KR20030006787A - Chitosan bead containing tricalcium phosphate for bone substitute - Google Patents

Abstract

PURPOSE: Provided is a keto acid bead for alternative bones with tricalcium phosphate included which transmits growth of new bones into a gap between beads in transplanting into a bone damage part so that it improves bone-regeneration effect. CONSTITUTION: The keto acid bead for the alternative bones with tricalcium phosphate included is characterized by including 0.1-0.8wt% of tricalcium phosphate on a basis of the weight of keto acid desirably and including 0.2-0.7wt% of tricalcium phosphate more desirably. The size of the beads is 300-1200micrometer desirably and more desirably, that of the beads is 400-800micrometer.

Description

Chitosan bead containing tricalcium phosphate for bone substitute for tricalcium phosphate

The present invention relates to a bone substitute for bone tissue regeneration, and in particular to provide a bone substitute comprising tricalcium phosphate in the beads (bead) made of chitosan.

In general, autologous or allogeneic bone graft for bone tissue regeneration has been used for a long time and is still widely used. However, autologous bone has the disadvantage that the supply is insufficient, there is a risk of infection and surgery to obtain autologous bone. In addition, in case of allogeneic bone, there is a disadvantage that there is a possibility of inducing an immune response and transmitting a disease. Therefore, a sufficient amount of bone can be easily obtained, and there is an urgent need for the development of bone graft material without the possibility of immune response and disease transmission.

So far, developed materials include freeze-dried allograft and retinal bone tissue, reticular bone, carbon compound, apatite and non-absorbent hard tissue, and tricalcium phosphate Is reported as. However, materials such as bone powder have problems such as immune response and transmission of diseases, and synthetic materials are susceptible to infection and have a disadvantage in that they are accumulated after transplantation. In addition, since they do not occur spontaneously, but serve as artificially embedded skeletons, they remain unknown as they stimulate surrounding tissue cells to have tissue regeneration from the cellular level.

BACKGROUND OF THE INVENTION In the case of porous calcium phosphate ceramics containing hydroxyapatite as a main component, its chemical composition is similar to that of bone, which has attracted much attention as a biomaterial because of its excellent biocompatibility. However, since hydroxyapatite is too slow to degrade, it may be a barrier to newly formed bone tissue at the graft site. Recently, it is not only excellent in biocompatibility and bone affinity, but also after a certain period of time after surgery. There is a need for a biodegradable material that can be properly degraded in the human body and replaced with new bone in accordance with regeneration.

An ideal bone filler or graft material should be able to fill the bone injury site to prevent invasion of epithelial tissue and to allow bone tissue to conduct between particles and increase bone regeneration. The requirements of the bone replacement system are as follows. First, biocompatibility should not induce immune response, histotoxicity, or inflammatory response. Second, there must be adequate mechanical strength initially and must degrade as the tissue regenerates. Third, tissue and neovascularization must grow and enter into the pores between the particles. In addition, it should not be restricted to sterilization methods and should be easy to operate. In reality, however, no material satisfies all of these requirements, but only partially.

Korean Patent Publication No. 10-1999-0037004 discloses a method for preparing chitosan porous beads used as an immobilization carrier, heavy metal ion adsorbent, and chromatographic filler for enzymes and cells. In addition, Republic of Korea Publication No. 10-1998-005684 discloses a method for producing a sustained-release chitosan microcapsules. However, the chitosan beads disclosed above required a crosslinking reaction in order to maintain their shape, and therefore it is impossible to apply them to the living body as it is. In addition, there is no mention of the possibility of applying chitosan beads as an alternative to bone, and the possibility is not found anywhere.

Accordingly, the present inventors have studied diligently to prepare a bone substitute which is beneficial for bone regeneration while maintaining sufficient mechanical strength without crosslinking reaction, thereby completing chitosan beads including tricalcium phosphate.

The present invention, the above requirements of the bone replacement system, that is, firstly, should not be biocompatible to cause immune response, histotoxicity, inflammatory response, secondly, there must be an appropriate mechanical strength at the beginning, and must be degraded as the tissue is regenerated Third, tissue and neovascularization must grow and enter into the pores between particles, and fourth, to provide a bone replacement system that satisfies all the requirements that should not be limited to sterilization methods and easy to operate.

1 is a differential scanning micrograph of chitosan beads prepared by Example 1,

2 is a view showing the water content of the chitosan beads prepared in Example 1,

3 is a diagram showing the ex vivo degradation rate of chitosan beads prepared by Example 1,

Figure 4 is a diagram showing the cell activity of osteoblasts to the chitosan beads prepared by Example 1,

5 is a diagram showing the adhesion of osteoblasts to the chitosan beads prepared by Example 1,

Figure 6 is a photograph of a differential scanning microscope showing the appearance of osteoblast adhesion to chitosan beads prepared in Example 1.

The present invention relates to chitosan beads for bone replacement containing tricalcium phosphate. The tricalcium phosphate is preferably contained in an amount of 0.1 to 0.8 parts by weight with respect to the weight of chitosan, and more preferably 0.2 to 0.7 parts by weight. In addition, the size of the beads is preferably 300 ~ 1200 ㎛, more preferably 400 ~ 800 ㎛.

The term bone replacement agent of the present invention may also be expressed in terms of bone graft material, bone defect graft material, bone tissue graft material, bone tissue regeneration graft material and the like.

Chitosan, a polysaccharide, is used as a medical biomaterial, that is, as an artificial kidney membrane, a controlled release agent, an antiulcer agent, an anticancer material, an artificial organ material design, an artificial skin, and a bioabsorbable suture. In tissue regeneration, the N-acetylglucosamine residues of chitosan are known to be involved in the migration, adhesion, proliferation and differentiation of tissue cells. This is because it has a structure similar to that of N-acetyl-D-hexaamine that structurally plays a role in tissue regeneration. In addition, chitosan promotes the formation of growth factors in tissues and binds to fibroblast growth factor (FGF) and platelet-derived growth factor (PDGF-BB) in tissues, resulting in doubling of osteoblasts, ligaments, and fibroblasts. It imparts chemotactic and cellular proliferation to the arachnoid cells.

When the bone substitute chitosan beads of the present invention are applied to the bone defect site, new bone can be restored between the beads from the surrounding bone tissue and biodegraded after bone tissue regeneration is lost in the body.

The tricalcium phosphate contained in the bone substitute chitosan beads of the present invention increases the mechanical strength of the beads, and increases the biocompatibility and serves as a calcium source of new bone tissue. Since the chitosan beads for bone substitutes of the present invention contain tricalcium phosphate, they can have a suitable mechanical strength without undergoing a separate crosslinking reaction and can maintain their shape.

The chitosan beads for bone substitute of the present invention may further include the following materials to enhance cell adhesion and histocompatibility after adhesion. Growth factors such as platelet-derived tissue growth factor, insulin-like tissue growth factor, epithelial cell growth factor, fibroblast growth factor and transforming growth factor, and tetracycline, minocycline, flu Drugs such as birpropene; There are extracellular matrix (ECM) components (eg collagen, chondroitin sulfate) that can increase histocompatibility and promote differentiation into osteoblasts. When introducing the said additional substance, the content is preferable 0.2-0.7 weight part with respect to content of chitosan.

The chitosan beads for bone substitute of the present invention may first prepare a chitosan solution and then disperse tricalcium phosphate therein, and then prepare chitosan beads by an appropriate method. More specifically, preparing a chitosan solution, and then dispersing tricalcium phosphate therein, compressing the mixture with compressed air and dropping the mixture into a stirring coagulant through a nozzle to form chitosan beads, formed chitosan beads The chitosan beads for bone replacement according to the present invention may be prepared by a series of processes including separating the water from the coagulating solution and washing with water and lyophilizing the washed chitosan beads.

The manufacturing method of chitosan beads for bone substitutes of this invention is more concretely, for example as follows. First, a chitosan solution is prepared, and tricalcium phosphate is dispersed therein. The mixed solution is compressed into chitosan solution by compressed air using a nozzle diameter of 0.15-0.2 mm, and the chitosan solution is added dropwise as a coagulation solution to form chitosan beads. . The composition of the coagulation solution is an alkaline solution of 4% (w / v) NaOH aqueous solution / 20% (v / v) ethanol solution. The chitosan beads formed are filtered with a coagulation solution, washed with a large amount of distilled water until pH 7.4, and the chitosan beads are lyophilized. The concentration of chitosan is suitably 3 to 5% (w / w).

When the additional substance is introduced, the extracellular matrix component may be encapsulated by dispersing it in chitosan solution together with tricalcium phosphate to prepare chitosan beads, and the drug that exhibits tissue growth factor and bone regeneration effect after preparation of chitosan beads It can be encapsulated by the swelling rod method immersed in.

If the concentration of chitosan is less than 3% when preparing the chitosan solution, the viscosity of the chitosan solution is too low to form beads, and if it is 5% or more, the viscosity of the chitosan solution is too high to pass through the nozzle and the decomposition rate is too low, which is undesirable. not.

Beads prepared by the above method replenish damaged bone tissue after filling bones with bones, and surrounding bone tissue is conducted between the beads. As the bone tissue regenerates, chitosan beads are broken down by lysozyme. The size of the chitosan beads can be controlled by the pressure of the compressed air. By appropriately adjusting the size of the beads, it can be implanted into bone defects of various sizes.

Hereinafter, embodiments of the present invention will be described in detail, but various changes in embodiments will be possible within the spirit and scope of the present invention.

Example 1. Preparation of chitosan beads for bone replacement

Chitosan was added to an acetic acid solution diluted to a concentration of 4% (w / w) to prepare a 4% by weight chitosan solution, and then 50% tricalcium phosphate by weight of chitosan was dispersed. The chitosan solution was compressed with compressed air through a 0.15-0.2 mm nozzle while stirring the alkaline solution 4% (w / v) NaOH / 20% ethanol solution to drop the chitosan solution. The precipitated beads were filtered, washed with water until pH 7.4 and lyophilized.

Example 2 Preparation of Chitosan Beads for Bone Replacement

Chitosan was added to an acetic acid solution diluted to a concentration of 4% (w / w) to prepare a 4% by weight chitosan solution, and then 50% tricalcium phosphate by weight of chitosan was dispersed. It was dissolved by adding 10% collagen of chitosan weight. The chitosan solution was compressed into compressed air through a 0.15-0.2 mm nozzle while stirring the alkaline solution 4% (w / v) NaOH / 20% ethanol solution to drop the chitosan solution. The precipitated beads were filtered, washed with water until pH 7.4 and lyophilized.

Example 3 Preparation of Chitosan Beads for Bone Replacement

500 mg of the lyophilized chitosan beads prepared in Example 1 were immersed in 35 mg of tetracycline-dispersed medicinal solution for 24 hours and then lyophilized.

Experimental Example 1. Observation of the properties of chitosan beads for bone replacement

The properties of the chitosan beads prepared in Example 1 were observed in a differential scanning microscope and shown in FIG. 1.

As shown in FIG. 1, tricalcium phosphate is contained on the surface and the inside of the beads, and it is understood that the cells form a rough surface, which is suitable for attachment of cells.

Experimental Example 2. Confirmation of hydrophilicity of chitosan beads for bone replacement

In order to measure the hydrophilicity of the chitosan beads prepared in Example 1, the wetting in the phosphate buffer solution was measured and used as an index thereof. The results are shown in FIG.

Implants implanted into bone defects require adequate strength and integrity and should be able to absorb blood and tissue fluid. When left in the phosphate buffer solution for 2 hours increased to 75% of the dried weight has sufficient hydrophilicity, and therefore, it is considered to be excellent in tissue compatibility.

Experimental Example 3. In vitro degradation of chitosan beads for bone replacement

Chitosan beads were added to 0.1M phosphate buffer solution to which lysozyme was added, and the experiment was performed for a period of time in the laboratory while maintaining 15 rpm at 37 ° C. in a shaking water bath. The results are shown in FIG.

3 shows that chitosan beads were added to the lysozyme-added phosphate buffer solution for 14 days, and the weight of chitosan beads was reduced by 35%. It should be able to support the bone defect until bone tissue is regenerated (6 months).

Experimental Example 4. Cellular activity of osteoblasts against chitosan beads for bone replacement

Substituted gingival fibroblasts were treated with 0.25% trypsin-EDTA (Gibco) solution, followed by centrifugation to make cell suspension from the culture solution, and inoculated with a standard hemocytometer at 1 × 10 ⁵ cells per well and inoculated. After 24 hours, the culture medium was replaced, and after 48 hours, the culture medium was removed, and then washed with Hanks' balanced salt solution (Gibco). The chitosan beads prepared in Example 1 were sterilized and placed in each well to make 200 µl of the culture solution. These were incubated for 24 hours, and 50 µl of MTT (methylthiazole-2-YL-2,5-diphenyl tetrazolium bromide (Sigma)) dissolved in saline was added. The plate was shaken well and the absorbance was measured at 570 nm with an ELISA reader (THERMO max, Molecular devices, Bohannon, Calif., USA). The control group was used as a sample containing α-MEM culture wells, and all the experimental results were calculated as a percentage of the control group. The results are shown in FIG.

During the incubation period (7 days) cell activity was higher than the control (100%).

Experimental Example 5. Cell adhesion of chitosan beads for bone replacement

After placing the chitosan beads prepared in Example 1 in a 24 well plate, agarose was poured around the beads to fix the beads to prevent floating by addition of the culture. Twenty-four wells fixed with chitosan beads were sterilized for 10 minutes by UV radiation. Cell cultured crab cells were treated with 0.25% trypsin-EDTA (Gibco) solution, followed by centrifugation to form cell suspensions from the culture medium, and cultured according to the culture conditions by adding beads to 1 x 10 ⁶ cells per well using a standard hemocytometer. . In order to measure the number of cells proliferated and attached to chitosan beads, cells attached to the beads were detached by trypsin-EDTA treatment, suspended therein by adding α-MEN medium, and then a certain amount of isotonic solution was added, followed by osteoporosis in solution. The number of cells was calculated. The results are shown in FIG.

Approximately 10 ⁴ cell adhesion was shown. It can be seen that bone tissue regeneration proceeds as the bone cells adhere to the chitosan bead surface.

Experimental Example 6. Observation of adhesion pattern of chitosan beads for bone replacement

As follows, the state of the cells attached to the chitosan beads was observed under a differential scanning microscope. Cells attached to osteoblasts obtained by the operation in Experimental Example 4 were washed to remove unattached cells, and 25 (w / w)% glutaraldehyde was added to 0.1M phosphate buffered saline (PBS, pH 7.4). 2.5% of glutaraldehyde solution obtained by dilution was pre-fixed at 4 ° C. for 40 minutes. After pre-fixing, the resultant was washed with 0.1M phosphate-buffered saline solution, and then post-fixed at 0 ° C. for 40 minutes with 1% osmium tetrachloride solution obtained by dissolving osmium tetrachloride in 0.1M phosphate-buffered saline solution. After post-fixation, it was stored for 24 hours at -70 ℃ and lyophilized. The lyophilized samples were gold-palladium coated and observed under a differential scanning microscope. The results are shown in FIG.

The cytoplasm of most cells expanded radially and had a flat shape, and magnified observation showed that the attachment was stable within the initial 12 hours.

According to the present invention, chitosan adopts beads that form a skeleton, thereby allowing the growth of new bone into the gaps between the beads at the time of implantation into the bone defect, and also supporting the bone injury by containing tricalcium phosphate ceramic therein. The present invention provides chitosan beads for bone replacement, which can increase the bone regeneration effect by supplying calcium necessary for bone regeneration while having mechanical strength. In addition, it can be prepared by appropriately adjusting the size of the beads to be implanted in the bone defects of various sizes, by including an extracellular matrix in the bead can create an optimal environment that can effectively reproduce bone tissue.

Claims (11)

Chitosan beads for bone replacement containing tricalcium phosphate. The bone substitute chitosan beads according to claim 1, wherein the tricalcium phosphate is contained in an amount of 0.1 to 0.8 parts by weight based on the weight of the chitosan. The bone substitute chitosan beads according to claim 2, wherein the tricalcium phosphate is contained in an amount of 0.2 to 0.7 parts by weight based on the weight of the chitosan. The chitosan beads for bone replacement according to claim 1, wherein the beads have a size of 300 to 1200 µm. The chitosan beads for bone replacement according to claim 4, wherein the beads have a size of 400 to 800 µm. The chitosan bead for bone substitute according to claim 1, further comprising at least one component selected from the group consisting of a tissue growth factor, an extracellular matrix component, and a drug exhibiting a bone regeneration effect. The method of claim 6, wherein the tissue growth factor is at least one component selected from the group consisting of platelet-derived tissue growth factor, insulin-like tissue growth factor, epithelial cell growth factor, fibroblast growth factor and transforming growth factor. Substrate component is one or more components selected from the group consisting of collagen and chondroitin sulfate, and chitosan for bone replacement, wherein the drug showing the bone regeneration effect is one or more components selected from the group consisting of tetracycline, minocycline and flurbiprofen Bead. A method for producing chitosan beads for bone substitutes according to claim 1, wherein after preparing the chitosan solution, tricalcium phosphate is dispersed therein. The method according to claim 8, wherein after preparing the chitosan solution and dispersing the tricalcium phosphate therein, the mixture is compressed into compressed air and added dropwise to the stirring coagulant through a nozzle to form chitosan beads. A method of producing chitosan beads for bone replacement, comprising separating the beads from a coagulating solution and washing with water and lyophilizing the washed chitosan beads. The method of claim 8, further comprising dispersing the extracellular matrix component in the chitosan solution together with the tricalcium phosphate in the step of dispersing the tricalcium phosphate in the chitosan solution. The method of claim 8, further comprising the step of swelling and loading the prepared chitosan beads in a solution of the drug, which exhibits a tissue growth factor and a bone regeneration effect after the chitosan beads are manufactured. .