KR20080012479A - Injectable bone using autologous fibrin glue combined with nano-size polymers and method for the production thereof - Google Patents

Abstract

An injectable bone using autologous fibrin glue combined with nano-sized polymers and a production method for the same are provided to solve the problem of the autologous fibrin glue and to increase the hydrophile property of the material thereof. A method for the production of injectable bone using autologous fibrin glue combined with nano-sized polymers includes the steps of: preparing autologous fibrin gel; and mixing the autologous fibrin into nano-sized polymers. The injectable bone contains nano-sized polymers and the autologous fibrin glue. The injectable bone using autologous fibrin glue has the size that is able to be injected with a syringe. The autologous fibrin gel contains plaque, fibrinogen and thrombin. The size of the nano-sized polymer is 20-400nm. The polymer has the shape of nano-fiber or nano-particle.

Description

Injectable bone using autologous fibrin glue combined with nano-size polymers and method for the production approximately}

1 is an electron micrograph of an autologous blood gel containing a nano-sized polymer according to the present invention.

Figure 2 is a micrograph of the cultured bone cells cultured in the periosteum in the autologous blood gel containing a nano-sized polymer according to the present invention.

3 is an electron micrograph of a nanosize fiber prepared using a synthetic polymer.

Figure 4 is an electron micrograph of the nano-size particles produced using a synthetic polymer.

Figure 5 is a photograph of a comparative experiment applying the autologous blood gel containing nano- and macro-sized polymer according to the present invention to the titanium artificial tooth surface, respectively.

The present invention relates to the improvement and regeneration of repair of bone tissue and other tissues. More specifically, the present invention relates to autologous blood gels containing nano-sized polymers and methods for their preparation.

Bone grafts are often used to augment the natural regeneration process when bones have defects or injuries. These bone grafts should be biocompatible and ideally bone-forming, bone-conductive and bone-inductive, and should be easily manipulated by a surgeon prior to transplantation, and their strength and properties should be It must be able to remain intact.

The most commonly used method in biotissue engineering is a method of regenerating tissue using a three-dimensional porous support, a cell and a tissue regeneration factor. As a support body, the biodegradable polymer absorbed after a fixed time passes is used.

Recently, there has been a growing interest in tissue engineering methods using injectable gels. Tissue engineering methods using gels offer several advantages over conventional methods using three-dimensional porous scaffolds, which can be filled in any form of defects, and easily mix various components such as growth factors. Not only does it contain a variety of chemicals that are indispensable to the support, in particular, the gel form has the advantage that it can be easily injected without surgical procedures. For this reason, various injectable gel materials have been developed, and so far collagen gel, polyethylene oxide, alginate and the like have been used. However, they have been reported to have various disadvantages such as difficulty in controlling degradation rate, high shrinkage rate, difficulty in penetrating tissues, and immune response.

In addition, there are several patents which attempt to fill bone loss sites using bone putty, pastes and gels (US Pat. No. 5290558, US Pat. No. 50733733, US Pat. 5314476, US Pat. No. 6030635, US Pat. No. 7437018 and US Pat. No. 7067123 et al.). However, these patents do not disclose the use of nano-sized polymers for bone graft compositions with low shrinkage, adequate physical strength, and adequate dissolution rates.

Recently, fibrin gels obtained from blood have been commercialized and used in clinical practice. However, since these fibrin gels are manufactured using blood obtained from several people, there is a risk of infection or transmission of infectious diseases such as acquired immunodeficiency syndrome, and the use of bovine thrombin may cause hypersensitivity reactions such as clotting disorders. In addition to this, there is a difficulty in stopping full use when mad cow disease occurs. Therefore, platelet rich plasma using platelets of autologous blood is currently used in the clinic.

The present inventors prepared autologous blood gel by extracting fibrinogen and thrombin capable of producing high concentration of growth factor and high concentration of fibrin from autologous blood through preliminary studies, and tissue regeneration was performed using other biomaterials or platelets. It was confirmed that more effective than abundant plasma. If they are used as a biomaterial using the patient's own blood, there is no toxicity, excellent biocompatibility, does not cause an immune response, and has the advantage that it can be easily applied to the desired site as a gel form without surgical procedures or with minimal invasiveness. However, these autologous blood gels have disadvantages such as reduced volume due to shrinkage, inadequate physical strength, too fast decomposition rate, and the like.

Therefore, there is a constant need to develop autologous blood gels with low shrinkage, proper physical strength, and proper dissolution rate.

The present inventors prepared a bone graft material by mixing a nano-sized polymer in the autologous blood gel in order to solve the above problems of the prior art and autologous blood gel. When the bone graft material according to the present invention is used, not only the problem of autologous blood gel is solved, but also the hydrophilicity and adhesiveness of the material are increased to confirm that bone regeneration can be effectively performed at various types of bone destruction sites, and the present invention is completed. Was done.

The present invention provides a bone graft composition comprising a nano-sized polymer and autologous blood gel.

As the polymer used in the composition of the present invention, any polymer mainly used in biotissue engineering may be used, but is not limited thereto, and preferably, polylactic acid, polyglycolic acid, poly (lactic-co-glycolic acid). ), Poly (lactic-co-glycolic acid) -glucose, polyorthoester, polyanhydride, polydioxanone, polyamino acid, polyhydroxybutyric acid, polycaprolactone, polyalkylcarbonate, tricalcium phosphate and bio Glass or derivatives thereof, or mixtures of the above polymers can be used, more preferably tricalcium phosphate.

In the present invention, the term "nano size" is not limited to a specific range, but generally referred to in the art as referring to the size of tens or hundreds of nanometers. The polymer of the present invention has a nano size, preferably a diameter of several tens to several hundred nanometers, more preferably 20 to 400 nm.

The nano-sized polymer of the present invention may be prepared according to known methods or commercially available. Preferred nanosize synthetic polymers are nanoparticles or nanofibers.

In addition, the bone graft composition of the present invention comprises an autologous blood gel.

As used herein, the term “self” means that the tissue or cell originates or is derived from a recipient. The autologous blood gel is prepared using the blood of the patient himself and may include platelets, fibrinogen, and thrombin.

Platelets are a type of blood cell that is an important component of blood that plays an important role in blood coagulation and hemostatic action. The platelets are 2 to 3 ㎛ in diameter and 300,000 to 500,000 are present in 1 mm 2 of blood. Change accordingly. Platelet-induced growth factors such as platelet-derived growth factor (PDGF), transforming growth factor-beta (TGF-beta), and many other factors affect the healing process of bone. This is because they secrete growth factors.

Fibrinogen, also known as fibrinogen, is a protein in the globulin that plays a central role in blood coagulation. It is present in the plasma of vertebrates and is mainly produced in the liver. In humans, it contains 2 to 4 g in 1 L. Plasma concentration is easily increased and decreased physiologically and also in pathological cases such as leukocyte increase and fever, or infectious diseases such as tuberculosis and rheumatism fever, and when it increases, it increases not only the erythrocyte sedimentation rate but also the viscosity of blood. It has a lot of influence. Fibrinogen has the general properties of globulin, one of the most prone to salting out in plasma proteins, and almost all precipitates when the same volume of saturated saline is added. According to the electron microscope, the molecule has a dumbbell-like structure and has a molecular weight of about 330,000. The enzyme thrombin makes insoluble fibrin.

Thrombin, a proteolytic enzyme involved in blood coagulation, exists in the plasma as prothrombin. When bleeding causes platelets to break down and thromboplastin is released into the plasma, it is activated in the presence of calcium ions in the blood to become thrombin. It catalyzes the reaction of hydrolyzing soluble fibrinogen in the blood, which is the essence of blood coagulation, to change to insoluble fibrin. The substrate specificity for fibrinogen is very high and the four peptide bonds of fibrinogen are cut, between the arginine and glycine residues. The optimum pH is near neutral or slightly alkaline. Its main component is protein, which has a minimum molecular weight of 8000 and is easy to polymerize.

The bone graft composition according to the present invention may further include bone cells or growth factors for promoting growth of bone tissue. Examples of growth factors include cell attachment mediators, biologically active ligands, integrin binding sequences, bone morphogenic proteins, epidermal growth factor (EGF), fibroblast growth factor (FGF), and platelet-derived growth factor), IGF-1, IGF-2, TGF-β 1-3, vascular endothelial growth factor (VEGF), growth differential factor (GDF), and one or more growth factors may be included together.

In addition, the composition of the present invention may additionally include other biologically active agents other than growth factors. Such bioactive agents include, but are not limited to, organic molecules, inorganic substances, proteins, peptides, nucleic acids (eg, gene fragments, gene regulatory sequences and antisense molecules), nucleoproteins, polysaccharides, glycoproteins, and lipoproteins. Biologically active compounds that can be added to the compositions of the invention include, but are not limited to, anticancer agents, antibiotics, analgesics, anti-inflammatory agents, immunosuppressants, enzyme inhibitors, antihistamines, anticonvulsants, hormones, muscle relaxants, nutrients and vaccines, and the like. .

In the bone graft composition according to the present invention, the ratio of the autologous blood gel and the nano-sized polymer may vary depending on the purpose of use. Preferably, autologous blood gel may be included in an amount of 50% of the total weight of the composition. More preferably, autologous blood gels and nanosized polymers comprise 50% and 25% of the total weight of the composition, respectively.

The present invention also provides a method for producing a bone graft composition comprising the nano-sized polymer and autologous blood gel.

The bone graft manufacturing method comprises the steps of a) making an autologous blood gel; And b) mixing the gel with the nanosize polymer. As one preferred embodiment of the present invention, the method comprises the steps of: a) preparing thrombin at 1/5 of the volume of plasma solution obtained by collecting autologous blood and fibrinogen at 4/5 to make autologous blood gel; And b) mixing the autologous blood gel and the nanosized polymer. In another preferred embodiment, a) making an autologous blood gel; And b) mixing the autologous blood gel with a nano-sized polymer in an amount of 50% of the total weight of the composition.

The invention produced by the above method has the following features. In other words, by making the synthetic polymers nanoscale, the hydrophilicity and adhesiveness of the materials are increased and the surface area is increased, and the autologous blood gel used as a support for these nanoscale synthetic polymers is obtained from the blood of the patient's own body, and thus the immune response is increased. Not only does it occur, but platelets are included so that growth factors contained in platelets promote bone formation. In addition, the nano-sized synthetic polymer is well mixed with the autologous blood gel can be injected using a syringe so that the bone graft material can easily penetrate into the bone fracture site of various forms, thereby effectively regenerating bone.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the present invention is not limited thereto.

Example

Example  1: Preparation of Bone Graft Composition

As a specific embodiment of the present invention, the bone graft material was prepared by the following method. First, a certain amount of blood was collected to prepare autologous blood gel and centrifuged in a centrifuge. After centrifugation, the blood was divided into three layers, of which the lowest cell layer was discarded, and the platelet layer, which is a very light white intermediate layer, and the plasma layer, which was the supernatant, were taken together. The obtained plasma solution was divided by a ratio of 1: 4 to prepare thrombin using plasma corresponding to 1/5 and fibrinogen using plasma corresponding to 4/5. Thrombin was added to the plasma by adding 2.84 mM citric acid, and then centrifuged to discard the supernatant. A precipitate was obtained, and then, CaCl 2 (0.1M and 0.05M) was added to the precipitate to dissolve the precipitate, and 75 mM NaHCO 3 was added to the solution. The pH was adjusted to neutral. Fibrinogen was stored for 30 to 60 minutes at 0 ° C by adding 100 mg / ml of tranexaminc acid and 99% ethyl alcohol to the plasma, and centrifuged when the precipitate formed, and the supernatant was discarded. It was able to obtain by dissolving by placing at ℃.

The thrombin and fibrinogen prepared as described above were mixed immediately before using the autologous blood gel to prepare an autologous blood gel (fibrin gel).

After the fibrin was prepared as described above, the nano-sized polymer was prepared by dissolving the polymer solution in methylene chloride, which is a solvent, and then removing the solvent (Pharm Rel. 10, 750 (1993)), where fibrin, nanosized polymer and saline were mixed in a ratio of 2: 1: 1 and fibrin was added when the mixture was gelled.All reagents were Sigma, St. Louise, MO. The product was used.

Example  2 : Autologous blood gel  Measurement of Mixing Ratio of Nanopolymers

The volume change rate was measured according to the mixing ratio of autologous blood gel and nanopolymer. A quantity of gel was added to a 4 well flask and the volume change of the gels was measured under a condition of 5% CO 2, 37 ° C. in a culture vessel.

Volume change rate according to the mixing ratio of autologous blood gel and nanopolymer Autologous Blood Gel Volume of manufacture (μl) Volume after 10 days % Smaller 50% fibrin 450 375 17% 25% fibrin 420 280 33% 100% fibrin 480 430 15% 50% fibrin + 50% nanopolymer 240 220 9% 50% fibrin + 25% nanopolymer 340 300 12%

As can be seen in Table 1, when the nanopolymer is mixed in 50% autologous blood gel, there is little change in volume.

Example  3: Autologous blood gel  Effect of Cell Proliferation on Gel According to the Mixing Ratio of Nanopolymers

Bone cells cultured in the periosteum were cultured with a mixture of autologous blood gel and nanopolymer to measure the proliferative effect of the cells (FIG. 2).

400 μl of the mixture and 2 × 10 ⁵ osteocytes in a 4 well flask were placed in a culture vessel with 5% CO 2 and minimal essential medium (MEM) at 37 ° C. Incubate for 1 week using fetal serum (FBS, Gibco) and the addition of antibiotics. Cell proliferation after culture was observed by electron microscopy. In Figure 2 it can be seen that the appearance of living bone cells in the gel, it can be seen that the bone cells proliferate in the mixture of autologous blood gel and nanopolymers.

Example  4: hydrophilicity, adhesion of bone graft material, Adhesion  effect

The mixture of autologous blood gel and nanopolymer was applied to the titanium artificial tooth surface and compared with autologous blood gel and macro-sized polymer (Fig. 5).

A mixture of nanosized tricalcium phosphate polymers and macrosized tricalcium phosphate polymers with 50% autologous blood gel, injected into the surface of artificial teeth, wait 48 hours, and then cut into artificial teeth with polymer and highly curved surfaces. The contact area with the surface was compared. As can be seen in Figure 5, since the nano-sized polymer shows a larger contact area than the macro-sized polymer, it can be seen that the nano-sized polymer has excellent adhesion and adhesion to the artificial tooth surface.

The bone graft material comprising the nano-sized polymer and the autologous blood gel of the present invention, unlike the macro-sized bone graft material, is excellent in hydrophilicity, adhesiveness, and adhesion, so that bone grafts can be effectively filled in various types of bone fracture sites. It can be easily injected using a syringe when applied to a bone defect, with minimal or no tissue incision and detachment.

In addition, the bone graft material including the nano-sized polymer and autologous blood gel of the present invention, unlike other gelling materials can not only prevent immune response problems, but also contains a large amount of growth factors to help bone regeneration, thereby regenerating bone Can shorten the healing period.

In addition, the bone graft material comprising the nano-sized polymer and autologous blood gel of the present invention can be used as a scaffold in the preparation of bone, cartilage or skin substitute using tissue engineering.

Claims (16)

A bone graft composition comprising a nano-sized synthetic polymer and autologous blood gel. A bone graft composition comprising a synthetic polymer and autologous blood gel of a size that can be injected into a syringe. The bone graft composition according to claim 1 or 2, wherein the autologous blood gel comprises platelets, fibrinogen, and thrombin. The method of claim 1 or 2, wherein the synthetic polymer is polylactic acid, polyglycolic acid, poly (lactic-co-glycolic acid), poly (lactic-to-glycolic acid) -glucose, polyorthoester, poly Anhydride, polydioxanone, polyamino acid, polyhydroxybutyric acid, polycaprolactone, polyalkylcarbonate, tricalcium phosphate, bioglass and one or more mixtures selected from the group consisting of derivatives thereof A bone graft composition. The bone graft composition according to claim 1 or 2, wherein the size of the synthetic polymer is 20 to 400 nm. The bone graft composition of claim 1 or 2, wherein the synthetic polymer is in the form of nanofibers or nanoparticles. The bone graft composition according to claim 1 or 2, wherein the composition further comprises bone cells. The bone graft composition of claim 1 or 2, wherein the composition further comprises a growth factor. 8. The method of claim 7, wherein the growth factor is cell attachment mediator, biologically active ligand, integrin binding sequence, bone morphogenic protein, epidermal growth factor, EGF, fibroblast growth factor, PDGF (platelet-derived growth factor), IGF-1, IGF-2, TGF-β 1 to 3, vascular endothelial growth factor (VEGF) and growth differential factor (GDF), characterized in that the bone graft composition. The bone graft composition of claim 1, wherein the composition is injectable with a syringe. The bone graft composition according to claim 1 or 2, wherein the autologous blood gel is contained in 50% by weight of the total composition. a) preparing an autologous blood gel; And b) mixing the gel and the nano-sized polymer. The method for producing a bone graft composition according to claim 12, wherein thrombin is produced by 1/5 of the volume of plasma solution obtained by collecting blood, and fibrinogen is produced by 4/5 to prepare autologous blood gel. 13. The method of claim 12, wherein the autologous blood gel comprises platelets, fibrinogen and thrombin. The method of claim 12, wherein the autologous blood gel is mixed to contain 50% by weight of the total composition. The method of claim 12, wherein the synthetic polymer is selected from the group consisting of polylactic acid, polyglycolic acid, poly (lactic-co-glycolic acid), poly (lactic-co-glycolic acid) -glucose, polyorthoesters, polyanhydrides, A method for producing a bone graft composition, characterized in that selected from polydioxanone, polyamino acid, polyhydroxybutyric acid, polycaprolactone, polyalkyl carbonate, tricalcium phosphate and bioglass, or a derivative or mixture thereof.

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