medium, approximately 80% of the culture vessels were cooled, and then bone cell differentiation-inducing medium (10 mM β-glycerol phosphate (Sigma), 0.2 mM ascorbate) 2-phosphate (Sigma), 10 nM dexamethasone (Sigma) and DMEM medium containing 10% FBS (Gibco)) were incubated for 2 weeks with medium replacement at 3 day intervals. After incubation, cells were fixed with 4% paraformaldehyde solution and subjected to von Kossa and alkaline phosphatase staining. As a result, it was confirmed that mesenchymal stem cells were differentiated into bone cells from the increase of intracellular alkaline phosphatase activity and the accumulation of hydroxyapatite-type calcium, which is confirmed by poncosa staining, outside the cells.

Example 2: Adenovirus Vector Construction

Example 2-1. Cloning of BMP-2 Gene and Construction of BMP-2 Expressing Adenovirus

Human BMP-2 gene (bone morphogenetic protein 2; SEQ ID NO: 1) was cloned by PCR (polymerase chain reaction) using human fetal brain cDNA (TissueGene Inc., USA) and two primers. The 5 'primer was 5'-TCCCAGCGTGAAAAGAGAGACTGC-3' (SEQ ID NO: 8) and the 3 'primer was 5'-TTTTGCTGTACTAGCGACACCCACAACC-3' (SEQ ID NO: 9).

After PCR using a GC rich PCR system (Roche), cloning into the CRII-TOPO vector was performed using the TOPO TA cloning kit (invitrogen). The sequencing of the cloned vector confirmed that the BMP-2 gene was included in the vector, and only the BMP-2 gene was cut out using restriction enzymes. The recombinant BMP-2 gene and the recombinant pAd vector (Pohang University, Korea) cut with the restriction enzyme were bound using T4 DNA ligase (Roche), and then transformed into E. coli DH5α (Stratagene, USA). A recombinant pAD / BMP2 vector was constructed in which the BMP-2 gene was inserted into the recombinant pAd vector.

In addition, to confirm the efficient expression of BMP-2 in the adenovirus system, a GFP adenovirus vector was prepared by inserting a green fluorescent protein (GFP) gene instead of the bone morphogenic protein gene in the adenovirus vector preparation process. A cleavage map of the adenovirus vector into which the bone morphogenetic protein (BMP2) is inserted is shown in FIG. 3.

The vector recombinant pAD / BMP2 or GFP adenovirus vector was transformed into QBI-293A (Qbiogene) cells, an adenovirus packaging cell line by calcium phosphate precipitation (Jordan, Nucleic Acid Research, 24, 596-601 (1996)). After transduction, the cells were placed in an incubator and incubated for 48 hours under humid conditions including 37 ° C. and 5% CO ₂ . After incubation for 48 hours, only the culture medium was obtained, and the culture solution was filtered through a 0.45 μm filtration membrane to obtain a BMP-2 adenovirus solution or a GFP adenovirus solution. The adenovirus solutions were aliquoted and stored at -70 ° C.

Example 2-2. Cloning of CD Gene and Construction of BMP-2 and CD Expressing Adenoviruses

DNA was isolated from Escherichia coli K-12 MG1655 (ATCC 700926, Korea Research Institute of Bioscience and Biotechnology), followed by primer CD-F (5'-GAATTCAGGCTAGCAATGTCTCGAATAACGCTTTACAAA C-3 '; SEQ ID NO: 10) and CD-R (5). '-GGATTCTCTAGCTGGCAGACAGCCGC-3', SEQ ID NO: 11), 94 ° C. 5 minutes; 94 ° C. 30, 60 ° C. 40 seconds, 72 ° C. 1 minute, 27 cycles; PCR was performed at 72 ° C. for 7 minutes.

The PCR product was cloned using pGEM-T Easy Vector Cloning Kit (Promega), and then, p-GEM-CD vector containing the CD gene was produced by screening blue-white colonies using X-gal IPTG. It was. The prepared pGEM-CD vector was sequenced using a primer having a sequence of SEQ ID NO: 12 (5'-CATACGATTTAGGTGACACTATAG-3 ') and a primer having a sequence of SEQ ID NO: 13 (5'-ACCGGGAAACACCTATTGTG-3'). As a result, it was confirmed that the suicide gene contains CD.

The pGEM-CD plasmid and pcDNA3.1 (Clontech) were cut with EcoR I (BM) and Not I (BM) restriction enzymes, respectively, and the CD gene and cleaved plasmid pcDNA3.1 isolated from plasmid pGEM-CD were cut. After linking with a T4 DNA ligase (BM Co., Ltd.), the E. coli DH5α, which is competent cells, was transformed, cultured in an LB plate containing 50 ㎍ / ml of ampicillin, and then plasmid pcDNA3. .1-CD was obtained.

The plasmid was cut with Bam H I (BM) restriction enzyme, and recombinant pAD / BMP2 (Qbiogene, Inc.), an adenovirus vector capable of producing adenovirus containing BMP2 prepared in Example 2-1, was cut with Bgl II. Then, the vector recombinant pAD / BMP2-CD was produced by connecting with T4 DNA ligase. As a result of sequencing the vector recombinant pAD / BMP2-CD, it was confirmed that it contained a sequence matching the CD (gi: 298594), which is a suicide gene having the sequence of SEQ ID NO: 3.

The vector recombinant pAD / BMP2-CD was transduced into QBI-293A (Qbiogene) cells, an adenovirus packaging cell line, by calcium phosphate precipitation (Jordan, Nucleic Acid Research, 24, 596-601 (1996)). It was then placed in an incubator and incubated at 37 ° C. and wet atmospheric conditions containing 5% CO ₂ . After incubation for 48 hours, only the culture solution was obtained, and the culture solution was filtered through a 0.45 μm filtration membrane to obtain an adenovirus solution. The adenovirus solutions were aliquoted and stored at -70 ° C.

Example 3: Introduction of Foreign Genes into Mesenchymal Stem Cells

Example 3-1. Introduction of BMP-2 into Mesenchymal Stem Cells

The BMP2 adenovirus obtained in Example 2-1 was isolated from Example 1-1 and cultured to about 70% in mesenchymal stem cells (MSC) cultured in Example 1-2 in a 100 mm culture vessel. Solution or GFP adenovirus solution with a titer of 2 × ^{10 10} pfu / ml in 5 ml incomplete medium (99% α-MEM (Thermo) and 1 × antibiotic-antimycotic (Gibco)). Dilutions were added to 50 MOI (Multiplicity of infection) and 500 MOI. After adding the recombinant pAD / BMP2 vector adenovirus solution or green fluorescent protein (GFP) adenovirus solution and treating the cells for 2 hours, 5 ml of culture medium (89% α-MEM (Thermo), 1X antibiotic Antibiotic-antimycotic (Gibco) and 10% FBS (Hyclone) were added.

In order to confirm the transfection, the expression of GFP was confirmed. Specifically, the GFP adenovirus solution was transfected into 1X10 ⁶ cells of mesenchymal stem cells at 50 MOI and 500 MOI, and then the transfected mesenchymal stem cells were cultured for 48 hours. After the incubation, the GFP expressed in the mesenchymal stem cells in the culture medium was observed under a fluorescence microscope, and the results are shown in FIG. 4. As shown in FIG. 4, the higher the concentration of the GFP adenovirus solution, the stronger the fluorescence was confirmed, and the transfected mesenchymal stem cells were confirmed to be able to normally express GFP.

In addition, to confirm the transfection of the BMP2, it was confirmed whether the expression of BMP. Specifically, the BMP2 adenovirus solution was transfected into 1X10 ⁶ cells of mesenchymal stem cells at a concentration of 500 MOI, and then the transfected mesenchymal stem cells were cultured for 24 hours. After the culture, the amount of BMP2 expressed in mesenchymal stem cells in the culture medium was measured by enzyme-mediated immunoassay (ELISA). The measured BMP2 amount is shown in Table 1 below.

Table 1

	Cell No.	BMP2 amount (ng)
After 24 hours incubation	1 X 10 6 cells	386.25

As shown in Table 1, a sufficient amount of BMP2 was detected in the culture of mesenchymal stem cells transformed with the BMP2 adenovirus solution, and it was confirmed that the transfected mesenchymal stem cells can normally express BMP2. .

Example 3-2. Introduction of BMP-2 and CD into Mesenchymal Stem Cells

After separating in Example 1-1 and incubating the mesenchymal stem cells (MSC) cultured in Example 1-2 to about 70% in a 100 mm culture vessel, the recombinant pAD / obtained in Example 2-2 The BMP2-CD adenovirus solution was added diluted to 50 MOI in 5 ml incomplete medium (99% α-MEM (Thermo) and 1X antibiotic-antimycotic (Gibco)). After adding the recombinant pAD / BMP2-CD adenovirus solution and treating the cells for 2 hours, 5 ml of culture medium (89% α-MEM (Thermo), 1X antibiotic-antimycotic, Gibco) and 10% FBS (Hyclone)) were added. After addition of the culture medium, cells were cultured in a CO ₂ incubator (CO ₂ water Jacketed Incubator, ThermoForma ScientificTM) for two days to prepare a cell line MSC / BMP2-CD capable of expressing BMP2 and CD suicide genes.

Example 4: Confirmation of the effect of mesenchymal stem cells introduced CD

Example 4-1. Prodrug Cytotoxicity Assessment in MSC / BMP2-CD

After incubating 5,000 MSC / BMP2-CD prepared in Example 3-2 per well into a 6-well plate, the following day, 5-FC (Sigma), a 5-FU prodrug, was 0 to 10,000 μM. MTT analysis was performed after treatment for 2 weeks with changing medium every 2 days. As a result of performing the MTT analysis, it was confirmed that as the concentration of 5-FC increased, the cell death rate increased.

Example 4-2. Identification of Peripheral Effects on Mesenchymal Stem Cells Incorporated with CD Gene

In a 6-well plate, 10,000 cells per well were isolated in Example 1-1, and the mesenchymal stem cells (MSC) cultured in Example 1-2 or MSC / BMP2-CD prepared in Example 3-2 3,000 C6 / LacZ glioma cells were added and cultured for 24 hours. From the next day, the 5-FC was treated for 2 weeks while changing the medium every two days at a concentration of 0 to 1,000 Μm, and then X-gal staining and beta-galactosidase analysis were performed.

According to the results, the treatment with 1000 μM 5-FC confirmed that C6 / LacZ cells were killed in the wells containing MSC / CD cells. In addition, in wells containing MSC cells and C6 / LacZ cells (MSC), beta-galactosidase activity was hardly changed even when the concentration of 5-FC was increased, whereas MSC / BMP2-CD cells and C6 / In wells (MSC / CD) containing LacZ cells, the activity of beta-galactosidase decreased with increasing concentration of 5-FC, thus increasing the death rate of C6 / LacZ cells. In addition, it was confirmed that the cell death rate increased with increasing concentration of 5-FC in wells (MSC / CD) containing MSC / BMP2-CD cells and C6 / LacZ cells. From the above results, it was confirmed that the peripheral effect only appeared in the mesenchymal stem cells introduced CD.

Example 5; Osteoporotic Fracture Rat, Bone Defect Rabbit, and Bone Defect Mouse Models

Example 5-1. Induction of Osteoporosis from Female Rats

Sprague-Dawley female rats (Orient Bio Co., South Korea) were used to induce osteoporosis by the following method.

First, rats were anesthetized by injecting lumpun (3.5 mg / kg body weight) and ketamine hydrochloride (20 mg / kg body weight) into the abdominal cavity, and then shaving the hairs of the abdominal cavity and then disinfecting with povidone iodine (betadine). The abdominal cavity was dissected to remove both ovaries. Rats from which both ovaries were removed were continuously bred using AIN-76a feed (Feedrap Co., Ltd., Korea) without calcium and phosphorus. After inducing osteoporosis, calcium concentrations were measured from bone mineral density (BMD) and blood until 20 weeks at 2 week intervals. The results are shown in FIGS. 5 and 6.

Example 5-2. Fracture Induction in Rats Induced Osteoporosis

In Example 5-1, the rats from which both ovaries were removed were bred with AIN-76a feeds from which calcium and phosphorus were removed. Fractures were induced after 5 weeks of breeding the calcium and phosphorus-free AIN-76a feed.

First, lumpun (3.5mg / kg body weight) and ketamine hydrochloride (ketamin hydrochloride, 20mg / kg body weight) were injected into the abdominal cavity to anesthetize female rats bred with AIN-76a feed without calcium and phosphorus. , The hair of the right tibia was cut and sterilized with povidone-iodine (betadine). After dissecting the disinfected right tibia area, a fracture induction of the right tibia was induced using a fracture induction machine. The bone-induced bone fracture of the rat was used in vivo experiments to determine the effect of the bone regeneration composition containing mesenchymal stem cells and hyaluronic acid introduced BMP-2 gene.

Example 5-3. Induction of Bone Defects in Rabbits

New Zealand white rabbits weighing 3.0-3.5 kg (Hyundai Bio, South Korea) were selected for the study to induce bone defects.

First, lumpun (3.5 mg / kg body weight) and ketamine hydrochloride (20 mg / kg body weight) were injected into the muscle to anesthetize the white rabbit, and then the hair of the tibia was cut off and the site to be cut was povidone iodine. sterilized with (betadine). The sterilized tibial site was dissected to expose the tibia and then defects (0.5 cm wide and 1.5 cm high) were made on the tibial site using an orthopedic surgical instrument. Fig. 7 shows the result of photographing the defect site by X-ray.

Example 5-4. Induction of Bone Defects in the Nude Mouse Skull

Six-week-old BALB / c / nu nude male males (Sungkyunkwan University Experimental Animal Center, South Korea) were used to induce the following skull defects.

First, anesthetize nude mice by injecting lumpun (3.5 mg / kg body weight) and ketamine hydrochloride (20 mg / kg body weight) into the abdominal cavity, and then attaching ears and mouth to the stereotaxic frame to prevent the mouse from moving. Fixed. The incision site was disinfected with povidone-iodine (betadine) and the skull was exposed by vertically dissecting 1 cm of the parietal part. The exposed skull was made a defect (4 mm in diameter) using Trephan Burr. After the euthanasia of the mouse with a defect in the skull was collected tissue of the defect site, the results are shown in FIG.

Example 5-5. Induction of Bone Defects in the Rat Skull

Six-week old Sprague-Dawley male rats (Seoul National University Experimental Animal Center, South Korea) were used to induce the following skull defects.

First, anesthetize rats by injecting lumpun (3.5 mg / kg body weight) and ketamine hydrochloride (20 mg / kg body weight) into the abdominal cavity, and then fixing ears and mouth in the stereotaxic frame to prevent rats from moving. I was. The incision site was disinfected with povidone-iodine (betadine), and the skull was exposed by vertically dissecting about 2 cm of the parietal part. Defects (diameter 8 mm) were made using Trephan Burr on the exposed skull. The skull-deficient rats were used in an in vivo experiment to confirm the effect of the bone regeneration composition comprising mesenchymal stem cells and hyaluronic acid introduced BMP-2 gene.

Example 5-6. Induced Ectopic Bone Formation

Six weeks old BALB / c / nu nude male mice (Inha University Experimental Animal Center, South Korea) were used to induce ectopic bone formation as follows.

First, lumpun (3.5 mg / kg body weight) and ketamine hydrochloride (20 mg / kg body weight) were injected into the abdominal cavity to anesthetize nude mice, and then the site to be incised into the nude mouse dorsal incision with povidone iodine (betadine). Sterilized. About 1 cm of the disinfected dorsal part was incised. The dorsal skin incision was used in an in vivo experiment to confirm the effect of the bone regeneration composition containing mesenchymal stem cells and hyaluronic acid introduced BMP-2 gene.

Example 6: In vivo of mesenchymal stem cells expressing BMP-2 ( in vivo Confirmation of bone regeneration induction

Example 6-1. Preparation of the composition for bone regeneration

The mesenchymal stem cells expressing BMP-2 prepared in Example 3-1 were separated with 0.25% trypsin-EDTA (Gibco Co., Ltd.), followed by PBS (1 × 10 ⁵ to 5 × 10 ⁵ cells per 50 μl). Diluted with Sigma). In order to prepare a gel-type bone regeneration composition of the present invention, hyaluronic acid or Puratrix was added to the mesenchymal stem cell dilution expressing the BMP-2. The bone regeneration composition prepared by adding hyaluronic acid or Puratrix to the mesenchymal stem cell dilution solution was transplanted to a test animal alone or together with a support.

The hyaluronic acid was added for the stabilization and growth of cells, the hyaluronic acid used at this time has an average molecular weight (weight average molecular weight) of 50,000 Da (manufactured by Kolon Life Science, Inc.), the average molecular weight 110,000 Da (Kolon Life Science) Lot number PFL-1612E), an average molecular weight of 300,000 Da (manufactured by Kolon Life Sciences, Inc.), and an average molecular weight of 1,220,000 Da (lot number P-0307DE manufactured by Kolon Life Sciences, Inc.) were used. The hyaluronic acid concentration in the distilled water of 0.1% (w / v), 1% (w / v), 2% (w / v), 4% (w / v) and 6% (w / v), respectively It was prepared and used in the form of a hyaluronic acid solution added. The dose of hyaluronic acid is expressed in units of the content of hyaluronic acid added based on the total volume of the total bone regeneration composition, the dose of the hyaluronic acid is 0.05% (w / v), 0.5% (w / v) , 1% (w / v), 2% (w / v), 3% (w / v), 4% (w / v) and 5% (w / v).

In addition, the furametrics is a clinically available hydrogel, specifically, a sequence similar to arginine-glycine-aspartate (RGD), which is a sequence of a cell's integrin binding site. It is a hydrogel (3DM inc, Japan) consisting of 1% (w / v) synthetic peptide consisting of 16 amino acids (can-RADARADARADARADA-CNH2) having a repeating sequence of alanine-aspartate and 99% water. The dose of the Purametrics was used 0.5% (w / v) based on the total bone regeneration composition.

Example 6-2. Identification of the composition

The addition of hyaluronic acid to the composition for bone regeneration is to maintain the gel state at 4 ℃ to 50 ℃. Therefore, in order to confirm the type of hyaluronic acid that can be added to the bone regeneration composition, the viscosity of the bone regeneration composition according to the type and amount of the hyaluronic phase was measured, and the results are shown in Table 2 below.

Preparation of the bone regeneration composition was diluted with PBS to include 1 x 10 ⁵ to 5 x 10 ⁵ cells per 50 μl of the hyaluronic acid solution of each concentration prepared in Example 6-1 and Example 6-1 Mesenchymal stem cell dilutions were prepared by mixing the same amount.

The viscosity was measured using a Brookfield DV-II + Pro (LV) Viscometer at No. 64 spindle at room temperature and 12 rpm rotational speed. In Table 2, 'ND' means that the viscosity value is not measured, that is, the viscosity is higher than the measurement range.

TABLE 2

Content (% of concentration based on total composition)	Viscosity (cps)
	PFL-1612E (110,000 Da)	P-0307D (1,220,000 Da)
0.05	0-5	120
0.5	10-50	11450
One	150	ND
2	2050	ND
4	22745	ND
5	48250	ND

As confirmed in Table 2, when the average molecular weight is used hyaluronic acid of 110,000 Da, when the content of the hyaluronic acid is 0.5% (w / v) to 2% (w / v) the composition is a gel state It was confirmed that it is possible to maintain the gel state in the range of 0.05% (w / v) to 3% (w / v). However, when the average molecular weight is 1,220,000 Da, when the content of hyaluronic acid is 0.5% (w / v) or more, the viscosity of the composition is too high to maintain the gel state, or to maintain the gel state too high viscosity It was determined that the in vivo administration of the composition would not be easy.

Therefore, in subsequent experiments to identify the optimal hyaluronic acid for use in the composition, those having average molecular weights of 50,000 Da, 110,000 Da and 300,000 Da were used.

In addition, the composition containing 2% (w / v) of hyaluronic acid having an average molecular weight of 110,000 Da, which is found to be preferable in Table 2, was applied to the bone site maintained at an angle of 10 degrees at room temperature, and then It was confirmed whether the composition can be fixed in the implanted position, the results are shown in Figure 9

As shown in FIG. 9, the composition containing 2% (w / v) of hyaluronic acid having an average molecular weight of 110,000 Da was confirmed that the composition was not fixed and flowed to the first implanted bone even after 10 minutes at room temperature. It became. From the above results, it was confirmed that when the hyaluronic acid having the average molecular weight of 110,000 Da is used, both the purpose of ease of transplantation and the fixation of post-transplantation position can be achieved.

Example 6-3. Confirmation of hyaluronic acid optimal condition

In order to specify the type (average molecular weight) and content of hyaluronic acid to be used in the bone regeneration composition prepared in Example 6-1, the average molecular weight was 50,000 Da, 110,000 Da based on the results of Example 6-2. And hyaluronic acid which is 300,000 Da.

First, the bone regeneration composition containing the hyaluronic acid in an amount of 1% (w / v), respectively, was left with the support at room temperature for 20 minutes, and then cultured for 2 days and 7 days. The support is a beta-tricalcium phosphate (β-ricalcium phosphate, β-TCP, Kyungwon Medical Co., South Korea) of the granule type (granule) of 1 to 3 mm in diameter.

More specifically, after separating the mesenchymal stem cells expressing BMP-2 prepared in Example 3-1 with 0.25% trypsin-EDTA (Gibco Co.), 1 x 10 ⁵ to 5 x 10 ⁵ cells per 10 μl 10 μl of the diluted solution diluted with PBS (Sigma) and 10 μl of the hyaluronic acid solution of Example 6-1 were mixed, mixed with 0.03 g of the support, and allowed to stand at room temperature for 20 minutes. To be attached. After standing for 20 minutes, the culture medium (89% α-MEM (Thermo), 1X antibiotic-antimycotic (Gibco) and 10% FBS (Hyclone) at 2 days intervals During the exchange, the cells were incubated for 7 days in a humid atmosphere containing 5% CO ₂ and a temperature condition of 37 ° C. using a CO ₂ incubator (ThermoForma Scientific ^™ ). At one time point, color development was performed using MTT reagent, and the cell number was measured by electron microscopy.

The measured results were calculated based on the number of cells in the experimental group measured when left at room temperature for 20 minutes without the addition of hyaluronic acid, the calculation results are shown in Table 3 below.

TABLE 3

	After 20 minutes (%)	2 day culture (%)	7 days culture (%)
No addition	100	112	143
50,000 Da	108	121	162
110,000 Da	131	148	182
300,000 Da	112	119	165

As shown in Table 3, as a result of measuring the cell proliferation rate, it was confirmed that the hyaluronic acid addition group can have a remarkably superior effect compared to the no addition group, and the hyaluronic acid having an average molecular weight of 110,000 Da in the hyaluronic acid addition group. It was confirmed that the cell proliferation showed a significantly high proliferation rate when using.

Based on the results of Table 2 and the results of Table 3 of Example 6-2, an experiment on the optimal content of hyaluronic acid was performed.

The hyaluronic acid having an average molecular weight of 110,000 Da, which was found to be the most preferable in the results of Table 3, was 0.05% (w / v), 0.5% (w / v) and 1% (w) based on the total volume of the total bone regeneration composition. / v) and using a composition for bone regeneration comprising a content of 3% (w / v) was confirmed the cell proliferation rate with the support in the same manner as described above, the results are shown in Table 4 below.

Table 4

Addition amount (w / v)	After 20 minutes	2-day culture	7 days culture (%)
No addition	100	114	141
0.05%	121	137	165
0.5%	129	150	179
One%	119	141	168
3%	105	122	158

As shown in Table 4, as a result of measuring the cell proliferation rate, it was confirmed that the hyaluronic acid addition group can have a remarkably excellent effect as compared to the no addition group, and the hyaluronic acid having an average molecular weight of 110,000 Da in the hyaluronic acid addition group. Using 0.5% (w / v) was found to show the highest growth rate. In the case of using 0.05% (w / v) of hyaluronic acid having an average molecular weight of 110,000 Da, it showed an excellent effect in terms of cell proliferation rate, but the viscosity of the composition was low as confirmed in Example 6-2. It has been pointed out that a problem of low cell proliferation rate has been pointed out when using 3% (w / v) of hyaluronic acid having an average molecular weight of 110,000 Da.

Example 6-4. Transplantation of Mesenchymal Stem Cells

The fracture was induced by the method of Example 5-2 using a fracture induction machine for the right tibia of the osteoporosis-induced rat prepared in Example 5-1. After inducing the fracture, 2 days after the incision was closed, the average molecular weight of 110,000 was confirmed to be the most effective in Example 6-3 while checking the fracture site using a C-arm apparatus. 50 μl of a solution of hyaluronic acid containing 0.5% (w / v) of hyaluronic acid as Da and mesenchymal stem cells expressing BMP-2 prepared in Example 3-1 were separated with 0.25% trypsin-EDTA (Gibco). After the injection, 100 μl of the bone regeneration composition including 50 μl of a dilution diluted with PBS (Sigma) to contain 1 × 10 ⁵ to 5 × 10 ⁵ cells per 50 μl was injected into the fracture site. PBS was injected in equal amounts as a control. Experimental animals injected with the composition for bone regeneration and PBS were observed at the fracture site by radiation at intervals of 2 weeks, the results are shown in Figures 10 and 11.

As shown in FIG. 10 and FIG. 11, when the bone regeneration composition of the present invention containing mesenchymal stem cells expressing BMP-2 (MSC / BMP2) and hyaluronic acid was injected, a remarkably excellent bone regeneration effect was observed. As confirmed, it can be confirmed that the composition for bone regeneration of the present invention is effective for cell therapy.

Example 6-5. Confirmation of bone regeneration effect using experimental animals

Experiments related to the bone regeneration effect on hyaluronic acid confirmed the effect on the cell proliferation rate in Example 6-3 was performed using the rat skull defect model prepared in Example 5-5.

First, hyaluronic acid with different average molecular weight and content of hyaluronic acid confirmed the effect in the experiment on the cell growth rate in Example 6-3 to the defect site of the experimental animal made skull defect by the method of Example 5-5 After treatment with the drop for the bone regeneration composition containing a diluent for transplanting mesenchymal stem cells (MSC / BMP2) introduced BMP-2 (drop), the incision skin was closed. As a comparison group, the furametric solution of Example 6-1 was used.

More specifically, 10 μl of a hyaluronic acid solution containing 0.5% (w / v) of hyaluronic acid having an average molecular weight of 110,000 Da, which was found to be the most effective in Example 6-3 prepared in Example 6-1. And after separating the mesenchymal stem cells expressing BMP-2 prepared in Example 3-1 with 0.25% trypsin-EDTA (Gibco) PBS to contain 1 x 10 ⁵ to 5 x 10 ⁵ cells per 10 μl 20 μl of the composition for bone regeneration containing 10 μl of the diluted solution (Sigma Co., Ltd.) was dropped on the defect site, left for 10 minutes, and then the cut skin was closed.

To confirm the effect of the bone regeneration composition of the present invention, 20 μl of a hyaluronic acid solution containing 0.5% (w / v) of hyaluronic acid having an average molecular weight of 110,000 Da in place of the bone regeneration composition of the present invention. , 20 μl of mesenchymal stem cell culture medium containing 1 × 10 ⁵ to 5 × 10 ⁵ cells per 20 μl passaged in Example 1-2 and the mesenchymal expressing BMP-2 prepared in Example 3-1. Stem cells were removed with 0.25% trypsin-EDTA (Gibco) and 10 μl of dilution diluted with PBS (Sigma) to contain 1 x 10 ⁵ to 5 x 10 ⁵ cells per 10 μl and 0.5% of the Purametrics. (w / v) 20 μl of the composition containing 10 μl of the included Purametrics solution was treated in the same manner as described above.

After 6 weeks after the treatment, the skin of the closed defect site of each experimental animal was incised again to observe the defect site, and the results are shown in FIG. 12.

As shown in FIG. 12, when hyaluronic acid was treated alone, it was confirmed that there was almost no bone regeneration effect. In addition, when the hyaluronic acid and mesenchymal stem cells were treated, a fine bone regeneration effect was confirmed. From the above results, it was evaluated that treatment with hyaluronic acid itself or mesenchymal stem cells and hyaluronic acid alone was not excellent in bone regeneration effect, and thus no therapeutic effect could be expected. On the other hand, even when BMP-2-introduced transplanted mesenchymal stem cells and Purametrics were used in the same amount, the therapeutic effect was insufficient. Thus, a composition containing BMP-2-induced transplanted mesenchymal stem cells was simply prepared in a gel state. It was confirmed that only the bone regeneration effect can be achieved.

Compared to the above results, in the case of the composition using the combined mesenchymal stem cells and hyaluronic acid for transplantation BMP-2 of the present invention compared to the above results to the extent that all of the bone defect sites of bone defects of 8 mm are regenerated A remarkably good bone regeneration effect was confirmed. From the above results, a remarkably excellent effect related to bone regeneration of the bone regeneration composition of the present invention is confirmed in vivo using an experimental animal, and the bone regeneration composition of the present invention can be effectively used for cell therapy. It was expected to be.

In addition, in order to confirm the bone regeneration effect in an in vivo experiment according to the average molecular weight of hyaluronic acid, the hyaluronic acid having an average molecular weight of 50,000 Da, 110,000 Da and 300,000 Da is 0.5 in Example 6-1. 10 μl of the hyaluronic acid solution containing% (w / v) and mesenchymal stem cells expressing BMP-2 prepared in Example 3-1 were separated with 0.25% trypsin-EDTA (Gibco), and then 1 per 10 μl. 20 μl of the composition for bone regeneration, containing 10 μl of dilution diluted with PBS (Sigma) to include x 10 ⁵ to 5 x 10 ⁵ cells, was dropped on the defect site in the same manner as described above. After standing, the incised skin was closed.

After 6 weeks after the treatment, the skin of the closed defect site of each experimental animal was incised again to observe the defect site, and the results are shown in FIG. 13.

As shown in FIG. 13, it was confirmed that when the hyaluronic acid was treated together, the bone regeneration effect was superior to that when the mesenchymal stem cells for transplantation with BMP-2 were introduced alone.

Specifically, in the case of using hyaluronic acid having an average molecular weight of 50,000 Da, the cell culture results in the in vitro experiments confirmed in Example 6-3 were compared with the case of using hyaluronic acid having a similar average molecular weight of 300,000 Da. The effect was low, and from these results it was expected that the viscosity of the whole composition, together with the cell culture results, would also affect the bone regeneration effect in relation to the optimal range of hyaluronic acid.

In addition, it was confirmed that the composition using hyaluronic acid having an average molecular weight of 110,000 Da was also excellent in the cell culture results in the in vitro experiments confirmed in Example 6-3, and in vivo using experimental animals. In the experiment, only about 50% of the total bone defects were regenerated, compared to other hyaluronic acids having an average molecular weight of 10%, and in the composition using hyaluronic acid having an average molecular weight of 110,000 Da, all of the 8 mm skull defects were regenerated. Significantly good bone regeneration effect was confirmed.

From the above results, it was confirmed that the optimum molecular weight of hyaluronic acid of the composition for bone regeneration of the present invention was 110,000 Da.

In addition, in order to confirm the bone regeneration effect in vivo ( in vivo ) according to the content of hyaluronic acid, the hyaluronic acid having an average molecular weight of 110,000 Da as the optimum conditions identified above is 0.05% (w / v), 0.5% ( 10 μl of the hyaluronic acid solution containing w / v) and 1% (w / v) and mesenchymal stem cells expressing BMP-2 prepared in Example 3-1 were separated with 0.25% trypsin-EDTA (Gibco). After treatment, 20 μl of the bone regeneration composition containing 10 μl of dilution diluted with PBS (Sigma) to contain 1 × 10 ⁵ to 5 × 10 ⁵ cells per 10 μl was treated at the defect site in the same manner as described above. drop) and allowed to stand for 10 minutes before closing the incised skin.

After 6 weeks after the treatment, the skin of the closed defect site of each experimental animal was incised again to observe the defect site, and the results are shown in FIG. 14.

As shown in FIG. 14, it was confirmed that the bone regeneration effect was excellent when hyaluronic acid was treated together as compared with the case when the mesenchymal stem cells for transplantation with BMP-2 were introduced alone.

In addition, the hyaluronic acid was found to be excellent in the bone regeneration effect even when 0.05% or 1% is included, in vitro experiments confirmed in Example 6-3 when the hyaluronic acid is included 0.05% Compared to the case where 1% of hyaluronic acid contained similar cell culture results in, the effect of bone regeneration was lower. From these results, in relation to the optimal range of hyaluronic acid, the viscosity of the whole composition together with the cell culture results It was expected to affect the bone regeneration effect.

In addition, the hyaluronic acid was found to be excellent in the cell culture results in the in vitro experiments confirmed in Example 6-3 in the case of a composition containing 0.5%, even in in vivo experiments using experimental animals Compared to a composition containing hyaluronic acid at a different content in which only about 50% or less of the total bone defect area was regenerated, the hyaluronic acid was almost regenerated in the entire skull defect area of 8 mm, which is a bone defect area, in the composition containing 0.5% Significantly good bone regeneration effect was confirmed.

From the above results, it was confirmed that the optimal content of hyaluronic acid of the composition for bone regeneration of the present invention is 0.5% (w / v) based on the total volume of the composition.

Example 6-6. Mixed and living transplantation of scaffold and mesenchymal stem cells for transplantation

The bone regeneration composition comprising a diluent for transplanting mesenchymal stem cells (MSC / BMP2) into which BMP-2 was prepared in Example 6-1 and hyaluronic acid prepared in Example 6-1 was used at a bone defect site. In order to allow bone regeneration more effectively, scaffolds were used together with the composition for bone regeneration. As the support, beta-tricalcium phosphate (β-tricalcium phosphate, β-TCP, Kyungwon Medical Co., South Korea) having a granule type of 1-3 mm in diameter was used.

More specifically, after separating the mesenchymal stem cells expressing BMP-2 prepared in Example 3-1 with 0.25% trypsin-EDTA (Gibco Co.), 1 x 10 ⁵ to 5 x 10 ⁵ cells per 200 μl 200 μl of the diluted solution diluted with PBS (Sigma) and 200 μl of the hyaluronic acid solution of Example 6-1 were mixed with 1 g of the support, followed by standing at room temperature for 20 minutes. The mesenchymal stem cells were attached to the support.

In vivo transplantation of the scaffold to which the mesenchymal stem cells were attached was prepared according to Example 5-3. First, rabbits were anesthetized by injecting lumpun (3.5 mg / kg body weight) and ketamine hydrochloride (20 mg / kg body weight) into the muscle, and then cutting the hairs of the tibia and then cutting the povidone iodine (betadine). Disinfection). The sterilized tibial site was dissected to expose the tibia and then defects (0.5 cm wide and 1.5 cm high) were made on the tibial site using an orthopedic surgical instrument. After inserting 1 g of a scaffold attached with mesenchymal stem cells to the defect site, the incised skin was closed.

It was confirmed that after 6 weeks elapsed, the suture was bonded to the site where the support was inserted, and the bone regeneration effect was observed. From the above results, it was confirmed that the support prepared with beta-tricalcium phosphate can be used.

Example 6-7. Tissue fragmentation

The bone regeneration effect was confirmed through the change in the bone tissue of the rat of Example 6-4, which confirmed that the bone fracture effect was excellent through the radiation.

More specifically, the animals of Example 6-4 were euthanized at 28 and 91 days after the biotransplantation of the composition for bone regeneration, the right tibia was fixed in 10% formalin solution for 2 days, and 7 % Nitric acid was demineralized for 4 days. The demineralized tibia was made into a paraffin block, and the block was cut to a thickness of 3 μm to prepare a section. The sections were stained by Masson's trichrome staining (MT staining) and hematoxylin and eosin staining (H & E staining), and then observed by electron microscopy, and the results are shown in FIG. 15. .

As shown in FIG. 15, the bone regeneration of the defect region of the tibial tissue of the experimental animal sacrificed at the time of 91 days passed was superior to the time at 28 days, and normal at the time of 91 days passed. It was confirmed that the degree similar to the bone state of the state. From the results confirmed through experimental animals related to bone defects of the osteoporosis patients, it was confirmed that the bone regeneration composition of the present invention has an effective and significant therapeutic effect against bone defects caused by osteoporosis.

Example 6-8. Immunohistochemistry

The change in bone tissue of rats of Example 6-4, which confirmed that the bone fracture was excellent through the radiation and confirmed the excellent bone regeneration effect, was confirmed by immunohistochemical staining.

More specifically, the slide attached to the tissue section prepared in Example 6-7 was washed with 1 x PBS for 2 minutes. In order to reduce the nonspecific response to the washed tissue sections, 3% H ₂ O ₂ was treated and reacted at room temperature for 10 minutes. After the reaction, the osteocalcin antibody (Biogenex) diluted to 1: 200, which is the primary antibody, was treated to the tissue sections and reacted in a moist chamber at room temperature for 1 hour. After reacting the primary antibody, washed three times with 1 x PBS for 2 minutes each, and a secondary antibody (Histostain-plus kit) against the biotine-labeled rat immunoglobulin-G (IgG) to detect the osteocalcin primary antibody , Zymed), and then reacted for 20 minutes in a humid chamber at room temperature. After reacting the secondary antibody, it was washed three times for 2 minutes with 1 x PBS, and treated with HRP-labeled streptavidin to detect the secondary antibody, followed by a moist chamber at room temperature. The reaction was carried out for 10 minutes. After reacting with the RP-labeled straptavidin, it was washed three times with 1 × PBS for 2 minutes and treated with the substrate for HRP enzyme.

Bone-forming proteins stained with the human osteocalcin antibody in tissue sections of experimental animals in which bone regeneration compositions subjected to immunohistochemical staining were sacrificed at 28 and 91 days after biotransplantation. Mesenchymal stem cells expressing BMP-2 were identified, and the results are shown in FIG. 16.

As shown in FIG. 16, the bone regeneration of the defect region of the tibial tissue of the experimental animal sacrificed at the time of 91 days passed was superior to the time at 28 days, and normal at the time of 91 days passed. It was confirmed that the degree similar to the bone state of the state. In addition, unlike the control group treated only with the secondary antibody, it was confirmed that the staining is remarkably well when both the osteocalcin primary antibody and the secondary antibody is treated, and transplanted mesenchymal stem cells into which the BMP-2 treated at the defect site was introduced. It was confirmed that (MSC / BMP2) differentiated into functional mature bone cells by expression of BMP-2, a bone forming protein.

Example 6-9. Live transplantation of scaffolds for ectopic bone formation induced experimental animals

In order to more effectively regenerate bone at the bone defect site, the bone regeneration composition may include a scaffold to which the mesenchymal stem cells expressing the BMP-2 of Example 6-1 are attached. Transplanted into experimental animals induced ectopic bone formation. Beta-tricalcium phosphate (β-tricalcium phosphate, β-TCP, Kyungwon Medical Co., South Korea) of Example 6-6 was used as the support.

More specifically, 10 μl of a solution containing 1% (w / v) of hyaluronic acid having an average molecular weight of 110,000 Da of Example 6-1, and the mesenchymal stem expressing BMP-2 prepared in Example 3-1 Cells were separated by 0.25% trypsin-EDTA (Gibco) and then mixed with 10 μl of dilution diluted with PBS (Sigma) to contain 1 × 10 ⁵ to 5 × 10 ⁵ cells per 10 μl of the present invention. After adding 0.03 g of the support to the composition for bone regeneration, cells were attached to the support for 20 minutes at room temperature. In order to confirm the effects of the present invention, 20 μl of a solution containing 1% (w / v) of hyaluronic acid having an average molecular weight of 110,000 Da of Example 6-1 and an average molecular weight of 110,000 Da of Example 6-1 10 μl of a solution containing 1% of phosphorus hyaluronic acid (w / v) and mesenchymal stem cells expressing BMP-2 prepared in Example 3-1 were separated with 0.25% trypsin-EDTA (Gibco Co., Ltd.) 0.03 g of the support was added to 10 µl of the diluted solution diluted with PBS (Sigma) so as to contain 1 x 10 ⁵ to 5 x 10 ⁵ cells per µl, and the support was cultured under the above conditions.

In vivo transplantation of the scaffold to which the mesenchymal stem cells were attached was performed according to Example 5-6.

More specifically, after anesthetizing male 6-week-old BABI / c / nu nude mouse males (Inha University Experimental Animal Center, South Korea) of Example 5-6, the site to be incised to the nude mouse dorsal povidone iodine ( betadine). About 1 cm of the disinfected dorsal part was incised. 0.03g of the support to which the mesenchymal stem cells were attached to the mouse in which the skin of the dorsal part was cut and 0.03g of the two types of support prepared to confirm the effect of the present invention were inserted, and then the cut skin was sutured. .

After inserting the support, the animals were sacrificed at the time point of 4 weeks, and the bone regeneration effect was confirmed by changing the bone tissue as in Example 6-7.

Specifically, the animals were euthanized 28 days after the insertion of the respective supports, the right tibia was fixed in 10% formalin solution for 2 days and demineralized in 7% nitric acid for 4 days. The demineralized tibia was made into a paraffin block, and the block was cut to a thickness of 3 μm to prepare a section. The sections were stained by hematoxylin and eosin staining (H & E staining), followed by electron microscopy, and the results are shown in FIG. 17.

As shown in FIG. 17, it was confirmed that the test animal inserted with the support treated with the hyaluronic acid solution had no bone regeneration effect. In addition, even when treated with a mixture of hyaluronic acid and mesenchymal stem cells, it was confirmed that almost no bone regeneration effect. From the above results, it was evaluated that treatment with hyaluronic acid itself or mesenchymal stem cells and hyaluronic acid alone was not excellent in bone regeneration effect, and thus no therapeutic effect could be expected.

Unlike the support treated with the hyaluronic acid solution or the support treated with a mixture of hyaluronic acid and mesenchymal stem cells, the bone of the present invention containing the mesenchymal stem cells expressing the BMP-2 and hyaluronic acid having an average molecular weight of 110,000 Da When the support body treated with the regeneration composition was inserted, it was confirmed that sufficient bone tissue was formed. From the results confirmed through the experimental animal induced ectopic bone formation, the bone regeneration composition of the present invention has a significant bone regeneration effect, and treated with the bone regeneration composition, mesenchymal stem cells expressing BMP-2 It was expected that effective bone regeneration would be possible when inserting the attached support.

Through the above embodiment, it was confirmed that the bone regeneration composition of the present invention including the stem cell and hyaluronic acid into which the protein gene having bone formation function was introduced has an effective bone regeneration effect in the non-union fracture, bone defect and bone loss model. Based on the above facts, it is expected that the composition for bone regeneration of the present invention can be used in various therapies including cell replacement therapy and gene therapy for treating bone diseases.

The composition for bone formation of the present invention can be used in various therapeutic methods, including cell replacement therapy or gene therapy, for the purpose of treating bone diseases, and widely used as a material for verifying drug effects or for various studies in the development of new drugs. Can be used.

SEQ ID NO: 1 in the Sequence Listing attached herein is a sequence of BMP-2, which is an example of a protein having a bone forming function of the present invention, and the sequences set forth in SEQ ID NO: 2 to SEQ ID NO: 7 are sequences of suicide genes, SEQ ID NO: The sequences set forth in 8 through SEQ ID NO: 13 are the sequences of the primers used to prepare the vectors of the invention.

Claims (12)

1. A composition for bone regeneration comprising a stem cell into which a gene encoding a protein having a bone forming function is introduced or a cell differentiated from the stem cell and hyaluronic acid.
2. The method of claim 1,

   The bone-forming protein is a bone morphogenic protein (BMP), the stem cells are mesenchymal stem cells (mesenchymal stem cell) composition for bone regeneration.
3. The method of claim 2,

   The bone morphogenetic protein is at least one composition selected from the group consisting of BMP-2 (GenBank Accession No. AAF21646), BMP-3 (GenBank Accession No. NP_001192), and BMP-7 (GenBank Accession No. NP_001710). .
4. The method of claim 1,

   The stem cells are bone regeneration composition is a stem cell into which a gene encoding a protein having the bone formation function and a suicide gene is introduced.
5. The method of claim 4, wherein

   The suicide gene is a composition for bone regeneration is one or more selected from the group consisting of HSV-tk gene, CD gene, VZV-tk gene, CYP2B1 gene, XGPRT gene and PNP gene.
6. The method of claim 1,

   The hyaluronic acid has an average molecular weight of 50,000 to 300,000 Daltons for bone regeneration composition.
7. The method of claim 1,

   The content of the hyaluronic acid is a bone regeneration composition of 0.01 to 3% (w / v) based on the total composition.
8. The method of claim 1,

   The bone regeneration composition is a bone regeneration composition, characterized in that the gel state at 4 ℃ to 50 ℃.
9. A composition for the prevention, alleviation or treatment of bone diseases, comprising the composition for bone regeneration according to any one of claims 1 to 8.
10. The method of claim 9,

    The bone diseases include osteoporosis, osteoporotic fractures, diabetic fractures, bone defects, osteoplastic insufficiency, osteomalacia and resulting fractures, fracture defects, bone growth around graft correction, bone growth disorders, bone tumors, nonunion fractures and hereditary A composition for preventing, alleviating or treating at least one bone disease selected from the group consisting of defects of bone growth.
11. Preparing a vector containing a gene encoding a protein having a bone formation function;

    Introducing a gene encoding a protein that functions to form bone into mesenchymal stem cells using the vector; And

    Preparing a gel-like composition by mixing the mesenchymal stem cells or cells differentiated from the mesenchymal stem cells with hyaluronic acid

    Method for producing a composition for bone regeneration comprising a.
12. A gene expression cassette comprising a gene encoding a protein having a bone formation function, a promoter that determines whether transcription is initiated by a protein expressed by the gene encoding the protein having a bone formation function, and a suicide gene are linked in the transcription direction. Preparing a vector comprising;

    Introducing the gene expression cassette into the mesenchymal stem cells using the vector; And

    Preparing a gel-like composition by mixing the mesenchymal stem cells or cells differentiated from the mesenchymal stem cells with hyaluronic acid

    Method for producing a composition for bone regeneration comprising a.

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