KR20200007747A - A chitosan/TEMPO oxidized cellulose nanofiber hydrogel comprising fk506 for bone therapy and a method for manufacturing the same - Google Patents

Abstract

According to one embodiment of the present invention, a chitosan/tempo oxidized cellulose nanofiber hydrogel containing fk506 for bone treatments comprises fk506, chitosan, gelling agents and tempo oxide cellulose nanofibers. The chitosan/tempo oxidized cellulose nanofiber hydrogel containing fk506 for bone treatments according to the present invention is used to increase a sol/gel transition speed while reducing a decomposition rate of CS/TOCNF hydrogel because of a high content of NaHCO_3. In addition, the present invention induces growth and differentiation of MC3T3-E1, a pre-osteoblastic cell, in vitro at a 5nm, a low concentration of fk506 to introduce into CS/TOCNF hydrogel, and introduces CS/TOCNF hydrogel containing fk506 to a bone loss portion in vivo to induce bone regeneration.

Description

Chitosan / TEMPO oxidized cellulose nanofiber hydrogel comprising fk506 for bone therapy and a method for manufacturing the same}

The present invention relates to a chitosan / tempo oxidized cellulose nanofiber hydrogel for bone treatment comprising fk506 and a method for preparing the same.

Bone, a dynamic and highly vascular tissue, plays an important role in the human body, providing a framework to support exercise, protect soft tissues, store minerals such as calcium and phosphate, and harble the bone marrow. In addition, unlike other tissues, bone is capable of regenerating and repairing itself due to its self-balancing ability between bone resorption by osteoclasts and bone formation by osteoblasts. However, such remodeling is not always effective when the bone defect is large, and it is necessary to supply sufficient blood and adequate calcium and phosphorus at the fracture site to strengthen and harden the new bone. Therefore, it is very urgent to find a suitable clinical treatment for severe bone defects.

Bone grafts are one of the clinical bone defect treatment methods commonly used to promote bone repair. Depending on the advantages of bone formation, bone conduction and bone induction, bone grafts, including autologous grafts (bone grafts obtained from patients), allograft or xenograft grafts (bone grafts obtained from donors or animals) may be used in body parts. It is desirable to implant in to close the defect and promote treatment. Nevertheless, the use of bone grafts for surgery is not a complete cure. Bone grafts are still under study because of the high likelihood of secondary surgery, the risk of disease transmission, the difficulty of finding foreign bone reactions, host rejection, available bone sources, and the difficulty of forming shapes and depths suitable for missing cavities. Thus, new biomaterials have been developed that can replace bone grafts in recent decades. Such biomaterials may be used for elements such as nontoxicity, biocompatibility, biodegradability, porosity sufficient for cellular and vascular invasion, strong mechanical properties, structures that can create an environment for cell development, bone formation, bone conduction and bone induction. Designed according to

Hydrogels are potential biomaterials ideal for tissue regeneration applications. It is a swelling three-dimensional (3D) structural network that can mimic natural extracellular matrix and capture cells or growth factors in the network to promote cell infiltration, growth and differentiation at trauma sites. Injectable hydrogels in hydrogels are easy to fill, suitable for irregular shaped defects and can form gels in place under mild conditions in a non-invasive manner, pre-forming (pre- It is known to be a smart hydrogel that has advantages over hydrogels. In addition, infusion hydrogels that are particularly sensitive to temperature can be freely injected at room temperature or at low temperatures and can be converted into gel at body temperature without the addition of specific additives (UV, pH regulators or oxidizers) that protect against pests or phase transitions. It is the most suitable material for the treatment of defects.

However, the CS / TOCNF hydrogel developed by the inventor in the previous study was designed as an injectable thermosensitive hydrogel system with less toxicity and improved gelling properties compared to the chitosan hydrogel system. These systems are more biocompatible with MC3T3-E1 than chitosan hydrogels, making them suitable for clinical biomedical applications, especially bone therapy. However, the mechanical strength was very weak and had the disadvantage of slow gelation. It is also biocompatible but does not possess drugs, growth factors or factors that promote the treatment of bleb. Thus, the system has the disadvantage of slow treatment compared to other hydrogel-containing cells, drugs or growth factors. In addition, its rate of degradation was very fast at 1 month and did not provide enough time for bone repair. There has been a need for the development of systems with desirable properties such as strength, fast gelling, fast bone formation, and slow rate of degradation.

KR 10-181537 B1 (January 5, 2018)

It is an object of the present invention to provide a chitosan / tempo oxidized cellulose nanofiber hydrogel comprising fk506 for bone therapy, comprising fk506, chitosan, a gelling agent, and a tempo oxidized cellulose nanofiber.

Another object of the present invention is to prepare a chitosan solution by (a) injecting chitosan into the aqueous lactic acid solution, (b) preparing a tempo-oxidized cellulose nanofiber solution, (c) the chitosan solution Mixing the tempo oxidized cellulose nanofibers (TOCNF) solution with a solution to prepare a first mixture, (d) preparing a fk506 solution and then mixing the first mixture with a second mixture to prepare a second mixture, (e) gel It provides a method for producing a chitosan / tempo oxidized cellulose nanofiber hydrogel comprising fk506 for the treatment of bone, comprising the step of preparing a topical agent, and (f) dropping a gelling agent into the secondary mixture.

Chitosan / tempo oxidized cellulose nanofiber hydrogel comprising fk506 for bone therapy according to an embodiment of the present invention includes fk506, chitosan, a gelling agent, and a tempo oxidized cellulose nanofiber.

The gelling agent may include 0.1-1.0 M sodium bicarbonate (NaHCO ₃ ) and β-glycerophosphate (β-Glycerophosphate).

The hydrogel may be gelled in vivo.

The hydrogel increases the rate of conversion from sol to gel as the content of sodium bicarbonate increases.

The hydrogel is inversely proportional to the content of the sodium bicarbonate and the decomposition rate of the hydrogel.

The hydrogel enhances the differentiation of osteoblasts in progenitor bone cells in 2D culture.

The gelling agent may comprise sodium bicarbonate at a concentration of 0.5 M.

Fk506 promotes osteogenic differentiation and prevents infection.

The sodium bicarbonate delays the decomposition time and drug release time of the hydrogel.

According to another embodiment of the present invention, a method for preparing chitosan / tempo oxidized cellulose nanofiber hydrogel comprising fk506 for bone treatment may include (a) preparing a chitosan solution by adding chitosan to an aqueous solution of lactic acid, and (b) tempo. Preparing a solution of (Tempo-oxidized cellulose nanofiber), (c) mixing the chitosan solution and the tempo-oxidized cellulose nanofiber (TOCNF) solution to prepare a primary mixed solution, (d) fk506 solution Preparing a secondary mixture by mixing with the primary mixture, (e) preparing a gelling agent, and (f) dropping the gelling agent into the secondary mixture.

In the step (c), the TOCNF solution and the chitosan solution may be mixed at a volume ratio of 1: 2 to 4 (v / v).

The step (e) may be prepared by dissolving 60% (w / v) β-glycerophosphate (β-Glycerophosphate) powder in an aqueous solution of sodium bicarbonate (NaHCO ₃ ) at a concentration of 0.1 to 1 M.

The fk506 solution may dissolve the fk506 powder in ethanol.

In the step (f), the gelling agent may be added dropwise to the secondary mixed solution at a volume ratio of 1 to 3: 1 (v / v).

Using the method for producing chitosan / tempo oxidized cellulose nanofiber hydrogel containing fk506 for bone treatment according to the present invention, since the content of NaHCO ₃ is high, the sol / gel transfer rate is increased and the decomposition rate of CS / TOCNF hydrogel is increased. There is an effect that can lower the. In addition, even at a low concentration of fk506 of 5 nM, it is effective to induce the growth and differentiation of MC3T3-E1 cells, which are precursor bone cells in vitro (2D culture), and can be introduced into CS / TOCNF hydrogel. Introducing CS / TOCNF hydrogel comprising fk506 at the defect site has the advantage of inducing bone regeneration.

Figure 1 shows the manufacturing process of the CS / TOCNF hydrogel containing fk506.
2A to 2D show the gelation time, viscosity and flow behavior of CS / TOCNF hydrogel at 37 ° C. according to the gelling agent.
Figure 3 shows the Fourier change infrared (FT-IR) spectrum of the CS / TOCNF hydrogel according to the concentration of sodium bicarbonate contained in the gelling agent.
4A and 4B show CS / TOCNF hydrogels immersed in PBS containing lysozyme enzyme (1 mg / ml) according to the concentration of sodium bicarbonate (0M, 0.1M, 0.25M, 0.5M) included in the gelling agent. The remaining profiles and the optical photographs are shown in the profiles analyzed in.
Figure 5 is an MTT analysis of the effect of fk506 (5 nM, 15 nM, 25 nM, 50 nM and 100 nM) on the proliferation of progenitor bone cells MC3T3-E1 after 1, 3, 5 and 7 days of culture .
Figure 6 shows the cumulative release rate of fk506 of CS / TOCNF hydrogel with 0.5 M sodium bicarbonate in the gelling agent by UV spectroscopy.
Figure 7 shows the implantation of a CS / TOCNF hydrogel containing fk506 in a critical-sized rat skull defect.
Figure 8 shows the staining results with Masson's Trichrome 3, 6 weeks after the transplantation of CS / TOCNF hydrogel containing fk506 in the animal model.

Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings. First of all, it should be noted that in the drawings, the same components or parts denote the same reference numerals as much as possible. In describing the present invention, detailed descriptions of related well-known functions or configurations are omitted in order not to obscure the subject matter of the present invention.

As used herein, the terms "about", "substantially", and the like, are used at, or in close proximity to, numerical values as are indicative of preparation and material tolerances inherent in the meanings mentioned, and are intended to be accurate or to facilitate understanding of the invention. Absolute figures are used to prevent unfair use by unscrupulous intruders.

EMBODIMENT OF THE INVENTION Hereinafter, this invention is demonstrated in detail.

Chitosan / tempo oxidized cellulose nanofiber hydrogel comprising fk506 for bone therapy according to an embodiment of the present invention includes fk506, chitosan, a gelling agent, and a tempo oxidized cellulose nanofiber.

The tacrolimus (fk506) promotes osteogenic differentiation and prevents infection.

The tacrolimus (Tacrolimus, fk506) is an immunopotentiator that can prevent allograft rejection and increase the survival rate of organ transplant patients after heart or heart transplantation. It can also have osteogenic differentiation and promote bone formation in vivo and ex vivo.

Therefore, chitosan / tempo oxidized cellulose nanofiber hydrogel comprising fk506 according to an embodiment of the present invention promotes bone formation, shortens the bone treatment period, and has an effect of preventing infection during bone formation in the body. have.

Nanocellulose is a biomaterial candidate. Nanocellulose can be used in regenerative medicine and wound healing applications such as tissue engineered meniscus, scaffolds such as blood vessels, ligaments and tendon substitutes, which have significant physical properties, special surface chemistry, biodegradability and bioavailability. This is due to good biological properties such as suitability and low toxicity.

In order to make cellulose nanofibers, it is necessary to separate cellulose fibers of wood. Cellulose fibers were difficult to separate with high efficiency because the fibers and the fibers are strongly bonded, but the functional catalyst 2,2,6,6-tetramethyl-piperidine-1-oxyl (2,2,6,6 When oxidized with -tetramethyl-piperidin-1-oxyl (TEMPO), the cellulose fibers can become uniform cellulose nanofibers. The TEMPO-oxidized cellulose nanofibers (TOMNF) are biodegradable from wood biomass. In addition, since it has various excellent properties such as high crystallinity, excellent heat resistance, and high transparency, it can be used for polymer composite materials, medical engineering materials, and membranes.

Tempo-oxidized cellulose nanofibers obtained by TEMPO mediated oxidation of nanocellulose are novel potent biobased nanomaterials. TOCNF has a high crystallinity and water solubility to form hydrogels to form a 3D environment capable of cell growth and differentiation.

The gelling agent may include 0.1-1.0 M sodium bicarbonate (NaHCO ₃ ) and β-glycerophosphate (β-Glycerophosphate).

Specifically, the gelling agent may include 0.5 M sodium bicarbonate.

The β-glycerophosphate is a material that can be used as a general gelling agent. However, when the β-glycerophosphate alone is used, there is a disadvantage in that the gelation is slow, and in the case of the hydrogel using the conventional β-glycerophosphate alone, the gel is slowed, which results in a defect or premature dissolution from the defect. There is this.

The sodium bicarbonate is a weak base that enhances gels and promotes gelation without affecting cell activity.

In addition, the gelling agent containing sodium bicarbonate has the effect of delaying the decomposition time and drug release time of the hydrogel.

The chitosan / tempo oxidized cellulose nanofiber hydrogel comprising fk506 according to an embodiment of the present invention was confirmed to increase the rate of change from sol to gel as the content of sodium bicarbonate contained in the gelling agent increased.

Chitosan / tempo oxidized cellulose nanofiber hydrogel comprising fk506 according to an embodiment of the present invention may have a form of a gel (gel) at 30 ℃ ~ 37 ℃, more specifically have a form of gel at 37 ℃ Can be. That is, it may be gelled in vivo.

Chitosan / tempo oxidized cellulose nanofiber hydrogel containing fk506 according to an embodiment of the present invention is inversely proportional to the content of sodium bicarbonate and the rate of hydrogel degradation.

That is, an increase in the amount of titanium bicarbonate contained in the gelling agent decreases the rate of degradation of the CS / TOCNF hydrogel and increases the time for the CS / TOCNF hydrogel to heal bone.

In one embodiment of the present invention, it was confirmed that CS / TOCNF hydrogel containing fk506 or fk506 enhances the differentiation of osteoblasts in progenitor bone cells in vitro in 2D culture.

Since fk506 has been described above, a description thereof will be omitted.

According to another embodiment of the present invention, a method for preparing chitosan / tempo oxidized cellulose nanofiber hydrogel comprising fk506 for bone treatment may include (a) preparing a chitosan solution by adding chitosan to an aqueous solution of lactic acid, and (b) tempo. Preparing a solution of (Tempo-oxidized cellulose nanofiber), (c) mixing the chitosan solution and the tempo-oxidized cellulose nanofiber (TOCNF) solution to prepare a primary mixed solution, (d) fk506 solution Preparing a secondary mixed solution by mixing with the primary mixed solution, (e) preparing a gelling agent, and (f) dropping a gelling agent into the secondary mixed solution (Drop by drop). Include.

Step (a) is a step of preparing chitosan solution by adding chitosan to the lactic acid aqueous solution.

Specifically, step (a) may be prepared by dissolving chitosan in 1% (v / v) lactic acid aqueous solution to prepare a 3% (w / v) chitosan (CS) solution.

Step (b) is a step of preparing a tempo-oxidized cellulose nanofiber solution.

Specifically step (b) is 2,2,6,6-tetramethyl-piperidine-1-oxyl (2,2,6,6-tetramethyl-piperidin-1-oxyl, TEMPO) -oxidized cellulose in distilled water A homogeneous suspension of nanofibers (TOCNF) can be diluted and an aqueous hydrochloric acid solution can be added to prepare a 0.4% (v / v) TOCNF solution containing TEMPO-oxidized cellulose nanofibers (TOCNF).

The term "dropping" means dropping a drop of liquid or the like.

The " suspension " refers to a suspension system in which solid fine particles are dispersed in a liquid, called a suspension or suspension.

The term "homogeneous" refers to having a physical and chemical property identical to that of other parts, even if any part is taken in one substance in a certain state, and is also called uniformity.

Step (c) is a step of preparing a primary mixed solution by mixing the chitosan solution and the tempo oxidized cellulose nanofibers (TOCNF) solution.

Specifically, step (c) may mix the TOCNF solution and the chitosan solution in a 1: 2 to 4 volume ratio (v / v), preferably in a 1: 3 volume ratio.

More specifically, the 3% (v / v) chitosan (CS) solution, the TOCNF solution and the chitosan solution were mixed in a 1: 3 volume ratio (v: v) at room temperature to prepare a primary mixed solution (CS / TOCNF). do.

Step (d) is a step of preparing a second mixture by preparing a fk506 solution and then mixing the mixture.

The fk506 solution is prepared by dissolving fk506 powder in ethanol.

The prepared fk506 solution was added to the primary mixed solution to prepare a secondary mixed solution (CS / TOCNF containing fk506).

The concentration of fk506 is contained at a concentration of 45-55 μg / ml with respect to the secondary mixture.

Step (e) is a step of preparing a gelling agent.

Specifically, the step of preparing the gelling agent (e) by dissolving 60% (w / v) β-glycerophosphate (β-Glycerophosphate) powder in an aqueous solution of sodium bicarbonate (NaHCO ₃ ) of 0.1 ~ 1 M concentration It can manufacture.

The sodium bicarbonate is prepared by dissolving in water and filtering, and then dissolving β-glycerophosphate in sodium bicarbonate solution of various concentrations.

Step (f) is a final step of preparing a CS / TOCNF hydrogel containing fk506 by dropping a gelling agent in the secondary mixture.

Specifically, the gelling agent is added dropwise to the secondary mixed solution at a volume ratio of 1 to 3: 1 (v / v), and continuously stirred at 4 ° C. in an ice bath to prepare CS / TOCNF hydrogel including fk506.

As described above, using chitosan / tempo oxidized cellulose nanofiber hydrogel containing fk506 and a method of preparing the same, the content of NaHCO ₃ is high, so the sol / gel transition rate is increased and the degradation rate of CS / TOCNF hydrogel is lowered. There is an effect that can be done. In addition, even at a low concentration of fk506 of 5 nM, it is effective to induce the growth and differentiation of MC3T3-E1 cells, which are precursor bone cells in vitro (2D culture), and can be introduced into CS / TOCNF hydrogel. CS / TOCNF hydrogel containing fk506 may be introduced at the defect site to be used for bone regeneration.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples are merely to illustrate the present invention, and the content of the present invention is not limited to the following examples.

Example Preparation of Hydrogels

The manufacturing process of the specific hydrogel is shown in FIG.

A 3: 1 volume ratio (CS: TOCNF, v / v) of 3% (w / v) chitosan (CS) solution and 0.4% (w / v) tempo-oxidized cellulose nanofiber (TOCNF) solution ) To prepare a primary mixed solution (CS / TOCNF) and then cooled.

Next, fk506 powder was dissolved in ethanol to prepare a fk506 solution, and then added to the first mixed solution to prepare a second mixed solution. The fk506 solution mixed in the secondary mixture was used at a concentration of 50 μg / ml.

Finally, the secondary mixed solution and the gelling agent (β-GP Solution) was added dropwise in a ratio of 2: 1 (mixing solution: gelling agent, v: v) and the hydrogel was continuously stirred at 4 ° C. in an ice bath. Prepared.

The final hydrogel was left at 4 ° C. before use.

Here, the gelling agent (β-GP Solution) is dissolved in water and filtered in 60% (w / v) β-glycerophosphate (β-Glycerophosphate) in 0.1M ~ 1M sodium bicarbonate aqueous solution (NaHCO ₃ Solution) concentration; GP) Powder was prepared by dissolving it.

Experimental Method

1. Gelation time measurement

The gelation time of CS / TOCNF hydrogel was measured according to the concentration of the gelling agent.

Determination of the gelation time of CS / TOCNF hydrogel according to the concentration of the gelling agent was performed by a vial inversion test method (H. Y. Zhou et al., 2008). In addition, vials containing 1 ml CS / TOCNF pre-gel solution were incubated in a 37 ° C. water bath to promote sol-gel transfer. The gel time according to the concentration of the gelling agent was recorded until the flow of solution stopped when the vial was turned upside down.

2. Viscosity Characterization and Flow Behavior Measurement

Viscosity of CS / TOCNF hydrogel was measured according to the concentration of the gelling agent in the liquid state (4 ° C.) and gel state (37 ° C.) using a Brookfield viscometer ((RVDV, USA).

7.1 ml of hydrogel was placed in a cylindrical vessel and stirred continuously with spindle 21. Samples were measured for 3 minutes at 100 RPM at 4 ° C and 150 RPM at a temperature of 37 ° C. Viscosity was measured every 30 seconds.

3. Fourier Transform Infrared Spectroscopy (FT-IR) Analysis

FT-IR spectra of CS / TOCNF hydrogels before and after the addition of sodium bicarbonate were measured using a Fourier Transform Infrared Spectrometer (Nicolet iS10-Smart iTR, Thermo Fisher Scientific, USA) ^It was measured in the ^-1 frequency range.

4. Degradation of CS / TOCNF Hydrogel

Degradation of CS / TOCNF hydrogels using various gelling agents was evaluated by immersion in PBS / lysozyme solution (1 mg / ml) at 37 ° C., followed by morphology, size, and weight of the hydrogel with continuous shaking for 8 weeks.

At regular intervals, samples were removed from the PBS / lysozyme solution and weighed. The PBS / lysozyme solution was replaced every 48 hours with fresh solution to keep the enzyme running and the sample dry. The remaining hydrogel mass percentage was calculated according to the following formula.

Hydrogel Remaining Mass (%) = (Wt / Wi) × 100 (%)

Here, Wi is the initial weight of each sample before immersing in PBS / lysozyme solution, Wt is the weight of the sample over time (t) after immersion in PBS / lysozyme solution.

5. Culture of In Vitro Cells

MC3T3-E1 cells (American Type Culture Collection, Manassas, VA), mouse precursor bone cells, were added with 10% fetal bovine serum (FBS, Mediatech Inc, USA) and 1% penicillin-streptomycin (PS, Welgene, South Korea). Culture in one alpha minimal medium (α-MEM, Welgene, South Korea). MC3T3-E1 cells were placed in a humidified incubator of 5% carbon dioxide (CO ₂₎ at 37 ° C. In addition, 10 nM dexamethasone (Sigma-Aldrich, South Korea), 50 μg / ml ascorbic acid 2-phosphate (Sigma-Aldrich, South Korea) and 10 mM β-glycerol phosphate were added to osteogenic differentiation medium of MC3T3-E1 cells. (Sigma-Aldrich, USA) was supplemented and the medium was changed every two days.

6. Biocompatibility Testing of fk506

MC3T3-E1 cells were seeded in 48-well culture plates until they reached a confluence of 70-80%. The medium was then removed and replaced with fk506 solutions of different concentrations (5 nM, 15 nM, 25 nM, 50 nM, and 100 nM) prepared by diluting the fk506 stock (12 mM) in the medium. Wells inoculated with cells in medium without fk506 were used as controls. After 1, 3, 5 and 7 days of incubation, MTT solution (5 mg / ml in PBS) was added at a ratio of 1:10 (v / v) per well and incubated again at 37 ° C.

After 4 hours of incubation all solutions in the wells were removed and DMSO was added to the wells to dissolve formazan salt. Cell viability was determined by measuring the optical density of the solution at a wavelength of 595 nm using an ELISA reader (Infinite F50, Tecan, Austria). Every two days the medium was reduced by half.

7. Release of in vitro fk506

Release of fk506 from CS / TOCNF hydrogel (selective sample) using 0.5 M sodium bicarbonate in the gelling agent was confirmed. After gel formation, 1 ml of gel containing fk506 was immersed in 1.5 ml of PBS and incubated at 37 ° C. under constant shaking conditions. Every extraction solution was collected at regular time, replaced with fresh PBS, and the fk506 content released was measured and analyzed. Ultraviolet absorbance was read by BioDrop (UK) at a wavelength of 220 nm. The amount of fk506 released to the extract was determined based on a standard calibration curve of known fk506 concentrations.

8. In Vivo Research

18 Sprague Dawley male rats (200-250 g, 9 weeks old) were raised and used for the experiments with the approval of the Animal Ethics Committee of Soonchunhyang University. Mice were anesthetized with diethyl ether (Daejung, South Korea) and placed in a procumbent position. The surgical site of the rat head was shaved and brushed with povidone-iodine solution for 70% alcohol and antibacterial purposes for hygiene purposes. A midline incision of about 25 mm was made to expose the skull. The periosteum was then inverted, forming a circular full thickness bone defect (diameter 8 mm, depth 1 mm) through a trepin drill in the frontal center of the skull. The saline feed was continuous during the drilling time. Defects were filled by injecting various groups of hydrogels (with or without fk506) and the defect site is considered a control when no hydrogels are implanted. The incision is then closed and wiped with povidone iodine. Healing was observed at 3 and 6 weeks after transplantation.

[Experiment result]

Experimental Example 1, NaHCO ₃  Gelation and Flow Behavior Analysis of CS / TOCNF Hydrogel According to Concentration

2A to 2D show the gelation time, viscosity and flow behavior of CS / TOCNF hydrogel at 37 ° C. according to the gelling agent.

The effect of sodium bicarbonate (NaHCO ₃ ) concentration on the gelation time of CSF / TOCNF hydrogel was observed at physiological temperature.

As shown in FIG. 2A, the gelation time of the hydrogel gradually decreased from 130 seconds to 25 seconds when the concentration of NaHCO ₃ of the gelling agent increased from 0 M to 1 M. This means that the higher the concentration of NaHCO ₃ contained in the gelling agent, the faster the gelation proceeds.

When NaHCO ₃ is added to the gelling agent, as the NaHCO ₃ content increases (0 M, 0.1 M, 0.25 M and 0.5 M), the viscosity of the hydrogels tends to decrease. As shown in FIG. 2B, the viscosity of the hydrogel at 37 ° C. was about 233 Pa.s for 0 M NaHCO ₃ and decreased to 149.6 Pa.s and 181.9 Pa.s for 0.1 M and 0.5M NaHCO ₃ , respectively. . However, for 0.25 M NaHCO ₃ the viscosity of the hydrogel shows about 350 Pa · s due to the repulsive force between charges.

In addition, the flow behavior of the CS / TOCNF hydrogel at 37 ° C. is shown in FIGS. 2C and 2D. Referring to Figures 2C and 2D, the viscosity and shear stress of all CS / TOCNF hydrogel showed a tendency to gradually decrease with increasing shear rate. This can be said that the flow behavior of CS / TOCNF hydrogel is a dedicated hearthinning model suitable for bone treatment use by the chief executive. This is because most injectable hydrogels have low viscosity for easy injection (through a syringe) and are designed according to a free flowing solution strategy that can fill cavity and cavity of any shape in vivo. .

Experimental Example 2 FT-IR Spectrum Analysis of CS / TOCNF Hydrogel

Figure 3 shows the Fourier change infrared (FT-IR) spectrum of the CS / TOCNF hydrogel according to the concentration of sodium bicarbonate contained in the gelling agent.

Referring to FIG. 3, no new peak was observed in the spectrum of the hydrogel including the NaHCO ₃ -added gelling agent, and it was confirmed that the chemical structure of the hydrogel did not change due to the addition of NaHCO ₃ . In addition, the absorption bands of OH and NH shifted slightly to wave-number as NaHCO ₃ content gradually increased in the gelling agent. This means that the hydrogel-forming combination of chitosan and a positive charge (NH ₃ ⁺⁾ is neutralized. This reduces the electrostatic repulsive force and strain between the chitosan molecules, thereby improving hydrophobic interactions of acetyl groups and promoting gelation, which explains why the gelation time is shortened as the NaHCO ₃ content increases.

Experimental Example 3 Analysis of Degradation Profile of CS / TOCNF Hydrogel According to NaHCO3 Content

4A and 4B show CS / TOCNF hydrogels immersed in PBS containing lysozyme enzyme (1 mg / ml) according to the concentration of sodium bicarbonate (0M, 0.1M, 0.25M, 0.5M) included in the gelling agent. Remaining amounts and optical photographs are shown with the profile analyzed in.

CS / TOCNF hydrogels are degradable in vivo and their degradability depends on the change in NaHCO ₃ content of the gelling agent. The degradation of CS / TOCNF hydrogels was assessed by measuring hydrogel mass loss and observing hydrogel erosion.

4A and 4B show that the mass of CS / TOCNF hydrogel with no gelling agent or NaHCO ₃ or small amounts (0.1 M and 0.25 M) is significantly reduced by at least 80% after 6 weeks. Increasing the concentration of NaHCO ₃ to 0.5 M resulted in a loss of mass and its morphology that did not change significantly compared to the CS / TOCNF hydrogel, which was almost degraded and only a small fraction remained. As a result, NaHCO ₃ contained in the gelling agent with increasing concentrations resulted in a decrease in the rate of degradation of CS / TOCNF hydrogels, in particular 0.5 M NaHCO ₃ , which is very stable and suitable for use in bone healing. Has been proven.

Experimental Example 4 in vitro biocompatibility analysis according to fk506 concentration

Figure 5 is an MTT analysis of the effect of fk506 (5 nM, 15 nM, 25 nM, 50 nM and 100 nM) on the proliferation of progenitor bone cells MC3T3-E1 after 1, 3, 5 and 7 days of culture .

To assess the effect of fk506 on cell activity, MC3T3-E1 cells, which are precursor bone cells, were seeded in wells containing fk506 at different concentrations (5 nM, 15 nM, 25 nM, 50 nM and 100 nM), MTT assays were performed in incubators for 1, 3, 5 and 7 days. The optical density measured confirmed the viable cell number. As shown in FIGS. 5A and 5B, the number of viable cells increased after 3 days, 5 days and 7 days incubation at all fk506 concentrations, meaning that fk506 was not toxic and did not interfere with cell proliferation. In addition, the optical density at low fk506 concentrations (5 nM, 15 nM and 25 nM) was higher than that of the control (medium treated cells), meaning that fk506 enhanced cell growth at low concentrations.

Experimental Example 5. Drug Release Analysis of fk506 of CS / TOCNF Hydrogel

Figure 6 shows the cumulative release rate of fk506 of CS / TOCNF hydrogel with 0.5 M sodium bicarbonate in the gelling agent by UV spectroscopy.

The cumulative release rate of fk506 from CS / TOCNF hydrogel with 50 μg / ml of fk506 was determined by UV spectroscopy.

The fk506 is introduced to promote osteogenic differentiation and to prevent infection.

Referring to FIG. 6, the initial release of fk506 was 66.5% of the original amount after the first hour and increased to 89.9% after the next 24 hours. The cumulative release rate was progressive until 1 day after the initial few hours of initial release. Thus, the addition of NaHCO ₃ delayed the fk506 release rate.

Experimental Example 6. Analysis of bone regeneration effect in vivo

CS / TOCNF hydrogels containing fk506 were injected and filled into bone defects to promote bone regeneration.

Figure 7 shows the transplantation of the CS / TOCNF hydrogel containing fk506 in the mouse cranial defect of the critical size.

As shown in FIG. 7, the hydrogel was injected in solution and rapidly transformed into a solid upon contact with body temperature. Rapid gelling of the hydrogel has the effect of limiting the diffusion of the solution from the injection site.

Figure 8 shows the results of staining using Masson's Trichrome 3, 6 weeks after transplantation of CS / TOCNF hydrogel containing fk506 in the animal model (HB: host bone, arrow: new bone, S : sample).

Referring to FIG. 8, the transplantation can be observed at low magnification, and the defect interface was observed at high magnification, and stromal cell formation in CS / TOCNF hydrogel containing fk506 compared to the control and CS / TOCNF hydrogels. The formation of new bone at the periphery of the graft was confirmed (after 3 weeks), and the hydrogel remained 6 weeks after the transplant.

This means that implanting a hydrogel containing fk506 according to an embodiment of the present invention to a defect site has an effect of lowering the decomposition rate of the hydrogel containing fk506 and inducing bone formation for a long time. .

As mentioned above, although this invention was demonstrated in detail using the preferable Example, the scope of the present invention is not limited to a specific Example and should be interpreted by attached Claim. In addition, those of ordinary skill in the art should understand that many modifications and variations are possible without departing from the scope of the present invention.

1

Claims (14)

A chitosan / tempo oxidized cellulose nanofiber hydrogel comprising fk506 for bone therapy, comprising fk506, chitosan, a gelling agent, and a tempo oxidized cellulose nanofiber. In claim 1,
The gelling agent,
A chitosan / tempo oxidized cellulose nanofiber hydrogel comprising 0.1-1.0 M sodium bicarbonate (NaHCO ₃ ) and β-glycerophosphate. In claim 1,
The hydrogel is chitosan / tempo oxidized cellulose nanofiber hydrogel, characterized in that the gel in vivo (In vivo). In claim 2,
The hydrogel is chitosan / tempo oxidized cellulose nanofiber hydrogel, characterized in that the rate of conversion from sol to gel increases when the content of the sodium bicarbonate increases. In claim 2,
The hydrogel is chitosan / tempo oxidized cellulose nanofiber hydrogel, characterized in that the content of the sodium bicarbonate is inversely proportional to the decomposition rate of the hydrogel. In claim 1,
The hydrogel is chitosan / tempo oxidized cellulose nanofiber hydrogel, characterized in that to enhance the differentiation of osteoblasts in progenitor bone cells during 2D culture. In claim 2,
The gelling agent is a chitosan / tempo oxidized cellulose nanofiber hydrogel, characterized in that it comprises sodium bicarbonate at a concentration of 0.5 M. In claim 1,
The fk506 promotes osteogenic differentiation and prevents infection, characterized in that chitosan / tempo oxidized cellulose nanofiber hydrogel. In claim 1,
The sodium bicarbonate is a chitosan / tempo oxidized cellulose nanofiber hydrogel, characterized in that the delay time and the drug release time of the hydrogel. (a) preparing chitosan solution by adding chitosan to an aqueous solution of lactic acid,
(b) preparing a tempo-oxidized cellulose nanofiber solution,
(c) mixing the chitosan solution and the tempo oxidized cellulose nanofiber (TOCNF) solution to prepare a primary mixed solution,
(d) preparing a fk506 solution and then mixing the first mixed solution to prepare a second mixed solution,
(e) preparing a gelling agent, and
(F) a method for producing a chitosan / tempo oxidized cellulose nanofiber hydrogel comprising fk506 for bone treatment, comprising the step of dropping a gelling agent in the secondary mixture. In claim 10,
In step (c),
Method for producing a chitosan / tempo oxidized cellulose nanofiber hydrogel, characterized in that the TOCNF solution and the chitosan solution is mixed in a 1: 2 to 4 volume ratio (v / v). In claim 10,
In step (e),
Chitosan / Tempo oxidized cellulose nanofiber hydros prepared by dissolving 60% (w / v) of β-glycerophosphate powder in an aqueous solution of sodium bicarbonate (NaHCO ₃ ) at a concentration of 0.1 to 1 M. Method for preparing gels. In claim 10,
The fk506 solution is a method for producing chitosan / tempo oxidized cellulose nanofiber hydrogel, characterized in that fk506 powder is dissolved in ethanol. In claim 10,
Step (f),
A method for producing chitosan / tempo oxidized cellulose nanofiber hydrogel, wherein the gelling agent is added dropwise to the secondary mixed solution at a volume ratio of 1 to 3: 1 (v / v).

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