KR20190035051A - Porous polymer microsphere for the prevention or treatment of bone diseases or cartilage diseases, and preparation method thereof - Google Patents

Abstract

The present invention relates to a porous polymer microsphere for preventing or treating bone diseases or cartilage diseases, and a method for manufacturing the same. The porous polymer microsphere according to the present invention can inject a drug effective for regeneration of damaged bone tissues or cartilage tissues directly into the affected part requiring treatment. In addition, since the drug is slowly released, a desired amount of drug can be delivered to the affected part over a long time, an excellent regeneration effect can be directly expressed in bone tissues, and there is no side effects since the porous polymer microsphere is composed of biodegradable materials. In addition, the porous polymer microsphere according to the present invention can provide customized medical care by controlling the rate of release of the drug through the control of porosity and the concentration of the carried drug according to the state of the affected part.

Description

TECHNICAL FIELD [0001] The present invention relates to a porous polymer microsphere for preventing or treating bone diseases or cartilage diseases, and a preparation method thereof,

The present invention relates to a porous polymer microsphere for prevention or treatment of bone diseases or cartilage diseases, and a method for producing the same.

Bone (bone) is a solid tissue that supports the body and constitutes the internal skeleton and protects the organs. Bone tissue is a solid tissue that supports the body. It constitutes the internal skeleton and protects organs. Physiologically, it is an important tissue that maintains homeostasis so that calcium concentration in blood is constant in vivo. To maintain this homeostasis, osteoblast-induced osteogenesis and osteoclast-induced bone resorption must be maintained in order to maintain this homeostasis. Osteoporosis is one of the causes of osteoporosis, osteoclastic hyperalgesia is a cause of decreased bone mass and bone strength, leading to increased risk of fracture. As reported by the Korean Society of Bone and Joint Surgery (Osteoporosis Diagnosis and Treatment Guidelines, 2013), about 50% of osteoporotic femoral epiphysis patients can not restore their ability to function and independence before fracture, and 25% And an average mortality rate of 20% a year. Therefore, the Bone Metabolism Society as a treatment for osteoporosis presents various treatment methods. In the first case of female hormone therapy, estrogen was injected by patch, gel or oral administration method to prevent osteoporosis caused by postmenopausal estrogen deficiency. However, it was reported that bone mineral density was significantly increased, but edema, mastitis, And the like. Next, bisphosphonates, the most widely prescribed drug in the world, are injected via oral and intravenous injection. Bisphosphonate is a synthetic substance that does not exist in human body. It moves to bone, enters osteoclast and inhibits differentiation and action. However, bisphosphonates for oral use are known to cause esophagitis and stomach ulcers, cause jawbone necrosis during long-term use, and cause intravenous injection of headache, muscle pain, and renal failure. Another treatment is parathyroid hormone (PTH). Although PTH is composed of 84 amino acids, PTH1-34, which is composed of 34 amino acids of amino terminal, has been approved in USA, Europe and Korea. PTH is a small dose that promotes bone formation during intermittent dosing, but it should be noted that maintaining high concentrations of PTH increases bone resorption. In addition, adverse reactions may cause nausea, headache, leg cramps, and temporary hypercalcemia. The treatment methods reported in the Korean Society of Bone and Joint Biology have been used as osteoporosis drugs, but new therapies are still needed because of the possibility of adverse reactions.

Osteoporosis is caused not only by the direct osteoclast activation but also by bone metastasis of cancer cells. Various cancer cells such as breast cancer, prostate cancer, and colon cancer are transferred to bone (bone), and lungs and liver are known as the third most common transfer sites. The bone metastasis of these cancers not only results in a simple metastasis but also affects osteoclasts that are involved in the osteoblast and bone uptake involved in bone formation in the bone marrow and ultimately leads to excessive bone resorption, (Yoneda T, Tanaka S, Hata K. Role of RANKL / RANK in Primary and Secondary Breast Cancer, World J Orthop 2013 18; 4 (4) : 178-85, doi: 10.5312 / wjo.v4.i4.178).

A representative treatment for osteoporosis developed so far is bisphosphonates, which inhibits bone loss by weakening the functions of osteoclasts (bone resorption-related cells). Merck's Fosamax, Procter Gamble And 'Actonel' of the company. However, these treatments do not promote the formation of bones, but as a decomposition inhibitor, they can lead to additional fractures by making the bone fragile during continuous use. Therefore, there is a need for a substitute treatment that can not be a fundamental treatment (Datamonitor Research Reports, 2007).

On the other hand, since articular cartilage has no blood vessels, nerves and lymphatic tissues, damaged articular cartilage is difficult to regenerate spontaneously when damaged. Therefore, even in case of small damage in joint cartilage, wound progression and joint degeneration may occur. Therefore, in order to restore and maintain the function of bone and cartilage tissue, various methods have been developed.

Cartilage is a tissue that originated from the mesoderm, like the tibial tissue, and it forms an endoskeleton with the bone. In addition, when cartilage damage is severe, not only the cartilage but also the bone tissue under the cartilage may be damaged. Although the cartilage and bone are organically functioning tissues, conventional tissue engineering treatments of cartilage and bone treated them individually. Specifically, existing clinical methods of regenerating cartilage include (1) inducing differentiation of stem cells into chondrocytes, (2) bone, cartilage tissue, autologous or allogeneic cartilage tissue, (Cartilage, periosteum) or a compound capable of inducing cartilage on the surface of a cartilage defect site, (4) a method of transplanting cartilage cells into a cartilage defect site by cartilage cell transplantation, A method of inducing regeneration, and a method of regenerating only cartilage are used.

However, as described above, since the cartilage and the bone are tissues in which the cartilage and the bone are organically operated, the conventional method of treating the cartilage and the bone separately has weak binding force with the bone and cartilage and takes a long time to recover.

Recently, stem cells, which have the ability to self-replicate and can differentiate into various tissues and have a large amount of cells that can be easily harvested without donor dysfunction, are recognized as ideal cell sources for cell therapy, However, there is still a lack of clear knowledge about the factors and environment for cartilage formation.

Stem cells for articular cartilage regeneration are characterized by autoproducing ability and differentiating ability to differentiate into cells constituting specific tissues. Recently, they have been proposed as a new cell source for application to articular cartilage treatment. Therefore, theoretically, it is possible to solve the limitations of conventional cell therapy using chondrocytes and apply it to the degeneration and damage of the articular cartilage as a whole. In addition, adult mesenchymal stem cells and mesenchymal precursor cells have an advantage in that they have no ethical problems and no in vivo rejection reaction at allograft.

However, all adult mesenchymal stem cells do not completely differentiate into chondrocytes at the same time, and therefore, a method of inducing differentiation into homogeneous chondrocytes is necessary.

In order to promote the formation of bone / cartilage or regeneration of bone / cartilage tissue, there is a method of directly injecting the drug into the oral administration or wound of the drug, but the oral administration of the drug requires The amount of the drug delivered to the affected area is only a small fraction, and thus the regeneration effect is not significant. In the case of directly injecting the drug into the affected area, there is a problem of side effects because a large amount of drug is injected at once.

As a method for solving these problems, a tissue engineering approach using a biodegradable porous polymer scaffold has emerged as an alternative. Biodegradable porous polymer scaffold mimics a natural extracellular matrix, and is known to be very favorable for the formation and differentiation of osteoid / chondrocytes and the regeneration of bone / cartilage tissue. They have the advantage of high cell adhesiveness, but they have poor mechanical properties and have a problem of slow formation and differentiation rate of bone / cartilage cells or regeneration rate of bone / cartilage tissue.

Therefore, it is required to develop a new therapeutic drug that exhibits the effect of promoting osteoclast / chondrocyte formation and differentiation, or promoting the regeneration of bone / cartilage tissue, without side effect.

KR 10-1105285 B

The present invention provides a porous polymer microsphere on which a drug capable of preventing or treating bone diseases or cartilage diseases is mounted.

Another object of the present invention is to provide a method for producing the porous polymer microspheres.

According to an aspect of the present invention, there is provided a biodegradable polymer scaffold comprising a three-dimensional net structure having pores having a size of 5 to 100 μm connected to each other; And a drug for treating a bone disease or a cartilage disease contained in the net structure of the biodegradable polymer scaffold. The present invention also provides a porous polymer microsphere for preventing or treating bone diseases or cartilage diseases.

According to the present invention, the porous polymer microsphere may contain 2 to 30 parts by weight of the drug relative to 10 parts by weight of the biocompatible polymer.

According to the present invention, the porous polymer microspheres may have a porosity of 10 to 90% and a particle size of 200 to 1000 탆.

According to the present invention, the porous polymer microsphere may have a porosity of 60 to 90%, a pore size of 10 to 40 μm, and a particle size of 200 to 500 μm.

According to the present invention, the drug for the treatment of bone diseases or cartilage diseases includes osteogenic protein-2, epidermal growth factor, osteogenic protein-7, cilastatin, nystatin, lovastatin but are not limited to, lovastatin, somatostatin, pravastatin, simvastatin, fluvastatin, atorvastatin, cervastatin, ulinastatin, rosuvastatin ( rosuvastatin, pitavastatin, glucosamine, chondroitin, mevastatin, cartogenin, transforming growth factor-beta 1, transformation But are not limited to, growth factors-beta 3, 6-bromoindirubin-3-oxime, sulfonamide, purmorphamine, mevinolin, genistein, Icariin, melatonin, But are not limited to, metformin, alendronate, zoledronate, etidronate, clodronate, tiludronate, pamidronate, Neridronate, olpadronate, ibandronate, risedronate, prostaglandins, dexamethasone, osteogenic protein-4, insulin-like growth factor-1 ( insulin-like growth factor-1, fibroblast growth factor-2, fibroblast growth factor-18, fibroblast growth factor, or a mixture thereof.

According to the present invention, the biocompatible polymer is selected from the group consisting of polyglycolic acid (PGA), polylactic acid (PLA), polylactide-co-glycolide (PLGA), polycaprolactone (PCL), polyamino acid, Any one selected from phosphazene, polyiminocarbonate, polyphosphoester, polyanhydride, polyorthoester, polyhydroxyvalerate, polyhydroxybutyrate, hyaluronic acid, cellulose, heparin, collagen, alginate and chitosan; Polymers in which two or more of these are blended; And copolymers of two or more thereof.

According to the present invention, the above-mentioned bone diseases can be used for the treatment and prophylaxis of osteoporosis, osteoporosis, osteoporosis, osteoporosis, osteoporosis, osteoporosis, bone necrosis, fracture, excessive osteoclast osteoporosis, , Metastatic bone cancers, rheumatoid arthritis, rheumatoid arthritis, rheumatoid arthritis, fibrotic bone disease, intractable bone disease, metabolic bone disease, renal osteodystrophy, periodontal disease, Paget disease, Wherein the cartilage disease is selected from the group consisting of degenerative arthritis, inflammatory arthritis, osteochondromatosis, distortion, bursitis, menstruation, Meniscal tear, meniscus cyst, collateral ligamentrupture, anterior cruciate ligament injury, posterior horn, Posterior cruciate ligament injury, intra-articular vitreous, exfoliation osteochondritis, plica syndrome, genu varum, knock-knee and pronation And a snapping knee.

According to the present invention, the porous polymer microspheres may be injected into a living body to release a drug for treating bone diseases or cartilage diseases.

The present invention also relates to a biocompatible polymer comprising: 1) a biocompatible polymer; And a drug for the treatment of bone diseases or cartilage diseases in an organic solvent to prepare a polymer solution; 2) adding the polymer solution and gelatin aqueous solution and homogenizing to prepare an emulsion; 3) The emulsion is used as a discontinuous phase, and a polyvinyl alcohol solution is used as a continuous phase in a fluidic device to treat a biocompatible polymer, bone disease or cartilage disease Preparing a polymer microsphere comprising a drug and a gelatin; And 4) adding the polymer microspheres to water at a temperature of 38 to 50 DEG C and stirring to elute the gelatin from the polymer microspheres, thereby providing a method for preparing porous polymer microspheres for prevention or treatment of bone diseases or cartilage diseases do.

The porous polymer microspheres according to the present invention can inject a drug effective for regeneration of damaged bone tissue or cartilage tissue directly into the affected part requiring treatment. In addition, since the drug is slowly released, a desired amount of drug can be delivered to the affected part over a long period of time, an excellent regeneration effect can be directly expressed in the bone tissue, and the biodegradable material is free from side effects. In addition, the porous polymer microspheres according to the present invention can provide customized medical care by controlling the rate of release of the drug through the control of the porosity and the concentration of the loaded drug according to the state of the affected part.

1 is a scanning electron microscope image of a porous polymer microsphere (SIM / PMSs) of Example 1. Fig.
Figure 2 shows the results of the drug release behavior test of the porous polymer microspheres (SIM / PMSs) of Example 1.
3 is a scanning electron microscope image of microspheres having different pore sizes depending on the concentration of gelatin added during the production of the porous polymer microspheres.
FIG. 4 shows the results of measurement of cell proliferation in porous polymer microspheres having different pore sizes.

Hereinafter, the present invention will be described in more detail with reference to the drawings.

The present invention relates to a biodegradable polymer scaffold having a three-dimensional net structure in which pores having a size of 5 to 100 μm are connected to each other; And a drug for treating a bone disease or a cartilage disease contained in the net structure of the biodegradable polymer scaffold. The present invention also provides a porous polymer microsphere for preventing or treating bone diseases or cartilage diseases.

The porous polymer microspheres according to the present invention may be injected into a living body to release the drug for treating bone diseases or cartilage diseases. The release of the drug may be released through the pores of the porous polymer microspheres or may be released as the biodegradable polymer scaffold (hereinafter referred to as the polymer scaffold) decomposes in vivo.

The release of the drug can be regulated by the type of polymer forming the polymer scaffold, the density of the net structure, the degree of porosity, and the size of the pores. Therefore, the porosity and pore size of the porous polymer microspheres and the concentration of the incorporated drug can be appropriately adjusted depending on the severity of the affected part to be treated.

The porous polymer microspheres according to the present invention may contain 2 to 30 parts by weight of the drug relative to 10 parts by weight of the biocompatible polymer. If the content of the drug is less than the above range, the amount of the drug is too small, and if it exceeds the above range, the amount of the drug released in a short time is too much.

The porous polymer microspheres according to the present invention may have a porosity of 10 to 90% and a particle size of 200 to 1000 탆. If the size of the particles is less than the above range, the amount of the drug that can be carried is too small to be continuously discharged in the living body. Therefore, there is the inconvenience that the drug should be frequently administered. If the particle size exceeds the above range, Is not easy.

In particular, the porous polymer microspheres preferably have a porosity of 60 to 90%, a pore size of 10 to 40 μm, and a particle size of 200 to 500 μm. Within this range, the drug can be easily released in vivo.

The porous polymer microspheres according to the present invention may have a pore size of 5 to 100 mu m. If the size of the pores is less than the above range, the size of the pores is too small to release the drug, and if the size of the pores exceeds the above range, the drug is discharged at once. Particularly preferred pore sizes may be from 10 to 80 mu m.

The medicament for treating bone diseases or cartilage diseases according to the present invention is a composition for the treatment of osteopathy or cartilage disease, which comprises bone formation protein-2, epidermal growth factor, osteogenic protein-7, cilastatin, nystatin, lovastatin, somatostatin ), Pravastatin, simvastatin, fluvastatin, atorvastatin, cervastatin, ulinastatin, rosuvastatin, pitavastatin ( pitavastatin, glucosamine, chondroitin, mevastatin, carotogenin, transforming growth factor-beta1, transforming growth factor-beta3, 6-bromoindine- Amid, permorphamine, mebinolin, genistein, icarin, melatonin, metformin, alendronate, zoledronate, etidronate, clodronate, tyrrulonate, pamidronate, neridronate, Fibroblast growth factor-18, fibroblast growth factor-1, fibroblast growth factor-1, fibroblast growth factor-1, fibroblast growth factor-1, fibroblast growth factor-1, fibroblast growth factor- And mixtures thereof.

The biocompatible polymer of the porous polymer microspheres according to the present invention may be selected from the group consisting of polyglycolic acid (PGA), polylactic acid (PLA), polylactide-co-glycolide (PLGA), polycaprolactone (PCL) Polylactide, polyhydroxybutyrate, hyaluronic acid, cellulose, heparin, collagen, alginate and chitosan, among others, such as polylactate, polyphosphate, polyiminocarbonate, polyphosphoester, polyanhydride, polyorthoester, polyhydroxyvalerate, polyhydroxybutyrate, Any one selected; Polymers in which two or more of these are blended; And copolymers of two or more thereof.

Particularly preferred drugs for the treatment of bone diseases or cartilage diseases according to the present invention are simvastatin, pravastatin, lovastatin, mevastatin, pitavastatin, osteogenic protein-2, fibroblast growth factor, carthogenin, dexamethasone, alendronate, Pamidronate, risedronate, prostaglandin, or a mixture of two or more thereof.

The bone disease according to the present invention is useful for treating osteoporosis, osteoporosis, osteoporosis, osteoporosis, osteoporosis, osteoporosis, osteonecrosis, bone fracture, excessive osteoclast bone resorption, joint damage due to trauma, From the group consisting of rheumatoid arthritis, osteoarthritis, rickets, fibrous bone disease, intractable bone disease, metabolic bone disease, renal osteodystrophy, periodontal disease, Paget disease and metastatic bone cancers And the cartilage diseases are selected from the group consisting of degenerative arthritis, inflammatory arthritis, osteochondromatosis, distortion, bursitis, menstruation, Meniscal tear, meniscus cyst, collateral ligamentrupture, anterior cruciate ligament injury, posterior fossa, Posterior cruciate ligament injury, intra-articular vitreous, exfoliation osteochondritis, plica syndrome, genu varum, knock-knee and pronation A knee, and a snapping knee.

The porous polymer microspheres according to the present invention can be particularly effective for preventing or treating bone / cartilage-reducing diseases or bone / cartilage defect diseases by promoting the formation or differentiation of osteoblasts or cartilage cells.

In the present invention, the prevention or treatment of bone diseases or cartilage diseases may promote the regeneration of bone or cartilage tissue or promote inflammation by promoting the formation of bone / cartilage cells or promoting the differentiation of bone / cartilage cells have.

Meanwhile, the porous polymer microspheres for preventing or treating bone diseases or cartilage diseases according to the present invention include: 1) a biocompatible polymer; And a drug for treating bone diseases or cartilage diseases, in an organic solvent to prepare a polymer solution; 2) adding the polymer solution and gelatin aqueous solution and homogenizing to prepare an emulsion; 3) Using the emulsion as a discontinuous phase and flowing in a fluidic device using a polyvinyl alcohol solution as a continuous phase, a biocompatible polymer, a drug for treating bone diseases, and gelatin Preparing a polymer microspheres comprising the polymer microspheres; And 4) adding the polymer microspheres to water at 38 to 50 DEG C and stirring to elute the gelatin from the polymer microspheres.

The porous polymer microsphere is prepared by mixing 2 to 30 parts by weight of the drug with respect to 10 parts by weight of the biocompatible polymer, and dissolving the drug in an organic solvent to prepare a polymer solution.

The organic solvent may be one or more selected from dimethylsulfoxide, dichloromethane, tetrahydrofuran and N, N-dimethylformamide, but is not limited thereto.

Next, a polymer solution and an aqueous gelatin solution are added and homogenized to prepare an emulsion.

The gelatin aqueous solution may be a gelatin aqueous solution of 1 to 10% by weight. If the content of the gelatin is less than the above range, the content of gelatin is too low to develop pores of the microspheres. If the content of the gelatins is more than the above range, the preparation of the microspheres is not easy and the pore size is relatively large, .

According to the present invention, an emulsion stabilizer can be further included in the homogenization. The emulsion stabilizer may be emulsion stabilization, which is commonly used, and may preferably be polyvinyl alcohol.

According to the present invention, homogenization in the step 2) may be performed by adding 3 to 15 parts by weight of gelatin to 10 parts by weight of the biocompatible polymer. When the content of gelatin is less than the above range with respect to the biocompatible polymer, development of pores is not easy and it is difficult to achieve the desired porosity. If it exceeds the above range, pores are too large to support the drug.

According to the present invention, 0.5 to 3 parts by weight of the emulsion stabilizer may be added to 10 parts by weight of the biocompatible polymer. If the content of the emulsion stabilizer is out of the above range, it is not easy to form microspheres of the size desired in the present invention.

In addition, the amount of the drug discharged and the rate of release of the drug can be controlled by adjusting the content of the gelatin, the drug, or both of them relative to the content of the biocompatible polymer.

In addition, the homogenization may be homogenization using a homogenizer at 8,000 to 20,000 rpm for 30 seconds to 10 minutes, but the present invention is not limited thereto.

In the present invention, the biocompatible polymer and the drug for treating bone diseases or cartilage diseases are as described above.

Next, the desired polymer microspheres can be prepared by using the emulsion as a discontinuous phase, and using a polyvinyl alcohol as a continuous phase and flowing in a fluid device. When the prepared microspheres are stirred in water at 38 to 50 ° C, the gelatin components in the microspheres are released from the microspheres, and pores are formed in the gelatin, so that the porous polymer microspheres are formed.

At this time, the flow rates of the discontinuous phase and the continuous phase may be 0.2 to 1 mL / min, respectively, and preferably 0.5 mL / min. It is possible to manufacture the microspheres having the desired size and porosity at the above-mentioned flow rate because the degree of dispersion of the porosity is excellent.

The microspheres prepared by this method have a porosity of 10 to 90%, a pore size of 5 to 100 μm, a particle size of 200 to 1000 μm, and a porosity of 60 to 90% And may be a microsphere having a pore size of 10 to 40 mu m and a particle size of 100 to 400 mu m.

The effective amount of the porous polymer microspheres according to the present invention can prevent or treat bone diseases.

As used herein, the term "effective amount" refers to the amount of active ingredient or pharmaceutical composition that elicits a biological or medical response in a tissue system, animal, or human, as contemplated by a researcher, veterinarian, physician or other clinician, ≪ / RTI > inducing a reduction of the symptoms of the disease or disorder. The effective amount and the administration frequency for the active ingredient of the present invention can be changed according to the desired effect. Thus, the optimal dosage to be administered can be readily determined by those skilled in the art and will vary with the nature of the disease, the severity of the disease, the amount of active and other ingredients contained in the composition, the type of formulation, and the age, The age, body weight, sex, diet, time of administration, route of administration and fraction of the composition, duration of treatment, concurrent medication, and the like. In the preventive, therapeutic or ameliorative method of the present invention, in the case of an adult, it is preferable to administer at a dose of 0.001 g / kg to 1 g / kg once to several times a day.

The porous polymer microspheres according to the present invention may be prepared by using pharmaceutically acceptable and physiologically acceptable adjuvants in addition to the above-mentioned effective components. Examples of the adjuvants include vitamins, book colors, thickening agents, pectic acid, , Alginic acid, an organic acid, a carbonating agent, an excipient, a disintegrant, a sweetener, a binder, a coating agent, a swelling agent, a lubricant, a lubricant or a flavoring agent.

The porous polymer microspheres may further comprise at least one pharmaceutically acceptable carrier in addition to the above-described effective ingredients for administration, and may be suitably formulated into pharmaceutical compositions.

The form of the porous polymer microspheres may be any form of conventional pharmaceutical composition so long as it is in the form of a pharmaceutical, but preferably it may be an injectable solution (liquid solution), and may be an acceptable pharmaceutical carrier for a composition to be formulated into a liquid solution Sterilized and sterile water, sterile water, Ringer's solution, buffered saline solution, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol and one or more of these components may be used in combination. , Other conventional additives such as an antioxidant, a buffer solution, a bacteriostatic agent and the like may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like.

According to the present invention, the preferred concentration of the drug contained in the porous polymer microspheres may be from 1 ng / ml to 500 mg / ml, and may be varied depending on the type of drug contained. For example, if the type of the drug is for regeneration of cartilage or bone, the preferable concentration of the drug may be 1 pg / ml to 20 mg / ml, and if the kind of the drug is for inflammation suppression, It may be from 1 / / ml to 20 mg / ml, but it can be easily changed depending on the severity of the affected part.

Hereinafter, the present invention will be described in more detail by way of examples. It will be apparent to those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not limited to these embodiments.

Example

Example  1. Porous polymer Microsphere  Produce( Simvastatin  contain)

140 mg of polylactide-co-glycolide (PLGA) was dissolved in 7 g of dichloromethane to prepare a 2 wt% PLGA solution. 750 mg of gelatin was dissolved in 10 g of distilled water to prepare a 7.5 wt% gelatin aqueous solution. PLGA: Gelatin was mixed in a weight ratio of 7: 3 on the basis of the solid content, and dimethyl (1, 5 and 10 mM) (2.94 g, 14.7 g and 29.4 g) Simvastatin dissolved in sulfoxide was added and homogenized at 13,500 rpm for 1 minute using a homogenizer (Ultra-Turrax T-25 Basic, IKA) to prepare an emulsion.

The emulsion was used as a discontinuous phase in the fluid device, and the microspheres were prepared by flowing 0.5% by weight of polyvinyl alcohol in a continuous phase at a flow rate of 0.5 mL / min. Distilled water was added to the prepared microspheres and the mixture was stirred in distilled water at 40 ° C to elute the gelatin in the microspheres, thereby preparing simvastatin-containing porous polymer microspheres.

Example  2. Porous polymer Microsphere  Produce( Pravastatin  contain)

Porous polymer microspheres were prepared in the same manner as in Example 1 except that pravastatin was added instead of simvastatin.

Example  3. Porous polymer Microsphere  Manufacturing (containing mevastatin)

The procedure of Example 1 was followed except that mevastatin was added instead of simvastatin to prepare porous polymer microspheres.

Example  4. Porous polymer Microsphere  Manufacturing (containing alendronate)

The procedure of Example 1 was followed except that alendronate was added instead of simvastatin to prepare porous polymer microspheres.

Example  5. Porous polymer Microsphere  Manufacturing (containing dexamethasone)

The procedure of Example 1 was followed except that dexamethasone was added instead of simvastatin to prepare porous polymer microspheres.

Comparative Example . Porous polymer Microsphere  Manufacturing (Drugs Free )

140 mg of polylactide-co-glycolide (PLGA) was dissolved in 7 g of dichloromethane to prepare a 2 wt% PLGA solution. 750 mg of gelatin was dissolved in 10 g of distilled water to prepare a 7.5 wt% gelatin aqueous solution. PLGA: Gelatin was mixed at a weight ratio of 7: 3 based on the solid content and homogenized at 13,500 rpm for 1 minute using a homogenizer (Ultra-Turrax T-25 Basic, IKA) to prepare an emulsion.

The emulsion was used as a discontinuous phase in the fluid device, and the microspheres were prepared by flowing 0.5% by weight of polyvinyl alcohol in a continuous phase at a flow rate of 0.5 mL / min. Distilled water was added to the prepared microspheres and stirred in distilled water at 40 ° C to elute the gelatin in the microspheres, thereby preparing porous polymer microspheres.

Test Example  1. Morphology analysis

Test Example  1.1. Scanning electron microscope observation

In order to confirm the morphology of the porous polymer microspheres prepared by the method of Example 1, it was observed with a scanning electron microscope, and it is shown in FIG.

1 is an image showing a comparison between simvastatin-containing porous polymer microspheres (hereinafter referred to as "microspheres") according to Example 1 and porous polymer microspheres containing no simvastatin according to a comparative example. The surface and pore sizes of microspheres at each concentration showed a similar shape and showed a significant difference according to the concentration of drug.

In order to confirm the pore size change according to the concentration of the drug, microspheres were prepared by gradually increasing the concentration of simvastatin from 1 to 10%. The microspheres were measured with a scanning electron microscope and are shown in FIG. 1 and Table 1 below.

division Simvastatin concentration (mM) of Example 1 0 One 5 10 Pore width (㎛) 28.45 + 10.01 27.11 + - 8.12 26.00 ± 10.37 25.54 ± 8.22 Pore height (탆) 24.45 + - 7.03 25.19 + - 6.41 24.15 ± 9.89 24.22 + - 8.05

As shown in Table 1, it was confirmed that the pore size was increased according to the concentration of the drug.

Test Example  2. Drug release behavior

The drug release behavior of the microspheres prepared in Example 1 was confirmed. Specifically, each microsphere was placed in 1 ml of PBS buffer (pH 7.4) and incubated at 37 ° C with stirring at 100 rpm for 1 hour, 3 hours, 6 hours, 10 hours, 1 day, 3 days, 5 days , 7 days, 10 days, 14 days, 21 days, and 28 days) were measured. After the measurement, a new buffer was changed. The released drug was measured for absorbance at 250 nm, and its release was analyzed, which is shown in FIG.

Referring to FIG. 2, the drug release amount was increased depending on the concentration of simvastatin.

Test Example  3. Verification of osteoblast differentiation promotion effect

Test Example  3.1. Alkaline Phosphatase  Activity evaluation

Alkaline phosphatase activity, which is an early bone differentiation index of osteoblast, was measured to evaluate the osmotic effect of simvastatin-containing porous polymer microspheres according to Example 1.

1 x 10 < ⁵ > stem cells were dispensed into the microspheres of the respective Examples and Comparative Examples, and then differentiated and cultured for 3 days, 7 days, 10 days, and 14 days. After the cells were cultured in the place where the stem cells were dispensed, the cells were washed with PBS buffer, and the cells were injected with 1 x RIPA buffer and sonicated at 110 W for 1 minute at 4 ° C. Next, the lysed cells were centrifuged at 13,500 rpm for 3 minutes at 4 ° C, p-nitrophenylphosphate solution was added to the supernatant, and the cells were incubated at 37 ° C for 30 minutes. Then, 500 ml of 1 N The reaction was terminated with NaOH. Absorbance was measured at 405 nm and the results are shown in Table 2 below.

Classification (concentration of drug) Alkaline phosphatase activity (μM / min / protein protein) 3 days later After 7 days After 10 days Control group 1.96 1.97 1.97 Comparative Example 1.96 1.95 1.96 Example 1 1 mM 2.52 3.25 3.94 5 mM 2.96 3.57 4.12 Example 2 1 mM 2.47 3.13 3.78 5 mM 2.85 3.44 4.01 Example 3 1 mM 2.33 3.11 3.65 5 mM 2.79 3.36 3.94 Example 4 1 mM 2.24 2.87 3.27 5 mM 2.58 3.02 3.54 Example 5 1 mM 2.44 3.10 3.69 5 mM 2.85 3.42 3.97

As shown in Table 2, in the porous polymer microspheres according to Examples 1 to 5, the activity of alkaline phosphatase, which is an early bone differentiation index for stem cells differentiated into osteoblasts, was evaluated to be effectively increased as compared with Comparative Examples.

Test Example 3.2. Calcium deposition assessment

The effect of simvastatin-containing porous polymer microspheres according to Example 1 on the osteosynthesis of osteoblast was evaluated by measuring calcium deposition concentration, which is an index of late bone differentiation of osteoblasts.

Specifically, 1 x 10 < ⁵ > stem cells were dispensed into the microspheres of Example 1 and Comparative Example 1, and then differentiated and cultured for 7 days, 14 days, and 21 days. After incubation, the cells were washed with PBS buffer and the cells were removed from each nanoparticle with Tris-EDTA solution. The detached cells were centrifuged at 13,500 rpm for 1 minute, and then 0.1% Triton X-100 solution was added to the cells. The cells were stored at 65 ° C for 3 hours and sonicated to pulverize the cells, followed by centrifugation. After separating the supernatant, the absorbance at 612 nm was measured and compared using a QuantiChrom ™ Calcium Assay kit to analyze the degree of calcium deposition. The results are shown in Table 3 below.

Classification (concentration of drug) Calcium deposition concentration (/ / mL) After 7 days After 21 days Control group 9.7 28.31 Comparative Example 9.8 28.42 Example 1 1 mM 10.0 37.74 5 mM 10.6 42.35 Example 2 1 mM 9.9 34.15 5 mM 10.3 41.47 Example 3 1 mM 9.7 34.97 5 mM 10.1 42.12 Example 4 1 mM 9.8 33.14 5 mM 10.1 40.05 Example 5 1 mM 10.0 35.46 5 mM 10.5 41.89

As shown in Table 3, in the porous polymer microspheres of Examples 1 to 5, the calcium deposition concentration on osteoblast-differentiated stem cells was evaluated to be effectively increased as compared with the comparative example.

Test Example  4. Verification of chondrocyte differentiation promoting effect

Test Example  4.1. GAG  analysis

The chondrocyte differentiation of porous polymer microspheres containing simvastatin according to Example 1 was evaluated by measuring glycosaminoglycan (GAG), which is a differentiation marker of cartilage cells. Specifically, 1 x 10 < ⁵ > stem cells were dispensed into the microspheres of Example 1 and Comparative Example 1, and then differentiated and cultured for 7 days, 14 days, and 21 days. After incubation, the cells were washed with PBS buffer and the cells were removed from each nanoparticle with Tris-EDTA solution. The detached cells were centrifuged at 13,500 rpm for 1 minute, and 0.1% Triton X-100 solution was added to the cells. Ultrasonic waves were applied at 4 ° C for 1 minute to crush the cells and centrifugation. The DNA was isolated using the qG-DEX ™ IIc Genomic DNA Extraction Kit and the DNA concentration was measured at 260/280 nm via NanoDrop. Then, after the dimethylmethylene blue (DMMB) assay, the absorbance was measured at 525 nm, and the GAG was quantitatively analyzed and shown in Table 4 below.

Classification (concentration of drug) GAG / DNA concentration ([mu] g / mL) After 21 days Comparative Example 1.1 Example 1 1 mM 1.4 5 mM 1.7 10 mM 2.6 Example 2 1 mM 1.3 5 mM 1.6 10 mM 2.3 Example 3 1 mM 1.2 5 mM 1.6 10 mM 2.2 Example 4 1 mM 1.4 5 mM 1.7 10 mM 2.4 Example 5 1 mM 1.3 5 mM 1.6 10 mM 2.2

As shown in Table 4, it was evaluated that the GAG / DNA concentration of stem cells differentiated into chondrocytes in the porous polymer microspheres of Examples 1 to 5 was effectively increased as compared with the comparative example.

Test Example  5.2. Gene expression analysis

Chondrocyte differentiation of porous polymer microspheres according to the examples was evaluated through expression analysis of late differentiation marker genes (Aggrecan, COL1A1, COL2A1 and COL10A1) of chondrocytes. Specifically, 1 x 10 < ⁵ > stem cells were dispensed into the microspheres of Examples and Comparative Examples, and then differentiated and cultured for 21 days. After incubation, the cells were washed with PBS buffer and the cells were removed from each microsphere with Tris-EDTA solution. The removed cells were centrifuged at 13,500 rpm for 1 minute, then Trizol reagent solution and chloroform were added to the cells, and the cells were separated by applying ultrasonic waves at 4 ° C for 1 minute and then centrifuged. After separating the supernatant, RNA concentration was measured at 260/280 nm on NanoDrop using RNeasy Mini kit (RNeasy mini kit; Qiagen, Doncaster, VIC, Austraila). The isolated RNAs were analyzed by cDNA synthesis and real-time PCR using AccuPower RT-PCR PreMix (Bioneer, Korea) and the expression levels of the respective genes are shown in Table 5 below.

Classification (concentration of drug) Aggrecan /
GAPDH COL1A1 / GAPDH COL2A1 / GAPDH COL10A1 / GAPDH After 21 days After 21 days After 21 days After 21 days Comparative Example 1.0 1.0 1.0 1.0 Example 1 1 mM 1.5 1.3 1.3 1.9 5 mM 2.3 1.6 1.8 2.1 10 mM 3.3 2.0 2.2 3.4 Example 2 1 mM 1.4 1.2 1.2 1.8 5 mM 2.2 1.4 1.7 2.0 10 mM 3.1 1.8 2.0 3.2 Example 3 1 mM 1.2 1.3 1.2 1.7 5 mM 2.0 1.7 1.5 1.9 10 mM 3.1 1.9 1.9 2.9 Example 4 1 mM 1.5 1.4 1.3 1.8 5 mM 2.4 1.7 1.9 2.3 10 mM 3.4 2.2 2.4 3.5 Example 5 1 mM 1.4 1.3 1.2 1.8 5 mM 2.0 1.5 1.6 2.2 10 mM 2.9 1.9 1.9 3.0

As shown in Table 5, in Comparative Example, there was no expression of a gene related to chondrogenic differentiation. However, in the porous polymer microspheres of Examples 1 to 5 according to the present invention, chondrogenic differentiation into chondrocytes differentiated into chondrocytes (aggrecan, COL10A1, COL1A1, and COL2A1) were increased. Thus, the drug-loaded porous polymer microspheres of the examples according to the present invention proved to be effective for cartilage differentiation.

Test Example 6. Verification of Inflammation Inhibitory Effect

The inflammatory activity of the porous polymer microspheres according to Examples 1 to 5 was evaluated through the expression analysis of inflammatory marker genes.

1 x 10 < ⁵ > stem cells were dispensed into the microspheres of Examples and Comparative Examples, and then differentiated and cultured for 1 and 3 days. After incubation, Trizol reagent solution and chloroform were added, and the cells were separated by applying ultrasonic waves at 4 ° C for 30 minutes, followed by centrifugation. After separating the supernatant, RNA concentration was measured at 260/280 nm on NanoDrop using RNeasy Mini kit (RNeasy mini kit; Qiagen, Doncaster, VIC, Austraila). RNA was isolated and analyzed by real-time PCR using cDNA synthesis and real-time PCR using AccuPower RT-PCR PreMix (Bioneer, Korea) And TNF- [alpha] were measured and are shown in Table 6 below.

Classification (concentration of drug) TNF-α / GAPDH IL-6 / GAPDH COX-2 / GAPDH After 1 day 3 days later After 1 day 3 days later After 1 day 3 days later Comparative Example 4.3 5.0 4.2 4.4 4.0 4.7 Example 1 1 mM 3.8 3.5 3.3 3.0 3.6 3.4 5 mM 3.0 2.5 2.1 1.8 2.2 2.0 Example 2 1 mM 4.0 3.7 3.5 3.1 3.7 3.5 5 mM 3.4 2.6 2.3 2.0 2.4 2.2 Example 3 1 mM 3.9 3.6 3.4 3.2 3.8 3.6 5 mM 3.2 2.7 2.2 2.0 2.5 2.3 Example 4 1 mM 3.7 3.3 3.2 3.1 3.6 3.5 5 mM 3.1 2.6 2.0 1.8 2.1 1.9 Example 5 1 mM 3.8 3.5 3.5 3.1 3.8 3.4 5 mM 3.2 2.6 2.3 2.0 2.4 2.1

Referring to Table 6, while the inflammation is maintained in the comparative example, the inflammatory markers COX-2, IL-6 and TNF-a are reduced in dependence on the concentration of simvastatin in the porous polymer microspheres of the examples according to the present invention Respectively.

Test Example 7. Cell proliferation according to pore size of porous polymer microsphere

The porous polymer microspheres of the present invention are advantageous in that the pore size and the volume of drug loaded can be controlled according to the concentration of gelatin added during the manufacturing process. Therefore, the degree of cell proliferation in microspheres having different pore sizes was measured according to the gelatin concentration (1 wt%, 7.5 wt% and 15 wt%) in an aqueous gelatin solution added during the production of microspheres. 3 is a scanning electron microscope image of microspheres having different pore sizes according to the concentration of gelatin (1 wt%, 7.5 wt% and 15 wt%).

To do this, 1 × 10 ⁵ adult stem cells were carefully placed in porous particles with different pore sizes and cultured for 1 day, 3 days, and 7 days. After 1, 3 and 7 days of culture, the cells were washed with phosphate buffered saline. After washing, CCK-8 proliferation kit reagent (Tetrazolium salt reagent, Cell Counting kit-8, Dojindo, USA) was added and incubated for 1 hour. The culture was carefully transferred to a 96-well plate and absorbance was measured at 450 nm 4.

4, adult stem cells cultured on porous particles having different pore sizes according to the concentration of gelatin (1 wt%, 7.5 wt%, and 15 wt%) gradually progressed after 1 day, 3 days, and 7 days Cells were actively proliferated, but the porous particles made of gelatin (15 wt%) at a higher concentration than the other groups (1 wt% and 7.5 wt% of gelatin) showed a decrease in cell proliferation due to the relatively large pore size I could.

Hereinafter, the present invention will be described in more detail with reference to preferred embodiments. It will be apparent, however, to those skilled in the art that these embodiments are for further explanation of the present invention and that the scope of the present invention is not limited thereby.

Claims (23)

A three-dimensional net structure biodegradable polymer scaffold in which pores having a size of 5 to 100 탆 are connected to each other; And a drug for treating a bone disease or a cartilage disease contained in the net structure of the biodegradable polymer scaffold. The porous polymer microsphere for preventing or treating bone diseases or cartilage diseases. The method according to claim 1,
Wherein the drug is contained in an amount of 2 to 30 parts by weight based on 10 parts by weight of the biocompatible polymer. The method according to claim 1,
A porous polymer microsphere for preventing or treating bone diseases or cartilage diseases, characterized by having a porosity of 10 to 90% and a particle size of 200 to 1000 탆. The method according to claim 1,
A porous polymer microsphere for preventing or treating bone diseases or cartilage diseases, characterized by having a porosity of 60 to 90%, a pore size of 10 to 40 탆, and a particle size of 200 to 500 탆. The method according to claim 1,
Drugs for the treatment of bone diseases or cartilage diseases include osteogenic protein-2, epidermal growth factor, osteogenic protein-7, cilastatin, nystatin, lovastatin, somatostatin, pravastatin pravastatin, simvastatin, fluvastatin, atorvastatin, cervastatin, ulinastatin, rosuvastatin, pitavastatin, pravastatin, Glucosamine, chondroitin, mevastatin, carotenine, transforming growth factor-beta1, transforming growth factor-beta3,6-bromoindine-3-oxime, But are not limited to, morphamine, meblinolone, genistein, icarin, melatonin, metformin, alendronate, zoledronate, etidronate, clodronate, tirudronate, pamidronate, neridronate, , Ibandronate, risedronate, prostaglandin, dexamethasone, osteogenic protein-4, insulin-like growth factor-1, fibroblast growth factor-2, fibroblast growth factor-18, fibroblast growth factor, Wherein the porous polymer microspheres are used for preventing or treating bone diseases or cartilage diseases. The method according to claim 1,
The biocompatible polymer may be at least one selected from the group consisting of polyglycolic acid (PGA), polylactic acid (PLA), polylactide-co-glycolide (PLGA), polycaprolactone (PCL), polyamino acid, polylactide, polyphosphazine, Any one selected from carbonate, polyphosphoester, polyanhydride, polyorthoester, polyhydroxyvalerate, polyhydroxybutyrate, hyaluronic acid, cellulose, heparin, collagen, alginate and chitosan; Polymers in which two or more of these are blended; And a copolymer of two or more thereof. The porous polymer microsphere for preventing or treating bone diseases or cartilage diseases according to claim 1, The method according to claim 1,
The above-mentioned bone disease is caused by osteoporosis, osteoporosis, bone injury, traumatic joint damage, osteomalacia, rickets, osteoporosis, osteoporosis, osteoporosis, osteonecrosis, osteonecrosis, fracture, excessive osteoclast osteoporosis, And is selected from the group consisting of fibrotic bone disease, intractable bone disease, metabolic bone disease, renal osteodystrophy, periodontal disease, Paget disease and metastatic bone cancers,
The cartilage diseases include osteochondromatosis, distortion, bursitis, menstruation, meniscal tears, meniscus cysts, meniscal tears, The anterior cruciate ligament injury, the posterior cruciate ligament injury, the intra-articular vitreous, the exfoliation osteochondritis, the posterior cruciate ligament injury, Wherein the porous polymeric microspheres are selected from the group consisting of plica syndrome, genu varum, knock-knee and pronounced knee knee. Spear. The method according to claim 1,
A porous polymer microsphere for prevention or treatment of bone diseases or cartilage diseases, which is injected into a living body and releases a drug for treating bone diseases or cartilage diseases. 6. The method of claim 5,
The medicament for the treatment of bone diseases or cartilage diseases includes simvastatin, pravastatin, lovastatin, mevastatin, pitavastatin, osteogenic protein-2, fibroblast growth factor, cartogenin, dexamethasone, alendronate, pamidronate, , Prostaglandin, or a mixture of two or more thereof. The porous polymer microsphere for the prevention or treatment of bone diseases or cartilage diseases. 1) biocompatible polymers; And a drug for the treatment of bone diseases or cartilage diseases in an organic solvent to prepare a polymer solution;
2) adding the polymer solution and gelatin aqueous solution and homogenizing to prepare an emulsion;
3) For the treatment of biocompatible polymers, bone diseases or cartilage diseases by using the emulsion as a discontinuous phase and using a polyvinyl alcohol solution as a continuous phase in a fluidic device Preparing a polymer microsphere comprising a drug and gelatin; And
4) adding the polymer microspheres to water at 38 to 50 캜 and stirring to elute the gelatin from the polymer microspheres; and 4) a method for producing porous polymer microspheres for preventing or treating bone diseases or cartilage diseases. 11. The method of claim 10,
Wherein the biocompatible polymer is contained in an amount of 2 to 30 parts by weight based on 10 parts by weight of the biocompatible polymer. 11. The method of claim 10,
Wherein the gelatin is homogenized by adding 3 to 15 parts by weight of the gelatin to 10 parts by weight of the biocompatible polymer, to thereby prevent or treat bone diseases or cartilage diseases. 11. The method of claim 10,
The biocompatible polymer may be at least one selected from the group consisting of polyglycolic acid (PGA), polylactic acid (PLA), polylactide-co-glycolide (PLGA), polycaprolactone (PCL), polyamino acid, polylactide, polyphosphazine, Any one selected from carbonate, polyphosphoester, polyanhydride, polyorthoester, polyhydroxyvalerate, polyhydroxybutyrate, hyaluronic acid, cellulose, heparin, collagen, alginate and chitosan; Polymers in which two or more of these are blended; And a copolymer of two or more thereof. The method for producing a porous polymer microsphere for preventing or treating bone diseases or cartilage diseases according to claim 1, 11. The method of claim 10,
Wherein the step 2) further comprises 0.5 to 3 parts by weight of an emulsion stabilizer. 11. The method of claim 10,
Characterized by having a porosity of 10 to 90%, a pore size of 5 to 100 μm, and a particle size of 200 to 1000 μm. The porous polymer microspheres for prevention or treatment of bone diseases or cartilage diseases Way. 11. The method of claim 10,
Characterized by having a porosity of 60 to 90%, a pore size of 10 to 40 μm, and a particle size of 100 to 400 μm. The porous polymer microspheres for prevention or treatment of bone diseases or cartilage diseases Way. 11. The method of claim 10,
The above-mentioned bone disease is caused by osteoporosis, osteoporosis, bone injury, traumatic joint damage, osteomalacia, rickets, osteoporosis, osteoporosis, osteoporosis, osteonecrosis, osteonecrosis, fracture, excessive osteoclast osteoporosis, And is selected from the group consisting of fibrotic bone disease, intractable bone disease, metabolic bone disease, renal osteodystrophy, periodontal disease, Paget disease and metastatic bone cancers,
The cartilage diseases are selected from the group consisting of degenerative arthritis, inflammatory arthritis, osteochondromatosis, distortion, bursitis, menstruation, discoid meniscus rupture meniscal tear, meniscus cyst, collateral ligamentrupture, anterior cruciate ligament injury, posterior cruciate ligament injury, intra-articular vitreous (intra- characterized in that it is selected from the group consisting of an articular vitreous, exfoliation osteochondritis, plica syndrome, genu varum, knock-knee and pronounced knee knee Wherein the porous microspheres are used for preventing or treating bone diseases or cartilage diseases. 11. The method of claim 10,
Wherein the step 2) is performed by homogenization at 8,000 to 20,000 rpm for 30 seconds to 10 minutes with a homogenizer to prevent or treat bone diseases or cartilage diseases. 11. The method of claim 10,
A method for preparing porous polymer microspheres for the prevention or treatment of bone diseases or cartilage diseases, which comprises controlling the release and release rate of a drug by controlling the content of gelatin, a drug or both of them relative to the content of a biocompatible polymer. 11. The method of claim 10,
Wherein the gelatin aqueous solution is an aqueous 1 to 10 wt% gelatin solution. 11. The method of claim 10,
Wherein the flow rate of the discontinuous phase and that of the continuous phase are 0.2 to 1 mL / min, respectively, for preventing or treating bone diseases or cartilage diseases. 11. The method of claim 10,
Wherein the organic solvent is one or more selected from the group consisting of dimethylsulfoxide, dichloromethane, tetrahydrofuran and N, N-dimethylformamide. The porous polymer microsphere for prevention or treatment of bone diseases or cartilage diseases Gt; 11. The method of claim 10,
Drugs for the treatment of bone diseases or cartilage diseases include osteogenic protein-2, epidermal growth factor, osteogenic protein-7, cilastatin, nystatin, lovastatin, somatostatin, pravastatin pravastatin, simvastatin, fluvastatin, atorvastatin, cervastatin, ulinastatin, rosuvastatin, pitavastatin, pravastatin, Glucosamine, chondroitin, mevastatin, carotenine, transforming growth factor-beta1, transforming growth factor-beta3,6-bromoindine-3-oxime, But are not limited to, morphamine, meblinolone, genistein, icarin, melatonin, metformin, alendronate, zoledronate, etidronate, clodronate, tirudronate, pamidronate, neridronate, , Ibandronate, risedronate, prostaglandin, dexamethasone, osteogenic protein-4, insulin-like growth factor-1, fibroblast growth factor-2, fibroblast growth factor-18, fibroblast growth factor, Wherein the porous polymer microspheres are selected from the group consisting of polyvinylpyrrolidone and polyvinylpyrrolidone.

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