KR102738265B1 - Polymer scaffold containing incorporated BMP/PDRN nanoparticle for bone regeneration and preparation method thereof - Google Patents

Abstract

The present invention relates to a polymer scaffold for bone regeneration with a BMP/PDRN nanocomposite fixed thereon and a method for manufacturing the same. The polymer scaffold comprises a biodegradable polymer, thereby not only improving the low biocompatibility of a hydrophobic bioimplant, but also enhancing bone regeneration through the promotion of angiogenesis and endothelialization at the transplant site by the BMP/PDRN nanocomposite.

Description

Polymer scaffold containing incorporated BMP/PDRN nanoparticle for bone regeneration and preparation method thereof

The present invention relates to a polymer scaffold for bone regeneration with a BMP/PDRN nanocomposite fixed thereon and a method for manufacturing the same.

One of the goals of tissue engineering is to replace damaged tissue functions, and to do so, it is important to manipulate the interaction between biomaterials as a support and cells and growth factors to create an environment in which tissues or organs can regenerate. In addition, it must be non-toxic and biocompatible so that blood clotting or inflammatory reactions do not occur after transplantation.

Meanwhile, biodegradable synthetic polymers generally used as support materials are hydrolyzed in the body and completely decompose after a certain period of time. However, these synthetic polymers have a problem in that they are hydrophobic and thus have low biocompatibility. In addition, they have relatively poor physical properties compared to other polymers, and when they are biodegraded, acidic substances such as lactic acid and glycolic acid are generated, which causes inflammatory reactions and cytotoxicity in the human body. In order to solve the above problems, there is a method of suppressing the generation of acidic substances at the source by containing basic ceramic particles in biodegradable polymers, and bone regeneration support materials using natural polymers are being studied. However, it is difficult to control the support decomposition period, and there are problems such as deposition of decomposition products due to rapid decomposition rates.

Therefore, it is necessary to develop a polymer support that can protect bioactive substances for a certain period of time in the body and control the release rate of bioactive substances through immobilization of bioactive substances using biocompatible substances.
(Patent Document) Domestic Publication No. 10-2018-0047423

One aspect is to provide a polymer scaffold for bone regeneration with BMP/PDRN nanocomposites immobilized thereon.

Another aspect provides a method for preparing a polymer scaffold for bone regeneration with BMP/PDRN nanocomposite immobilized thereon.

One aspect provides a polymer scaffold for bone regeneration in which a nanocomposite including bone morphogenetic protein (BMP) and a bioactive substance is fixed to a porous scaffold including a biodegradable polymer, basic ceramic particles and an extracellular matrix.

In this specification, the term "polymer scaffolds" is an important element of tissue engineering, and serves as a kind of framework for forming a tissue in which cells are fused to regenerate damaged tissue. Since damaged tissue has an atypical damage form, there is a limit to regenerating the damaged area by simply injecting cells, and thus, it serves a role in enabling cells to settle, survive, and proliferate for effective tissue regeneration. In one specific example, the polymer scaffold may be a porous scaffold including a biodegradable polymer, basic ceramic particles, and an extracellular matrix, to which a nanocomposite including a bone formation protein and a bioactive substance is fixed. Since the polymer scaffold has a nanocomposite including a bone formation protein and a bioactive substance fixed on the porous scaffold, the release rate of the bone formation protein and the bioactive substance in vivo can be controlled.

The above bone morphogenetic protein (BMP) is a growth factor that induces the formation of bone and cartilage, and may be, for example, BMP1, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP10, BMP11, BMP15, etc.

The above-mentioned physiologically active substance may be a substance derived from DNA that promotes or inhibits physiological activity of a living organism to maintain life, and may be, for example, polydeoxyribonucleotide (PDRN), polynucleotide (PN), hydrolyzed DNA, etc. In one specific example, the PDRN may be a short deoxyribonucleotide polymer (DNA fragment of 50 to 2,000 bases) having a molecular weight of 50 to 1,500 KDa. In general, the physiologically active substance is directly injected into the living organism separately from the support when the polymer support is transplanted into the living organism. In this case, there is a problem that the physiologically active substance must be treated multiple times to maintain an appropriate concentration in the living organism due to the short half-life of the physiologically active substance and its rapid removal by enzymes existing in the living organism. In addition, when a bioactive substance is treated at a high concentration, there is a problem that the biocompatibility of the polymer support decreases due to cytotoxicity. However, the polymer support according to one aspect can maintain the release rate in the body constant for a long time because the bioactive substance is fixed on the porous support, so the inconvenience of having to separately administer the bioactive substance can be reduced, and side effects such as the initial release of all the encapsulated drugs can be prevented.

The term "nanoparticle" as used herein refers to a nanoparticle formed by ionic bonding of a bone formation protein and a bioactive substance, which may have a size of 10 to 1,000 nm. The nanocomposite may be fixed to the surface of a porous support including a biodegradable polymer, basic ceramic particles, and an extracellular matrix. In one specific example, the nanocomposite may include the bone formation protein and the bioactive substance in a weight ratio of 1:0.5 to 5. The bone formation protein and the bioactive substance may be mixed in a weight ratio of, for example, 1:0.5 to 5, 1:0.5 to 4.5, 1:0.5 to 4, 1:0.5 to 2, 1:1 to 4, 1:1 to 3, or 1:1.5 to 2.5. At this time, if the mixing ratio of the bone formation protein and the physiologically active substance is below or exceeds the above range, there is a problem that it is difficult to form a nanocomposite.

In another specific example, the nanocomposite may be included (immobilized) at a concentration of 0.1 to 10 ppm. The nanocomposite may be included at a concentration of 0.1 to 10 ppm, 0.1 to 8 ppm, 0.5 to 5 ppm, 1 to 4 ppm or 2 to 3 ppm. In this case, if the content of the nanocomposite is less than the above range, there is a problem that the physiological activity promoting or inhibiting effect by the physiologically active substance cannot be sufficiently exerted, and if it exceeds the above range, there is a problem that cytotoxicity may occur.

The biodegradable polymer may be, for example, polylactide, polyglycolide, polycaprolactone, polylactide-co-glycolide, polylactide-co-caprolactone, polyglycolide-co-caprolactone, polydioxanone, polydioxanone, polytrimethylene carbonate, polyglycolide-co-dioxanone, polyamide ester, polypeptide, polyolthoester, polymaleic acid, polyanhydride, polysebacinhydride, polyhydroxyalkanoate, polyhydroxybutyrate, polycyanoacrylate, or the like. In one specific example, the biodegradable polymer may be included in an amount of 5 to 100 wt% based on the total weight of the composition. The biodegradable polymer may be included in an amount of, for example, 5 to 100 wt%, 5 to 80 wt%, 5 to 50 wt%, 10 to 90 wt%, 15 to 70 wt%, 30 to 80 wt%, or 40 to 50 wt% based on the total weight of the composition. In this case, if the content of the biodegradable polymer is less than the above range, there is a problem in that the thermal stability and mechanical properties of the polymer support decrease. In another specific example, the number average molecular weight (Mn) of the biodegradable polymer may be 5,000 to 3,000,000 g/mol. The number average molecular weight of the biodegradable polymer may be, for example, 7,000 to 2,800,000 g/mol, 8,000 to 2,500,000 g/mol, or 10,000 to 2,000,000 g/mol.

The basic ceramic particles may be, for example, magnesium hydroxide, lithium hydroxide, beryllium hydroxide, sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium oxide, lithium oxide, beryllium oxide, sodium oxide, potassium oxide, calcium oxide, calcium carbonate, calcium carbonate, hydroxyapatite (HAp), tricalcium phosphate (β-TCP), magnesium ceramic, etc. In one specific example, the basic ceramic particles may be surface-modified. The basic ceramic particles may be surface-modified with a fatty acid or a polymer. The above fatty acids may be, for example, caprylic acid, capric acid, lauric acid, myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, elaidinic acid, vaccenic acid, linoleic acid, ricinoleic acid, linoleic acid, α-linoleic acid, arachidonic acid, eicosapentaenoic acid, erucic acid, stearic acid, DHA, octatecanoic acid, coconut oil, palm oil, cottonseed oil, safflower oil, soybean oil, olive oil, corn oil, sunflower oil, safflower oil, hemp oil, canola oil, and the like. In addition, the polymer is at least one monomer selected from the group consisting of, for example, L-lactide, D-lactide, D,L-lactide, glycolide, caprolactone, dioxanone, trimethylene carbonate, hydroxyalkanoate, peptide, cyanoacrylate, lactic acid, glycolic acid, hydroxycaproic acid, maleic acid, phosphazene, amino acid, hydroxybutyric acid, sebacic acid, hydroxyethoxyacetic acid and trimethylene glycol, such as poly-L-lactide, poly-D-lactide, poly-D,L-lactide, polyglycolide, polycaprolactone, poly-L-lactide-co-glycolide, poly-D-lactide-co-glycolide, poly-D,L-lactide-co-glycolide, poly-L-lactide-co-caprolactone, It may be poly-D-lactide-co-caprolactone, poly-D,L-lactide-co-caprolactone, polyglycolide-co-caprolactone, polydioxanone, polytrimethylene carbonate, polyglycolide-co-dioxanone, polyamide ester, polypeptide, polyorthoester, polymaleic acid, polyphosphazene, polyanhydride, polysebacinhydride, polyhydroxyalkanoate, polyhydroxybutylate, polycyanoacrylate, etc. In another specific example, the diameter of the basic ceramic particles or the surface-modified basic ceramic particles may be 1 nm to 1 mm. At this time, when the diameter of the basic ceramic particles exceeds the above range, there is a problem that precipitation occurs due to the weight of the basic ceramic particles, causing phase separation in the organic solvent.

In another specific example, the basic ceramic particles may be included in an amount of 5 to 50 wt% based on the total weight of the composition. The basic ceramic particles may be included in an amount of, for example, 5 to 50 wt%, 5 to 45 wt%, 5 to 30 wt%, 10 to 40 wt%, 10 to 30 wt%, 20 to 40 wt%, or 10 to 25 wt%. At this time, if the content of the basic ceramic particles is less than the above range, the acidic substance, which is a decomposition product of the polymer support, cannot be neutralized, which causes a problem of an inflammatory response and cytotoxicity in a living body, and if it exceeds the above range, there is a problem of inducing alkalinization of the environment around the polymer support.

The above extracellular matrix is a matrix protein derived from the tissue or cell of a human or animal, and may be in a complexly mixed state or an artificially separated single molecule state, and may have a protein with a denatured structure. The above extracellular matrix may be derived from, for example, vertebrates such as humans, pigs, cows, mice, sheep, horses, dogs or cats, and may be separated from bone, kidney, amniotic membrane, skin, brain, small intestinal submucosa, fascia or spinal cord meninges depending on the purpose. The above extracellular matrix may be appropriately selected depending on the structure or function. In one specific example, the above extracellular matrix may be a group having a fibrous structure, a group related to bone differentiation and osteogenesis, a glucosaminoglycan group or a proteoglycan group, etc. The group having a fibrous structure may be, for example, collagen fibers, elastin fibers, laminin, fibrinogen, fibronectin, gelatin, etc. At this time, the collagen may be, by type, I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIV, XV, XVI, XVIII, XIX, XX, XXI, XXII, XXIII, XXIV, XXV, XXVI, XXVII, XXVIII collagen, etc. In addition, the group related to the bone differentiation and osteogenesis may be, for example, osteonectin, osteopontin, vitronectin, vimentin, etc. In addition, the glucosaminoglycan group may be, for example, heparan sulfate, keratan sulfate, chondroitin sulfate, dermatan sulfate, heparin, low molecular weight heparin, hyaluronic acid, etc. In addition, the proteoglycan group may be, for example, decorin, biclycan, versican, tertican, perlecan, vicunin, neurocan, aggrecan, fibromodulin, lumican, etc. In another specific example, the extracellular matrix may be included in an amount of 0.1 to 20% with respect to the total weight of the polymer scaffold. The extracellular matrix may be included in an amount of, for example, 0.1 to 20 wt%, 0.1 to 15 wt%, 1 to 20 wt%, 1 to 15 wt%, 5 to 20 wt%, 5 to 15 wt%, 5 to 10 wt%, or 10 to 20 wt% with respect to the total weight of the polymer scaffold. At this time, when the content of the extracellular matrix is less than the above range, there is a problem that the effect of improving cell compatibility cannot be sufficiently exhibited, and when it exceeds the above range, there is a problem that the mechanical properties of the polymer scaffold decrease. In another specific example, the extracellular matrix may be decellularized, and may be decellularized by a physical or chemical method after culturing tissues or cells. Examples of physical decellularization methods include freeze-thaw, sonication, physical stirring, and the like, and examples of chemical decellularization methods include treating powder of animal-derived tissue with a storage solution containing water, anionic surfactant, nonionic surfactant, cationic surfactant, DNase, RNase, or trypsin. In the chemical decellularization method, a Tris-HCl (pH 8.0) solution may be used as the storage solution, and the anionic surfactant may be sodium dodecyl sulfate (SDS), sodium deoxycholate, Triton X-200, or the like. In addition, as the nonionic surfactant, Triton X-100, Tween 20 or Tween 80 can be used, and as the cationic surfactant, CHAPS, Sulfobetaine-10 (SB-10), Sulfobetaine-16 (SB-16), Tri-n-butyl phosphate, N-lauroyl-sarcosinate, IGEPAL CA-630, etc. can be used. In addition, the decellularization can be performed before or after performing the powdering process after collecting animal tissue, for example, bone tissue, or simultaneously with the powdering process. In another specific embodiment, the extracellular matrix may be a pulverized bone (mineral) from which bone cells have been removed, and the bone powder may be derived from one or more types of bone selected from the group consisting of autologous bone, allogeneic bone, xenogeneic bone, and synthetic bone (e.g., hydroxyapatite).

In one embodiment, a polymer scaffold for bone regeneration, in which a nanocomposite including a bone morphogenetic protein and a polydeoxyribonucleotide is fixed to a porous support including a biodegradable polymer, basic ceramic particles, and an extracellular matrix, was transplanted into a rat model with a calvarial defect, and it was confirmed that new blood vessels were formed and bone was regenerated at the defect site, thereby confirming that a bone tissue regeneration function can be implemented. Therefore, the polymer scaffold according to one aspect can be used for bone tissue regeneration by containing a bone morphogenetic protein and a polydeoxyribonucleotide, and can treat and regenerate damaged bone through growth, differentiation, and fusion processes via organic signal transmission with surrounding cells at the damaged site.

Another aspect provides a method for producing a polymer scaffold for bone regeneration, comprising the steps of: preparing a polymer solution by mixing a biodegradable polymer, basic ceramic particles, an extracellular matrix, and a solvent; preparing a porous scaffold by mixing a porous inducer into the polymer solution; and immobilizing a nanocomposite containing a bone-forming protein and a bioactive substance onto the porous scaffold. The specific contents of the polymer scaffold are as described above.

In the step of preparing the above polymer solution, the solvent may be, for example, alcohols such as methanol, ethanol, propanol, and butanol; aldehydes such as ammonia, dimethyl sulfoxide, dimethylformamide, acetronitrile, tetrahydrofuran, formaldehyde, glutaraldehyde, and acetaldehyde; alkanes such as dioxane, chloroform, heptane, hexane, pentane, octane, nonane, and decane; benzene ring-type solvents such as benzene, toluene, and xylene; ethers such as ether, dipropyl ether, petroleum ether, and methyl-t-butyl ether; ketones such as propanone, butanone, pentanone, hexanone, and heptanone; and common organic solvents such as methylene chloride, tetrafluoroisopropane, and carbon tetrachloride.

In the step of manufacturing the above porous support, the pore inducer may be ice particles. The pore size and porosity of the porous polymer support can be controlled by controlling the size and content of the ice particles. The size of the ice particles may be 10 to 500 ㎛. The size of the ice particles may be, for example, 10 to 500 ㎛, 10 to 450 ㎛, 10 to 400 ㎛, 10 to 300 ㎛, 30 to 500 ㎛, 30 to 300 ㎛, 30 to 250 ㎛, 50 to 500 ㎛, 50 to 400 ㎛, 50 to 300 ㎛, 50 to 200 ㎛, 100 to 500 ㎛, 100 to 300 ㎛, or 150 to 200 ㎛. At this time, if the size of the ice particles is less than the above range, there is a problem that the cell infiltration rate is low and the angiogenesis effect cannot be sufficiently exhibited, and if it exceeds the above range, there is a problem that the mechanical properties of the polymer support are reduced. In addition, the ice particles may be included in an amount of 100 to 2000 wt% with respect to the total weight of the polymer solution. The ice particles may be included in an amount of 100 to 2000 wt%, 100 to 1500 wt%, 100 to 1300 wt%, 100 to 1000 wt%, 100 to 500 wt%, 500 to 2000 wt%, 500 to 1500 wt%, 500 to 1000 wt%, 1000 to 2000 wt%, or 1500 to 2000 wt% with respect to the total weight of the polymer solution. At this time, if the content of ice particles is below the above range, there is a problem that pores are not sufficiently formed inside the polymer support, and if it exceeds the above range, there is a problem that the mechanical properties of the polymer support decrease. In addition, by manufacturing the polymer support by the freeze-drying method using the ice particles, it is possible to prevent the detachment of basic ceramic particles and extracellular matrix that may occur in the conventional salt forming method.

The step of immobilizing the nanocomposite including the bone formation protein and the bioactive substance on the porous support may be performed in an ultra-high concentration calcium-phosphorus solution. In one specific example, the concentration of the calcium-phosphorus solution may be 1 to 100 mM. The step of immobilizing the nanocomposite including the bone formation protein and the bioactive substance on the porous support may be such that the nanocomposite having a negative charge and a cation present in the calcium-phosphate ion solution is fixed to the porous support by electrostatic attraction. Specifically, the nanocomposite and an inorganic component such as calcium present in the calcium-phosphate ion solution may be deposited and fixed on the porous support. That is, the nanocomposite according to one aspect is fixed on the porous support, so that the initial and short-term release of the bioactive substance can be suppressed, and thus the removal of the bioactive substance by various enzymes present in the body can be prevented. In addition, since the release rate of the bioactive substance can be controlled, the stability and sustainability of the bioactive substance can be maintained, and suitability for the transplantation site can be ensured.

According to one aspect, the polymer scaffold can improve the low biocompatibility of hydrophobic bioimplant by including a biodegradable polymer, and can also promote angiogenesis and endothelialization of the transplant site by the BMP/PDRN nanocomposite. In addition, since the BMP/PDRN nanocomposite is fixed to the porous scaffold, the release amount and speed of the bioactive substance can be controlled.

Figure 1 is a schematic diagram of a polymer support according to one aspect.
Figure 2 shows the results of measuring the biocompatibility of a polymer support according to various aspects.
Figures 3a and 3b show the effect of polymer scaffolds on angiogenesis according to one aspect.
Figure 4 shows the effect of polymer scaffolds on bone regeneration according to the daily aspect.

Hereinafter, preferred examples are presented to help understand the present invention. However, the following examples are provided only to help understand the present invention more easily, and the content of the present invention is not limited by the following examples.

[Manufacturing example]

Manufacturing Example 1. Manufacturing of a nanocomposite containing bone-forming protein and bioactive substance

10 μg of Born Morphogenetic Protein 2 (BMP2) and 10 μg of polydeoxyribonucleotide (PDRN), a bioactive substance derived from DNA, were dissolved in 10 mM NaCl and reacted at 4°C for 1 hour to induce ionic bonding. Afterwards, unreacted materials were removed by centrifugation to produce a BMP/PDRN nanocomposite.

Manufacturing Example 2. Manufacturing of basic ceramic particles

2-1. Manufacturing of magnesium hydroxide particles

A sodium hydroxide solution was prepared by dissolving 10.8 g of sodium hydroxide in 300 ml of distilled water. A magnesium nitrate solution was prepared by dissolving 20 g of magnesium nitrate in 150 ml of distilled water. Thereafter, a reaction solution was prepared by dropping the magnesium nitrate solution into the sodium hydroxide solution at a rate of 30 drops/min using a dropping funnel. Thereafter, while flowing distilled water into the prepared reaction solution, the precipitated nano magnesium hydroxide particles were purified and then filtered to obtain nano magnesium hydroxide particles. The obtained nano magnesium hydroxide particles were vacuum-dried and stored.

2-2. Preparation of magnesium hydroxide particles surface-modified with fatty acids

Magnesium hydroxide particles surface-modified with ricinoleic acid were prepared. Specifically, 10 wt% of the magnesium hydroxide particles prepared in Manufacturing Example 2-1 and 90 wt% of ricinoleic acid were mixed based on the total weight of the mixture, and stirred for 12 hours at a glass reactor temperature of 70°C under a nitrogen atmosphere. Thereafter, ethanol was flowed into the stirred solution to remove unreacted substances, followed by purification and filtration to obtain magnesium hydroxide particles surface-modified with ricinoleic acid. The obtained fatty acid surface-modified magnesium hydroxide particles were vacuum-dried and stored.

2-3. Preparation of magnesium hydroxide particles surface-modified with polymer

Magnesium hydroxide particles surface-modified with L-lactide were prepared. Specifically, 80 wt% of the magnesium hydroxide particles prepared in Manufacturing Example 2-1 and 20 wt% of L-lactide based on the total weight of the mixture were mixed to prepare a reaction solution. Thereafter, 0.05 wt% of tin octoate (catalyst) based on the total weight of the reaction solution (magnesium hydroxide and L-lactide) was diluted in toluene and added, and the glass reactor containing the reaction solution was maintained at 70°C in a vacuum for 6 hours while stirring to completely remove toluene and moisture. Thereafter, the sealed glass reactor was stirred in an oil bath controlled to 150°C while performing a ring-opening polymerization reaction for 48 hours to prepare a polymer. After recovering the polymer, it was placed in a sufficient amount of chloroform solution for more than 1 hour to remove the homopolymer and unreacted residues, thereby producing magnesium hydroxide surface-modified with L-lactide.

2-4. Preparation of magnesium hydroxide particles surface-modified with fatty acids and polymers

Magnesium hydroxide particles surface-modified with ricinoleic acid and L-lactide were manufactured in the same manner as in Manufacturing Example 2-3, except that the magnesium hydroxide particles surface-modified with ricinoleic acid manufactured in Manufacturing Example 2-2 were used.

Manufacturing Example 3. Manufacturing of decellularized and powdered extracellular matrix

3-1. Production of extracellular matrix derived from bovine bone

Cortical bone of a cow was treated with 83% ethanol and 6% hydrogen peroxide solution to remove bone marrow and lipids. Thereafter, the bone from which the bone marrow and lipids were removed was cut into a block shape, and then further crushed and powdered using a freeze grinder. 15 ml of purified water was added per 1 g of bone powder, the bone powder was washed for 10 minutes, and the process of exchanging the purified water was repeated 3 times to remove debris from the bone powder. Thereafter, the bone powder from which the debris had been removed was placed in a stirrer, 15 ml of 0.6 N HCl solution was added per 1 g of bone powder, and the mixture was stirred at 24°C and 200 rpm for 6 hours, and washing was repeated until the pH of the stirred solution became 6.0 or higher. Thereafter, the stirred solution was disinfected by treating 83% ethanol and 6% hydrogen peroxide, the supernatant was removed, and the mixture was frozen and stored at -70°C for 12 hours to induce decellularization of the extracellular matrix. The decellularized extracellular matrix was freeze-pulverized to a size of approximately 50 μm using a freeze-pulverizer and then powdered to produce bone-derived extracellular matrix.

3-2. Preparation of extracellular matrix derived from bovine skin

After collecting bovine skin tissue, it was washed three times for 10 minutes using saline solution. After dehydrating the washed tissue with ethanol, it was placed in 1 L of 0.1% SDS solution per 10 g of tissue and stirred at 100 rpm for 24 hours. Thereafter, it was washed five times for 30 minutes at 100 rpm using distilled water, and 200 mL of DNAse at a concentration of 200 U/㎖ was added and stirred at 100 rpm for 24 hours at 37°C to remove adipocytes and genetic components present in the tissue. Thereafter, it was washed five times for 30 minutes at 100 rpm using distilled water, and the decellularized pure extracellular matrix from which the adipocytes and genetic components were removed was freeze-dried. Thereafter, the extracellular matrix was freeze-pulverized into particles of about 50 ㎛ in size using a freeze-pulverizer and then powdered to prepare extracellular matrix derived from bovine skin.

Pure extracellular matrix was obtained by decellularization by removing fat cells and genetic components present in the tissue.

3-3. Production of extracellular matrix derived from human skin

Human skin extracellular matrix was prepared in the same manner as in Manufacturing Example 3-2, except that human skin tissue was used.

3-4. Preparation of human adipose derived extracellular matrix

Human adipose-derived extracellular matrix was prepared in the same manner as in Manufacturing Example 3-2, except that human adipose tissue was used.

3-5. Preparation of extracellular matrix derived from pig kidney

A porcine kidney-derived extracellular matrix was prepared in the same manner as in Manufacturing Example 3-2, except that porcine kidney tissue was used.

3-6. Preparation of extracellular matrix derived from rat brain

A rat brain-derived extracellular matrix was prepared in the same manner as in Manufacturing Example 3-2, except that rat brain tissue was used.

[Example]

Example 1. Preparation of polymer scaffold with BMP/PDRN nanocomposite fixed thereon (1)

A polymer scaffold having BMP/PDRN nanocomposites fixed thereon was prepared. Specifically, 20 wt% of the magnesium hydroxide particles surface-modified with fatty acids of Preparation Example 2-2 and 50 wt% of the bone-derived extracellular matrix of Preparation Example 3-1 were mixed with an organic solvent based on the total weight of polylactide-co-glycolide (PLGA) (50:50) (molecular weight: 110,000 Da), to prepare a polymer solution. Thereafter, ice particles having a size of 100 to 200 ㎛ were uniformly mixed into the polymer solution, and then freeze-dried for 48 hours using a Teflon mold to prepare a porous scaffold. The porous scaffold was sterilized by immersion in 70% ethanol, washed with sterile distilled water to remove ethanol, and hydrated by immersion in physiological saline. Thereafter, the BMP/PDRN nanocomposite of Manufacturing Example 1-1 was fixed to the porous polymer scaffold through a polarity-based precipitation method in a calcium-phosphorus saturated solution to manufacture a polymer scaffold for bone regeneration.

Example 2. Preparation of polymer scaffold with BMP/PDRN nanocomposite fixed (2)

A polymer scaffold for bone regeneration was prepared in the same manner as in Example 1, except that polylactide-co-glycolide (PLGA) (50:50) (molecular weight: 40,000 Da) was used.

Example 3. Preparation of polymer scaffold with BMP/PDRN nanocomposite fixed (3)

A polymer scaffold for bone regeneration was prepared in the same manner as in Example 1, except that polylactide-co-glycolide (PLGA) (75:25) (molecular weight: 110,000 Da) was used.

[Comparative example]

Comparative Example 1. Preparation of polymer support without basic ceramic particles (1)

A polymer scaffold that does not contain basic ceramic particles was prepared. Specifically, 50 wt% of the bone-derived extracellular matrix of Manufacturing Example 3-1 was mixed with an organic solvent based on the total weight of polylactide (PLA) (molecular weight: 150,000 Da) to prepare a polymer solution. Thereafter, a polymer scaffold for bone regeneration that does not contain basic ceramic particles and has BMP/PDRN nanocomposites fixed thereto was prepared in the same manner as in Example 1.

Comparative Example 2. Preparation of polymer support without extracellular matrix (2)

A polymer scaffold that does not include an extracellular matrix was prepared. Specifically, 20 wt% of the magnesium hydroxide particles surface-modified with the fatty acid of Manufacturing Example 2-2 was mixed with an organic solvent based on the total weight of polycaprolactone (PCL) (molecular weight: 100,000 Da), to prepare a polymer solution. Thereafter, a polymer scaffold for bone regeneration that does not include an extracellular matrix and has a BMP/PDRN nanocomposite fixed thereto was prepared in the same manner as in Example 1.

Comparative Example 3. Preparation of polymer scaffold without BMP/PDRN nanocomposite immobilization

A polymer scaffold without BMP/PDRN nanocomposites was prepared. Specifically, 20 wt% of the magnesium hydroxide particles surface-modified with fatty acids of Preparation Example 2-2 and 50 wt% of the bone-derived extracellular matrix of Preparation Example 3-1 were mixed with an organic solvent based on the total weight of polylactide-co-caprolactone (PLC) (70:30) (molecular weight: 100,000 Da), to prepare a polymer solution. Thereafter, ice particles having a size of 100 to 200 ㎛ were uniformly mixed into the polymer solution, and then freeze-dried for 48 hours using a Teflon mold to prepare a porous scaffold. The porous scaffold was sterilized by immersion in 70% ethanol, washed with sterile distilled water to remove ethanol, and hydrated by immersion in physiological saline.

Comparative Example 4. Preparation of biodegradable polymer support (1)

Polylactide-co-glycolide (PLGA) (50:50) (molecular weight: 110,000 Da) was mixed with an organic solvent to prepare a polymer solution. Thereafter, ice particles of 100 to 200 μm in size were uniformly mixed into the polymer solution, and then freeze-dried for 48 hours using a Teflon mold to prepare a porous support. The porous support was sterilized by immersion in 70% ethanol, washed with sterilized distilled water to remove ethanol, and hydrated by immersion in saline solution.

Comparative Example 5. Preparation of biodegradable polymer support (2)

A biodegradable polymer support was prepared in the same manner as in Comparative Example 4, except that polylactide-co-glycolide (PLGA) (50:50) (molecular weight: 40,000 Da) was used.

[Experimental example]

Biocompatibility measurement

In order to evaluate the biocompatibility of polymer supports immobilized with BMP/PDRN nanocomposites according to the aspect of the invention, human-derived mesenchymal stem cells were cultured on the polymer supports of Examples 1 to 3 and Comparative Examples 3 to 5. Thereafter, cell growth rate and cytotoxicity were analyzed by staining with CCK-8 and Calcein AM/EthD-1, respectively.

Example 1 Example 2 Example 3 Comparative Example 3 Comparative Example 4 Comparative Example 5 Biocompatibility ○ ○ ○ △ X X

○: Cell death less than 10%, △: Cell death 10-30%, X: Cell death 30% or more

As shown in Table 1, it was confirmed that Examples 1 to 3 showed cell death of less than 10%, whereas Comparative Examples 4 to 5 showed cell death of 30% or more. In other words, it was confirmed that the polymer support according to one aspect had excellent biocompatibility.

Figure 2 shows the results of measuring the biocompatibility of a polymer support according to various aspects.

As a result, as shown in Fig. 2, Comparative Examples 1 to 4 and Example 1 showed an increase in cell proliferation rate over time, and it was confirmed that the cell proliferation rate was particularly remarkable in Example 1.

That is, it can be seen that the polymer support according to the aspect of the work has an excellent cell attachment rate because the BMP/PDRN nanocomposite is fixed to the polymer support.

Confirmation of angiogenic capacity

In order to confirm the angiogenic ability of polymer scaffolds immobilized with BMP/PDRN nanocomposites according to the aspect of the invention, the polymer scaffolds of Examples 1 to 3 and Comparative Examples 3 to 5 were transplanted into a rat model with a calvarial defect. After 8 weeks, an angiographic contrast agent was injected, and the volume and number of newly formed blood vessels around the defect site were measured.

Example 1 Example 2 Example 3 Comparative Example 3 Comparative Example 4 Comparative Example 5 Angiogenic capacity ○ ○ ○ △ X X

○: Excellent, △: Average, X: Bad

As shown in Table 2, it was confirmed that Examples 1 to 3 had superior angiogenesis performance compared to Comparative Examples 3 to 5.

Figures 3a and 3b show the effect of polymer scaffolds on angiogenesis according to one aspect.

As a result, as shown in Fig. 3a, it was confirmed that significantly more new blood vessels were generated around the defect site in Example 1 compared to Comparative Examples 3 to 4. Specifically, it was confirmed that new blood vessels were generated at a level similar to that of the normal control group. In addition, as a result of measuring the number of generated new blood vessels, as shown in Fig. 3b, it was confirmed that the number of new blood vessels statistically significantly increased in Example 1 compared to Comparative Examples 3 to 4, and that the defect site was recovered to a level similar to that of the normal control group.

That is, the polymer scaffold according to the aspect can promote angiogenesis and endothelialization of the transplant site by BMP/PDRN nanocomposite.

Confirmation of bone tissue regeneration ability

In order to confirm the bone tissue regeneration ability of the polymer scaffold immobilized with BMP/PDRN nanocomposite according to the aspect, the polymer scaffold of Example 1 was transplanted into a rat model with a skull defect. After 8 weeks, the bone defect area was photographed using Micro-CT to confirm the bone regeneration effect.

Example 1 Example 2 Example 3 Comparative Example 3 Comparative Example 4 Comparative Example 5 Bone tissue regeneration ability ○ ○ ○ △ X X

○: Excellent, △: Average, X: Bad

As shown in Table 2, it was confirmed that Examples 1 to 3 had superior bone tissue regeneration ability compared to Comparative Examples 3 to 5.

Figure 4 shows the effect of polymer scaffolds on bone regeneration according to the daily aspect.

As a result, as shown in Fig. 4, in the case of Comparative Example 3, no bone regeneration effect was observed at all in the defective area, and in the case of Comparative Example 3, a bone regeneration effect was observed partially. On the other hand, in Example 1, it was confirmed that new bone tissue was generated in the defective area, and the size of the defective area was significantly reduced.

Therefore, the polymer scaffold according to the aspect of the invention is excellent in bone tissue regeneration because the BMP/PDRN nanocomposite is fixed to the porous scaffold and the release rate of BMP/PDRN can be controlled.

The above description of the present invention is for illustrative purposes only, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical idea or essential characteristics of the present invention. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive.

Claims (10)

A polymer scaffold for bone regeneration in which a nanocomposite containing bone morphogenetic protein (BMP) and polydeoxyribonucleotide (PDRN) is fixed to a porous scaffold containing a biodegradable polymer, basic ceramic particles, and an extracellular matrix by electrostatic attraction,
The above nanocomposite is formed by ionic bonding of bone forming protein and polydeoxyribonucleotide,
A polymer scaffold for bone regeneration, wherein the bone formation protein and polydeoxyribonucleotide are mixed in a weight ratio of 1:0.5 to 5. A polymer scaffold for bone regeneration according to claim 1, wherein the bone formation protein is selected from the group consisting of BMP1, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP10, BMP11 and BMP15. delete A polymer scaffold for bone regeneration according to claim 1, wherein the biodegradable polymer is selected from the group consisting of polylactide, polyglycolide, polycaprolactone, polylactide-co-glycolide, polylactide-co-caprolactone, polyglycolide-co-caprolactone, polydioxanone, polydioxanone, polytrimethylene carbonate, polyglycolide-co-dioxanone, polyamide ester, polypeptide, polyolthoester, polymaleic acid, polyanhydride, polysebacinhydride, polyhydroxyalkanoate, polyhydroxybutyrate, and polycyanoacrylate. A polymer scaffold for bone regeneration according to claim 1, wherein the basic ceramic particles are selected from the group consisting of magnesium hydroxide, lithium hydroxide, beryllium hydroxide, sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium oxide, lithium oxide, beryllium oxide, sodium oxide, potassium oxide, calcium oxide, calcium carbonate, hydroxyapatite (HAp), tricalcium phosphate (β-TCP), and magnesium ceramics. delete A polymer scaffold for bone regeneration according to claim 1, wherein the nanocomposite is included at a concentration of 0.1 to 10 ppm. A step of preparing a polymer solution by mixing a biodegradable polymer, basic ceramic particles, an extracellular matrix, and a solvent;
A step of preparing a porous support by mixing a porous inducer into the above polymer solution; and
A method for producing a polymer scaffold for bone regeneration, comprising a step of fixing a nanocomposite containing a bone formation protein and a polydeoxyribonucleotide in a weight ratio of 1:0.5 to 5 to the porous scaffold by electrostatic attraction,
A method for producing a polymer scaffold for bone regeneration, wherein the nanocomposite is formed by ionic bonding of a bone formation protein and polydeoxyribonucleotide. delete A method for producing a polymer scaffold for bone regeneration according to claim 8, wherein the step of fixing the nanocomposite to the porous scaffold is performed in a calcium-phosphorus solution.