KR101533094B1 - Long Bone Filler for Tissue Engineering and Preparing Method thereof - Google Patents

Abstract

The present invention relates to a filler for regenerating long bones, comprising biocompatible polymers and drugs. Also, the present invention relates to a preparing method of the filler for regenerating long bones. The filler for regenerating long bones enables drugs included in a bone filler to be transferred to an affected part for a long time period when transplanting the bone filler in the affected part, whereby the filler can replace a conventional bone filler which discharges drugs only in a first stage. In addition, the filler uses stem cells having angiogenesis activity to perform an excellent bone regeneration, thereby being easily and safely transplanted.

Description

TECHNICAL FIELD [0001] The present invention relates to a long bone filler for tissue engineering,

The present invention relates to a bone filler for regeneration of iliac bone and a method for producing the same.

Generally, the damaged bone has the ability to regenerate itself, but in order to regenerate bone tissue, it is necessary that a support capable of growing cells, a physiologically active substance that induces differentiation and growth of cells, and a stem capable of differentiating into osteoblasts and osteoblasts Cells are essential.

Bone fillers are used for physically filling bone defect sites and promoting bone formation. They should have a porous support structure that is well biocompatible and capable of spreading nutrients or excrement for attachment, proliferation and differentiation of cells. It is also important to have mechanical strength similar to that of natural bone.

Bone fillers include autogenous bone, allogeneic bone, heterogeneous bone, and synthetic bone. The autogenous bone is taken from the patient's own bone and transplanted into the defect, which has no tissue rejection reaction, and has excellent bone induction, bone formation, and bone conduction. However, autogenous bone requires additional surgery for bone extraction, and the shape and amount of bone taken is extremely limited and the operation is complicated, resulting in a great physical burden on the patient. Next, allogeneic bone has bone regeneration effect by osteogenic protein known to have osteoinductive function by securing bone tissue from carcass, and there is no need for secondary surgery, and there is an advantage of having quick operation time and recovery period. However, there is a drawback that there is a difficulty in having a donor, the possibility of transmission of disease, and the difference in bone induction ability depending on the site of harvest and the age of the donor. The heterogeneous bone can be secured from bovine or pig, and it is easy to acquire bone conduction ability and bone tissue, but there is a risk of immune reaction or infection, and there is a disadvantage that it remains absent even after long term in vivo. In addition, the synthetic bone has no possibility of transmission of disease and there is no limit to the amount of the disease, and many researches have been carried out. The advantage of having less antigen reaction than allograft bone graft materials is that it has a small effect on bone regeneration, And is a bone filler that can not maintain shape or space after surgery and is deformed.

Long bones such as iliac bones are long, thick cylindrical bones of which both ends are spherical and filled with bone marrow. It takes a long time that it takes 8 to 12 weeks for these iliac bone to be lost and regenerated. Recently, a drug delivery system based on a scaffold has been developed and applied to regenerate the iliac bone. However, only a part of the drug is released within 2 weeks after the bone grafting, and the drug is not released until the iliac defect is regenerated. There is a problem that can not be done. Therefore, when the number of drug administration is increased and the drug is released only at the initial stage, the secondary and tertiary operations are required, which increases the physical and economic burden of the patient. In addition, there is a problem that the mechanical strength of the bone filler drops when an excessive amount of the drug is added to solve the initial release.

Thus, there has been a need for the development of an iliac filling agent that is biocompatible and capable of long-term release of the drug.

It should be understood that the foregoing description of the background art is merely for the purpose of promoting an understanding of the background of the present invention and is not to be construed as adhering to the prior art already known to those skilled in the art.

The present inventors have made efforts to develop a bone filler suitable for regeneration of iliac bone, which is different from the conventional bone filler, and which suppresses the initial release of the drug and promotes the release effect and takes a long time for bone regeneration. As a result, when a drug is homogeneously mixed with a biocompatible polymer to form a bone filler, the drug contained in the bone filler can be transferred to the local lesion in the long term, And confirmed that excellent bone regeneration can be achieved by using stem cells having neovascularization ability, thereby completing the present invention.

Accordingly, an object of the present invention is to provide a filler for iliac bone regeneration comprising a biocompatible polymer and a drug.

It is another object of the present invention to provide a method for preparing filler for iliac bone regeneration.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, the present invention provides a pharmaceutical composition comprising a biocompatible polymer and a drug containing vitamin D homogeneously mixed with the polymer, wherein the drug is capable of continuously releasing a long bone Provide a filler.

The present inventors have made efforts to develop a bone filler suitable for regeneration of iliac bone, which is different from the conventional bone filler, and which suppresses the initial release of the drug and promotes the release effect and takes a long time for bone regeneration. As a result, it was confirmed that if a drug is homogeneously mixed with a biocompatible polymer to form a bone filler, the drug can be released continuously for 2 to 3 months or more, and thus the damaged portion of the iliac bone can be effectively regenerated without additional drug administration or surgery Respectively.

As used herein, the term " biocompatible polymer "refers to a natural or synthetic polymer that is substantially non-toxic to the human body, chemically inert, and non-immunogenic.

According to a preferred embodiment of the present invention, the biocompatible polymer includes a gel-forming polymer, a biodegradable polymer, an insoluble polymer, and an enteric polymer, and more preferably, a biodegradable polymer is used.

Examples of the gel-forming polymer include hydroxypropylmethylcellulose, hydroxypropylcellulose, carboxyvinyl polymer, polyvinyl alcohol, chitosan, hyaluronic acid, alginic acid, pectin, carrageenan, chondroitin sulfate, sulfuric acid textur, collagen, gelatin, Complexed chitin, fibrin, dextran, agarose, pullulan and the like; Examples of the biodegradable polymer include polyesters, polyethylene glycol, polyacrylamide, polymethacrylic acid, polymaleic acid, polyvinyl alcohol, polyethylene oxide, polyvinylpyrrolidone, polyglycolic acid, polylactic acid, Tide-glycolide copolymer, polycaprolactone, albumin, gelatin, collagen and the like; Examples of the insoluble polymer include ethylcellulose, cellulose acetate and the like; Examples of the enteric polymer include butyric acid acetate, carboxymethylcellulose, methacrylic acid-methyl methacrylate copolymer, and the like.

The drug containing vitamin D (preferably vitamin D3) to be mixed with the polymer is preferably homogeneously mixed with the biocompatible polymer at 0.05 to 1.00% by weight based on the total weight of the bone filler, so that continuous drug release is possible.

According to a preferred embodiment of the present invention, the drug contained in the bone filler of the present invention may be continuously released for 2 to 3 months or more, and taking 8 to 12 weeks for regeneration of the iliac bone, It is possible to effectively regenerate the defective part of the ilium without administration or surgery.

According to a preferred embodiment of the present invention, the present invention may further include a drug other than the vitamin D. Additional drugs include, but are not limited to, bisphosphonates, calcitonin, salcatonin, elctatonin, fluorine, menatetrenone, alfacascolid, calcium phosphate, dexamethasone, alendronate, risedronate, ibandronate, zoledronate Porphyrronate, Parathyroid Hormone, Strontium, Denosum Map, Cathethine K Inhibitor, Terpyparidide, Lomosuccine, Calcitriol, Cholecalciferol, Raloxifene, Tibolone, Ipriflavone, Alphalcysidone, Estrogen Receptor modulators, and the like.

The term "long bone " as used in the present invention means a bone of a vertebrate having a length of 4 cm or more and is characterized by having a sturdy structure for supporting the body unlike cartilage. In the present specification, the iliac bone includes the skull, the clavicle, the femur, the humerus, the radial, the ulna, the tibia, the fibula, and the like.

According to a preferred embodiment of the present invention, the present invention may additionally include stem cells having angiogenic activity.

Unlike cartilage, the strength of the iliac bone is very important, and if it does not have adequate strength in regeneration with bone filler, it may cause osteoporosis, . In particular, effective treatment of iliac defect sites requires induction of neovascularization unlike cartilage regeneration. Therefore, when stem cells are used, the neovascularization capability can be improved and rapid restoration of the intestinal tract of appropriate strength can be induced.

The stem cells are not limited as long as they possess neovascularization ability, and preferably mesenchymal stem cells, more preferably, bone marrow mesenchymal stem cells are used.

According to another aspect of the present invention, there is provided a process for producing a biocompatible polymer, comprising the steps of: (a) dissolving a biocompatible polymer in a solvent; (b) loading a drug comprising vitamin D into the dissolved biocompatible polymer; (c) adding a porous material to the drug-loaded biocompatible polymer and injecting the drug into a mold to form a bone filler; (d) compressing and drying the formed bone filler; (e) porosity formation of the dried bone filler; And (f) drying the porous formed bone filler. The present invention also provides a method for preparing a filler for long bone regeneration.

According to a preferred embodiment of the present invention, the manufacturing method of the present invention further comprises (g) sowing the stem cells into the porous formed bone filler.

The manufacturing method of the present invention will be described in accordance with the respective steps as follows:

(a) a polymer dissolution step for dissolving a biocompatible polymer in a solvent

In the present invention, a biocompatible polymer is dissolved in a solvent. In this case, the solvent used should be a solvent capable of dissolving the biocompatible polymer, and it is preferable to use a solvent that does not dissolve the drug. The biocompatible polymer is added to the solvent in an amount of 10 to 40 w / v%, preferably 20 to 30 w / v%, and the mixture is stirred. Most preferably 25 w / v%.

The solvent is not limited as long as it can dissolve the biocompatible polymer and is preferably an organic solvent such as ethanol, methanol, hexane, chloroform, acetone, methylene chloride, dimethylsulfoxide, toluene, tetrahydrofuran, methyl ethyl ketone, Hexafluoroisopropanol, ether, dimethyl carbonate, cyclohexane, xylene, styrene and the like can be used. More preferably, methylene chloride, chloroform and the like are used.

(b) loading the drug comprising vitamin D into the polymer

Vitamin D and / or a drug comprising it is then loaded into the biocompatible polymer dissolved. Dissolving the drug in a solvent capable of dissolving the drug in the sustained-release polymer solution dissolved in the solvent, adding the biocompatible polymer in an amount of 0.01 to 1.00 w / v% based on the dissolved solution, and mixing the mixture using a high-pressure homogenizer good. At this time, it is preferable to use a solvent which does not dissolve the sustained-release polymer, preferably alcohol, acetone, methanol, distilled water or the like.

(c) Porogen  Addition and injection into the mold Filler  Forming step

A porous forming material is added to a solution in which the biocompatible polymer and drug are mixed. The porous forming material which can be used is added in advance to form a porous structure in the finished bone filler and can be removed by using an elution external fluid or the like in the future. Preferably, sodium chloride, sugar, paraffin, gelatin or the like is used.

At this time, the size of the porous forming material may be about 90 to 600 mu m, preferably 250 to 425 mu m. At this time, it is preferable to use a material capable of eluting in the exudate liquid. Then, it is put into a mold (for example, a silicon mold).

(d) Filler  Compression and drying step

Next, the silicone mold containing the biocompatible polymer, drug, and porous forming material is compressed and dried. The method of compression and drying is not limited, but it is preferably possible to pressurize at a pressure of 50 to 70 kgf / cm < ² > at room temperature for 12 to 48 hours using a press machine, and then dried.

(e) Filler Porosity formation  step

The dried bone filler obtained above is collected in a silicon mold and immersed in the eluate to elute the porous forming material to form pores. When the porous forming material is eluted, it is preferable to use a solution of the sustained-release polymer and an exuding liquid which does not dissolve the drug but dissolves only the porous forming material. Preferably, distilled water, xylene, hexane and the like can be used. At this time, it is preferable to replace the eluted external fluid every 4 to 7 hours, preferably 48 to 72 hours.

(f) Perforated  goal Filler  Drying step

The porous filler is frozen at 4 to 7 mTorr, -70 to -100 ° C for 12 to 48 hours, and then dried. It is preferable to dry it for at least one week in order to sufficiently remove the residual solvent, and it is preferable to sterilize by using iogas, gamma rays, alcohol or the like.

(g) Perforated  goal On fillers  Steps to seed stem cells

After the stem cells are inoculated into the sterilized bone filler, the cells are preferably cultured for 2 to 7 days at 20 to 40 ° C and 1 to 10% CO ₂ , and more preferably for 3 days.

The iliac filling agent prepared by the process according to the present invention can be manufactured such that the release of the drug is released more efficiently than the conventional bone filling agent and the bone regeneration due to stem cells is promoted.

The features and advantages of the present invention are summarized as follows:

(i) The present invention provides a filler for iliac bone regeneration comprising a biocompatible polymer and a drug.

(Ii) In addition, the present invention provides a method for producing filler for iliac bone regeneration.

(Iii) Using the filler for iliac bone regeneration according to the present invention, the drug contained in the bone filler can be transferred to the local lesion for a long period of time, thereby replacing the bone filler which releases the drug only in the early stage. The present invention relates to an osteoinductive filler for tissue engineering,

1 is a graph showing release behavior of a drug from a bone filler.
FIG. 2 is a photograph (PLGA: A (von Kossa), Vitamin D3 / PLGA / MSCs: B) of the bone filler (Vitamin D3 / PLGA / MSCs) H & E), C (von Kossa), D (Alizalin red).
FIG. 3 is a photograph (A: PLGA, B: Vitamin D3 / PLGA / MSCs) of a bone filler according to a comparative example taken after in vivo implantation.
FIG. 4 is a graph showing the bone formation density after 3, 6, and 10 weeks after implantation of the bone filler (Vitamin D3 / PLGA / MSCs) of the present invention.
FIG. 5 is a photograph (A: Vitamin D3 / PLGA / MSCs, B: Vitamin D3 / PLGA) of a three-dimensional CT image taken 20 weeks after implantation of a bone filler in vivo.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

1. Polymer dissolution step to dissolve biocompatible polymers that can be used in a solvent in a sustained release form

1 g of a polylactide-glycolide copolymer as a biocompatible polymer is prepared and placed in a glass vial. At this time, the polylactate-glycolide copolymer had a molar ratio of 75:25 and an average molecular weight of 90,000 g / mole. Then, 4 ml of methylene chloride as a solvent was added, and the mixture was stirred for 5 minutes.

2. Drug loading step to load the drug into the dissolved polymer

Add the vitamin D3 drug dissolved in pure alcohol to the solution in which the polylactide-glycolide copolymer is dissolved. At this time, the vitamin D3 drug was added to the polylactide-glycolide copolymer solution at 0.086 w / v%, and the two solutions were mixed using a high-pressure homogenizer.

3. Bone filler forming step to form bone filler using biocompatible polymer and drug as silicon mold

To the solution containing the biocompatible polymer and drug, 9 g of sodium chloride as a porogen is added. The size of sodium chloride was 250 to 425 탆. Thereafter, a solution containing a biocompatible polymer, a drug, and sodium chloride is put into a silicone mold having a diameter of 5 mm and a thickness of 40 mm.

4. Step of compressing and drying the formed bone filler

The silicone mold containing the biocompatible polymer, vitamin D3 and sodium chloride was pressurized at a pressure of 60 kgf / cm ² for 24 hours using a press (MH-50Y, CAP 50 tons, Japan) and then dried.

5. Pore formation step of dried bone filler

In order to form pores in the dried bone filler, the supernatant was added to the third distilled water, changed every 6 hours, and then sodium chloride was eluted for 48 hours.

6. Drying the porous bone filler

The porous filler was frozen at 5 mTorr, -80 ℃ for 24 hours and lyophilized. In order to remove the residual solvent methylene chloride sufficiently, it was dried in a vacuum oven at 25 ° C for at least one week and sterilized by gamma irradiation.

7. Sterile bone On fillers  Stem cell seeding step to seed stem cells

The sterilized bone filler was placed on the bottom of the culture plate. Here, bone marrow mesenchymal stem cells were seeded at a concentration of 10 × 10 ⁵ cells / well and cultured for 3 days. At this time, the culture was carried out at 37 ° C and 5% CO ₂ .

Comparative Example

For the same sample, a polylactide - glycolide copolymer bone filler without addition of drugs and stem cells was used.

Experimental Example  1: Drug Release Experiment

The drug release behavior of vitamin D3 released from aqueous solution of polylactide-glycolide copolymer bone filler with vitamin D3 prepared in the above example was compared and analyzed. Since vitamin D3 is a lipid-soluble drug, a drug called dexamethasone was used to produce a bone filler and the release test was conducted.

The drug release experiment was carried out by placing the polylactide-glycolide copolymer support prepared in the above example in a 50 ml glass vial and adding 50 ml of a phoshphate buffered saline (PBS, pH 7.4). The buffer solution used was PBS, which is very similar to the environment in the human body. 2, 3, 4, 5, 6, 7, 8, 9, and 10 days while stirring the 50 ml glass vial containing the bone filler at 37 DEG C and 50 rpm, 10 days, 14 days, 18 days, 21 days, 28 days, 35 days, 42 days, 49 days, 56 days, 63 days, 70 days, 77 days, 84 days, 91 days, 98 days). 100 μl of the buffer solution was collected at predetermined time intervals, and 100 μl of a new buffer solution was further added thereto. The measurement results are shown in Fig.

As shown in Fig. 1, it was confirmed that the amount of vitamin D3 released from the polylactide-glycolide copolymer increases with time. It has been confirmed that sustained release of unresolved drugs has occurred in bone fillers containing existing drugs.

Experimental Example  2: Histological evaluation

To evaluate the in vivo bone formation ability of polylactide-glycolide copolymer bone filler with vitamin D3 and stem cells, the bone filler was implanted into the bone defect of the skull of 4-week-old SD rats and recovered at 4 weeks And fixed with 4% formalin (Sigma-Aldrich, USA). Then, the block was cut into 4 ㎛ thick and fixed on a slide glass to be stained with H & E (hemetoxylin & eosin), von kossa, and Alizalin red.

As shown in Fig. 2, bone formation was observed in both vitamin D3 and stem cell-loaded bone filler (BD) rather than in the drug-loaded bone filler (A), and significantly better bone formation .

Experimental Example  3: Goals On fillers  In vivo bone formation Formability  Rating 1 - Measure

In vivo bone formation was evaluated using a rat skull defect model at 8 weeks after transplanting the bone filler prepared in the above example. As a comparative example, only the bone filler without drug loading was implanted under the same conditions, and the results are shown in FIG.

As shown in FIG. 3, it was confirmed that the bone-filling agent (B) containing the drug and the stem cells had better bone formation ability than the bone-filling agent (A) not loaded with the drug.

Experimental Example  4: Goals On fillers  In vivo bone formation Formability  Rating 2 - BMD  Measure

The following experiment was conducted to evaluate the in vivo bone forming ability of the bone filler prepared in the above Examples. Bone density of PLGA, PLGA / MSCs, Vitamin D3 / PLGA and Vitamin D3 / PLGA / MSCs were measured after 3, 6 and 10 weeks after implantation in 12 week old New Zealand white rabbits. Is shown in Fig.

As shown in FIG. 4, when PLGA bone filler alone was implanted, bone formation was found to be less than 50% over time. Vitamin D3 / PLGA bone filler showed better bone formation over time, MSCs bone filler showed nearly 100% bone formation.

Experimental Example  5: Goals On fillers  In vivo bone formation Formability  Rating 3 - 3D CT

The following experiment was conducted to evaluate the in vivo bone forming ability of the bone filler prepared in the above Examples. Fig. 5 shows photographs taken after three-dimensional CT measurement after 20 weeks after implantation of Vitamin D3 / PLGA and Vitamin D3 / PLGA / MSCs bone filler into 12 week-old New Zealand White rabbits.

As shown in FIG. 5, after 20 weeks, it was confirmed that Vitamin D3 / PLGA / MSCs bone filler had better bone formation than Vitamin D3 / PLGA bone filler.

The osteoinductive agent for tissue engineering of the present invention was confirmed to be suitable as a long-clavicle filling agent due to its excellent bone regeneration ability, while releasing the drug over a long period of time with a bone filler including drugs and stem cells.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (17)

delete delete delete delete delete delete (a) dissolving a polymer in a solvent to dissolve the biocompatible polymer;
(b) loading the drug comprising vitamin D3 into the dissolved biocompatible polymer while homogeneously loading the biocompatible polymer in an amount of 0.01 to 1.00 w / v% based on the dissolved solution;
(c) adding a porous forming material having an average particle size of 90 to 600 탆 to the drug-loaded biocompatible polymer and injecting the porous forming material into a mold to form a bone filler;
(d) pressing the formed bone filler at a pressure of 50 to 70 kgf / cm ² at room temperature for 12 to 48 hours and then drying;
(e) immersing the dried bone filler in the exudate fluid and eluting the porous material for 48 to 72 hours to form a porous body; And
(f) freezing the porous formed bone filler under conditions of -70 to -100 DEG C under 4 to 7 mTorr for 12 to 48 hours and then drying for at least a week to remove residual solvent
(g) culturing stem cells having neovascularization capability in said porous formed bone filler for 2 to 7 days at 20 to 40 캜 and 1 to 10% CO ₂ after sowing
Wherein the filler is a filler.
delete The method of claim 7, wherein the solvent is selected from the group consisting of ethanol, methanol, hexane, chloroform, acetone, methylene chloride, dimethylsulfoxide, toluene, tetrahydrofuran, methyl ethyl ketone, ethyl acetate, phenol, hexafluoroisopropanol, Wherein the filler is selected from the group consisting of dimethyl carbonate, cyclohexane, xylene, and styrene. delete delete delete delete delete delete delete delete

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