Patent Application
20210213163
Present invention relates to a bone regeneration material having antimicrobial property and a method for producing the antimicrobial bone regeneration material property. The antimicrobial property is imparted by using inositol phosphate.
Conventionally, artificial bone made of calcium compounds such as hydroxyapatite (hereinafter abbreviated as HAp), tricalcium phosphate (hereinafter abbreviated as TCP) has been used as a material for bone regeneration. Recently, a type of material that uses the self-regeneration repair function of the human body by filling a scaffold containing a bone formation promoting factor in a defect portion has been actively used (Patent Document 1).
Since implantation of materials into a human body requires a surgery, there is a risk of bacterial infection after surgery. As a measure to solve this problem, it is possible to impart antimicrobial property to the material to be implanted or to administer antibiotics. Metals such as silver, zinc and copper are used as means for imparting antibacterial properties. In particular, silver has excellent bactericidal activity in ionized form, does not produce resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA), and is widely and suitably used because silver has a broad antimicrobial spectrum.
As a method of providing silver on the bone regeneration material, if the material is a metal material such as titanium, the surface coating can be performed by spraying silver metal by using a flame spraying method. However, when the bone regeneration material is composed of biodegradable fibers, it is difficult to use this method because of the effect of high temperature and difficulty of coating to complex shapes.
Recently, there has been developed an implant material for bone regeneration of a type in which biodegradable fibers contain particles of a calcium phosphate compound, and after the material is implanted in vivo, calcium phosphate is eluted together with decomposition and absorption of the biodegradable fibers to promote bone formation. As a method of imparting antimicrobial properties to this type of bone regeneration material, there has been proposed a design in which silver is carried on calcium phosphate particles contained in biodegradable fibers, and the biodegradable fibers are degraded in the body to dissolve the calcium phosphate particles and the silver contained therein is eluted to exert antimicrobial properties (Non-Patent Document 1). However, silver ions should be eluted early after implantation of the bone regeneration material, since the risk of bacterial infection is most serious early after surgery. In the method of Non-Patent Document 1, since silver is contained inside the fiber, there is a possibility that silver ions in an amount necessary for antimicrobial are not eluted early after the operation.
On the other hand, eluted silver ions have antimicrobial properties that kill bacteria, but at the same time they may develop cytotoxicity against osteoblasts and surrounding cells that need to proliferate. Since both antimicrobial and cytotoxicity are more effective as the silver ion concentration increases, it is important to simultaneously satisfy these two mutually conflicting requirements when providing an antimicrobial property to artificial bone.
[Patent Document 1] U.S. Pat. No. 6,162,916
[Non-Patent Document 1] Flexible, silver containing nanocomposites for the repair of bone defects: antimicrobial effect against E. coli infection and comparison to Tetracycline, Journal of Materials Chemistry 2008 Oliver D. Schneider et al. etc.
Under the circumstances as described above, there has been a need for a highly safe bone regeneration material which exhibits antimicrobial properties early after surgery by carrying silver on a bone regeneration material containing biodegradable fibers and eluting silver ions after the bone regeneration material has been implanted in vivo, and which is less likely to develop cytotoxicity, and a method of manufacturing the same.
In order to solve the above-mentioned problems, the inventors of the present invention have intensively studied, and as a result, have noticed that a portion of the particles of the calcium compound embedded in the fiber is exposed on the surface of the biodegradable fiber containing a considerable amount of the particles of the calcium compound. It has been envisioned that controlled antimicrobial properties can be imparted to the exposed calcium compound particles in the intended amounts by chelating calcium ions (Ca2+) and silver ions (Ag+) of the calcium compound via hydroxyl groups (OH−) of inositol phosphate.
Based on the above idea, inventors of the present invention reached a method for producing a bone regenerating material having antimicrobial properties comprising biodegradable fibers,
Further, inventors of the present invention reached materials for bone regeneration having antimicrobial properties including biodegradable fibers.
Preferably, the inositol phosphate used herein is IP6.
Preferably, the biodegradable resins used herein are poly-L lactic acid (PLLA) or lactic acid-glycolic acid copolymer (PLGA).
Preferably, the calcium compound used in the present invention is β-phase tricalcium phosphate or calcium carbonate.
Preferably, the bone regenerating material used in the present invention is formed in a cotton wool like structure.
Since the bone regeneration material to which the antimicrobial property of the present invention is imparted carries silver on the surface of the biodegradable fiber, the bone regeneration material exhibits effective antimicrobial properties against bacterial infection in an early stage after surgery.
In the bone regeneration material imparted with the antimicrobial property of the present invention, it is possible to appropriately control the balance between the antimicrobial property and the cytotoxicity by adjusting the amount of silver fixed to the surface of the biodegradable fiber by adjusting the concentration of the silver ion solution and the amount of the calcium compound particles contained in the biodegradable fiber.
Since silver is carried only on the surface of the biodegradable fiber in the bone regeneration material imparted with the antimicrobial property of the present invention, elution of silver ions is limited to an early stage after the operation. Therefore, there is little concern that the silver ions will be eluted for a long period of time after the surgery, thereby causing cytotoxicity.
The bone regeneration material having cotton wool like structure that is imparted with the antimicrobial property contains a large amount of calcium compound particles serving as an osteogenic factor, and has controlled antimicrobial/cytotoxicity, and thus is an excellent implant material having high osteogenic ability and high safety.
Hereinafter, embodiments of the present invention will be described with reference to the drawings.
The biodegradable fibers of silver-bearing bone regeneration materials of the present invention are produced by spinning polylactic acid or lactic acid-glycolic acid copolymers, such as poly L lactic acid (PLLA) or lactic acid-glycolic acid copolymer (PLGA) as suitable matrix resins using an electrospinning process. In order to allow the calcium compound particles to be contained in a partially exposed state, outer diameter of the fiber is preferably 10 to 100 μm, more preferably 20 to 50 μm. By spinning in a state in which the calcium compound particles are uniformly dispersed and contained in the spinning solution used in the electrospinning method, the particles of the calcium compound can be uniformly dispersed in the biodegradable fiber. When amount of particles of the calcium compound contained in the biodegradable fiber is 40 to 70% by weight, particles of the calcium compound are exposed on the surface of the fiber.
As the material of silver-bearing bone regenerating material of the present invention, biodegradable fibers spun by an electrospinning method using a spinning solution containing calcium compound particles can be suitably used. Electrospun biodegradable fibers are deposited in a cotton wool like structure on a collector of an electrospinning device and collected to form a cotton wool-like bone regenerating material. The material is manufactured and sold under ReBOSSIS trademark by one of the applicants of the present application, for example, and is widely used in actual clinical practice as a bone defect filler material excellent in handleability for an operator.
The inositol phosphoric acid used to support silver on the bone regeneration material of the present invention refers to inositol phosphorylated with a hydroxyl group, and includes inositol trisphosphate (IP3 C6H15O15P3), inositol pentachyphosphoric acid (IP5 C6H17O21P5), and phytic acid (IP6 c6H18O24P6). Phytic acid (IP6) is inexpensive and has the largest number of hydroxyl groups to be chelated, so that it can be used particularly suitably.
As the calcium compound used in the bone regeneration material of the present invention, calcium phosphate, calcium carbonate, and silicon eluting calcium carbonate are suitably used. β-phase tricalcium phosphate (β-TCP) is particularly preferred in terms of osteogenic potential. It is preferable that the size of the calcium compound particles is 1 to 4 μm in order to contain the calcium compound particles in a biodegradable fiber in a state in which a part of the particles is exposed on the surface of the fiber.
Referring to
When the bone regenerating material of the present invention is implanted into a body, the biodegradable fiber 1 is dissolved over time, resulting in release of inositol phosphate 3. When silver ion 4 is dissolved in a state that it is chelate bonded with inositol phosphate 3 in the presence of calcium ions, bonding between inositol phosphate and silver is cut and silver ion 4 is replaced with calcium ion, because chelate of inositol phosphate 3 is more stable (chelating stability is higher) when it is bonded with Ca2+ than when it is bonded with Ag+. As a result, silver ion is eluted and exerts an antimicrobial effect.
When a biodegradable fiber is immersed in an aqueous silver nitrate solution, a negatively charged functional group (carboxyl group, carbonyl group, or the like) of the biodegradable resin is bonded to Ag+ and silver is attached to the surface of the fiber, but since the ionic bond between the silver ion and the functional group of the biodegradable resin is weak, the silver attached to the surface of the resin is not fixed, and is detached by cleaning with pure water. As a result, it is considered that only silver immobilized by chelating with the calcium compound particles via phytic acid remains on the surface of the biodegradable fiber after washing.
6 well plate was loaded with 0.15 g samples of fluffy bone-regenerating materials (ReBOSSIS® PLLA 30 wt %/βTCP40 wt %/silicon eluting calcium carbonate 30 wt %) and immersed in 1000 ppm concentration 6 ml IP6 solutions and left for 24 hours at room temperature (or 37° C.) humidified condition. Thereafter, IP6 solution not adsorbed to the fiber was recovered and removed. Phosphate ion concentrations of IP6 solution prior to immersion and the recovered solution were measured by inductively coupled plasma-emission spectroscopy. FIG. 7 shows the results of calculating IP6 adsorbed amounts by the phosphate ion concentrations before and after immersion. As shown in
According to the procedures shown in
According to the procedures shown in
By selecting the concentration of the silver nitrate aqueous solution within the range examined in this study, the amount of silver loaded per 1 g of bone regeneration material can be controlled from 0 to 50 mg, and it has been revealed that the amount of silver ions loaded increases as the concentration of the immersed silver nitrate aqueous solution increases.
IP6 surface-modified bone regeneration materials (ReBOSSIS) were immersed in an aqueous silver nitrate solution (concentrations 0, 1.25, 2.5, 5.0 mM) to support silver to produce samples IP6_ReBO(0), IP6_ReBO(1.25), IP6_ReB)(2.5), and IP6_ReBO(5.0), and the antimicrobial properties of each sample were evaluated by two methods: the I. Shake method and the II. Inhibition Circle method.
Antimicrobial Assessment by I. Shake Method
Medium: LB medium (1×, 1/10×)
Samples: LB medium, IP6 surface-modified to silver-loaded
IP6_ReBO(0)IP6_ReBO(1.25); IP6_ReBO(2.5); IP6_ReBO(5.0)
Escherichia coli (E. coli)
Bacterial count: 1×10 5 cells/tube
Preparation of Microbial Solution
1) Control
9 ml of LB culture medium is placed into the 50 ml centrifuge pipe, and a further 1 ml of the fungal solution prepared in 1×105 CFU/ml is added to produce the suspension.
2) Sample
Prepare 9 ml of extract, add 1 ml of bacterial suspension prepared at 1×10 5 CFU/ml, and prepare a suspension.
Set a 50 ml centrifuge tube filled with a microbial solution in a shaker at 37° C. and start culture. After 24 hours, the bacterial suspension was collected from a 50 ml centrifuge tube and the turbidity was measured using a spectrophotometer.
IP6_ReBO(0), IP6_ReBO(1.25) and IP6_ReBO(2.5) of materials for regeneration of fluffy bones (ReBOSSIS®),
0.15 g of a sample of IP6_ReBO (5.0) was filled into a mold former and pressure-molded to prepare a disc sample piece. After sterilizing the sample pieces, each of the samples was evaluated for antimicrobial properties by using the inhibition circle method.
Specifically, the aforementioned disc sample pieces were placed on LB-agar medium, to which top agar containing E. coli prepared to be 1×106 CFU/plate was overlaid. Antimicrobial properties were evaluated by observing the formation of inhibition circles after 48 hours of incubation at 37° C. and comparing the relative antimicrobial rates (
In addition, samples IP6_ReBO(5), IP6_ReBO(10), and IP6_ReBO(20) were prepared by immersing in an aqueous silver nitrate solution (concentration: 5.0, 10, 20 mM) to support silver, and the same experiment was conducted, and it was found that although the areas of the blocking bands of samples IP6_ReBO(10) and IP6_ReBO(20) were about the same, the areas were larger than those of IP6_ReBO(5). This result suggests that although the silver ion to be immobilized increases depending on the concentration of the silver nitrate aqueous solution, since the amount of silver ion immobilized reaches the amount of silver ion necessary for the antimicrobial action at a concentration of 10 mM or more of the silver nitrate aqueous solution, even if the silver ion is immobilized further, a large change does not occur in the antimicrobial level.
Animal experiments were performed using samples of silver-loaded bone regeneration materials (ReBOSSIS) using IP6 to evaluate biocompatibility. 4.1 mm diameter defect was made using drills in the right and left paw tibiae of male Japan white rabbits weighing about 3 kilograms and samples (silver-loaded cotton-shaped bone regeneration materials at 0, 5, and 10 mM aqueous silver nitrate concentrations, respectively) were implanted. At the time of implantation, the blood and the sample material which came out at the time of making the defect were mixed and then implanted. The implantation period was 4 weeks, and then number of each was 3 (
Antimicrobial tests were performed in in vivo environments using “models of mouse superficial gluteus infection”. Antimicrobial cotton-shaped artificial aggregate (Ag+ion concentration: 0, 1, 5 mM anti-microbe processing) was embedded in shallow gluteal muscles of mouse organisms with light-emitting stapler (MSSA)1×10 CFU 2 μl (each n=5). The luminescence MSSA in the mice on days 1 and 3 after implantation was measured by light imaging (IVIS) and bacterial growth changes in the mice were observed.
Observation results of bacterial growth changes in mice in animal experiment 2 are shown in
Although the preferred embodiments for carrying out the present invention have been described above, the present invention is not limited thereto, and various modifications can be made within the scope of Technology idea of the present invention.
1. Biodegradable fiber
2. Calcium compound particle
3. Inositol phosphate
4. Silver ion
5. Matrix resin
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2019/019238 | 5/15/2019 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62672618 | May 2018 | US |
