BR102013000771B1 - fiber filling to fill bone defects - Google Patents

Abstract

fiber filling to fill bone defects. a fiber filling is revealed to fill bone defects with a flocculating three-dimensional structure. the fiber filler includes several fibers which contain a biodegradable resin as a main component and a siloxane. the outer diameter of the various fibers of the filler is from about 0.05 pm to about 30 pm. the bulk density of the fiber filler is about 0.005 to 0.3 g / m³.

Description

FILLING FIBERS FOR FILLING BONE DEFECTS CROSS REFERENCE TO RELATED ORDERS

This application is a continuation application in part of US Patent Application number 12 / 707,302 filed on February 17, 2010, which claims priority from Japanese Patent Application number 2009-163320, filed on July 10, 2009. This application claims the priority of these two previous applications and Japanese Patent Application number 2011-021790, filed on February 3, 2011. The content of these three previous applications is incorporated into this document as a reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to bioactive materials that are useful as bone repair materials for filling bone defects and can be used in the fields, such as maxillofacial surgery and orthopedic surgery. More specifically, the present invention relates to a filling of fibers to fill in bone defects. The fiber filler has a three-dimensional structure, and comprises a biodegradable bioresorbable resin.

BACKGROUND OF THE INVENTION

Some materials, when grafted or implanted in bone defects, react with the bone and are chemically combined directly with the bone. These materials are called bioactive materials and are further classified into superficial bioactive materials, characterized by the fact that the reaction occurs only on the surface of the materials, and bioabsorbable materials, characterized by the fact that the reaction occurs, even inside the

2/33 materials and materials are gradually replaced with bone. Exemplary marketed surface bioactive materials include hydroxyapatite ceramics (for example, APACERAM ™ trade name provided by Hoya Corporation, Japan); and examples of marketable bioabsorbable materials include beta-phase tricalcium phosphate ceramics (eg, OSferion ™ trade name provided by Olympus Terumo Biomaterials Corp., Japan).

Calcium carbonate (CaCO ₃ ) and gypsum (CaSO ₄ H ₂ O) are also known to be resorbable. These substances, however, have low strength and hardness and are difficult to machine. In contrast, biodegradable polymers such as poly (lactic acid (s)), poly (glycolic acid (s)), copolymers thereof, and polycaprolactones are highly flexible and are easy to machine. Biodegradable polymers, however, do not have osteogenic capacity (bone formation capacity), due to their biodegradability being derived from the phenomenon that they are degraded in vivo and are discharged from it. In addition, there are some reports that some of the biodegradable polymers can affect the surrounding tissues, because they are typically degraded into lactic acid or glycolic acid upon degradation and therefore show acidity. Under such circumstances, investigations have been made to provide composite materials between these inorganic compounds and organic compounds to allow composite materials to have both osteogenic and bioresorbable capacity and still have mechanical properties

3/33 improved. For example, Unexamined Japanese Patent Application Publication (JP-A) Number 2001-294673 discloses a process for preparing a resorbable material, combining a poly (lactic acid) and a calcium carbonate. Specifically, this document refers to a process for the synthesis of a bioresorbable material, by mixing a calcium carbonate vaterite phase as a main component with a biodegradable polymeric compound, such as a poly (lactic acid), the vaterite being highly soluble in water between such calcium carbonates. This technique is also advantageous in that the pH is always maintained around neutrality, because even when the poly (lactic acid) is decomposed to be acidic, the acidity is neutralized by the buffering effects of the calcium carbonate as dissolved.

In this unprecedented aging society, bone defects should desirably be cured as soon as possible, since it is very important to maintain and ensure mastication and exercise performance to maintain health. In order to improve osteogenic capacity, the incorporation of a bioresorbable membrane has been attempted, a factor such as an inducer of bone formation (see Publication of Japanese Patent Application (JP-A) number H06 (1994) -319794), or a proliferating factor or a bone morphogenic protein (see Publication of Unexamined Japanese Patent Application (PCT Application Translation) (JP-A) number 2001-519210 and Publication of Unexamined Japanese Patent Application (JP-A) number 2006-187303). However, these factors are difficult to deal with. Consequently, demands have been made to develop

4/33 a resorbable material with superior bone reconstruction capacity, to allow the bone to self-heal more reliably and more quickly.

In view of recent research and technology trends for related biomaterials, the mainstream of research has been shifted from a design of materials for gluing a material with bone to a design of materials for bone regeneration; In these researches, the role of silicon in bone formation has received much attention, and several materials doped with silicon have been devised (TSURU Kanji, OGAWA Tetsuro and Ogushi Hajime, Recent Trends of Bioceramics Research, Technology and Standardization, Ceramics Japan, 41, 549553 (2006)). For example, it has been reported that controlled silicon release 15 can act on cells to promote bone formation (Maeda Η., T. Kasuga, and LL Hench: Preparation of Poly (L-lactic acid) -PolysiloxaneCalcium Carbonate Hybrid Membranes for Guided Bone

Regeneration, Biomaterials, 27, 1216-1222 (2006)). Independently, when compounds of a poly (lactic acid) with one of the three types of calcium carbonate (calcite, aragonite and vaterite) are prepared and soaked in a simulated body fluid (SBF), the poly (lactic acid) compound , with vaterite forms a hydroxyapatite 25 presenting bone-like composition and dimensions within a short time between the three composites (H. Maeda, T. Kasuga, M. Nogami, and Y Ota, Preparation of Calcium Carbonate Composite and Their Apatita -Forming Ability in Simulated Body Fluid, J. Ceram. Soc. Japan, 30 112, S804-808 (2004)). These results demonstrate that ο

5/33 use of vaterite, which can gradually release silicone, is believed to be the key to obtaining a material that produces faster bone reconstruction.

To use a material to fill in bone defects, the affected area (bone defect) is incised and a dense or porous material, having a size that fills the affected area sufficiently, is directly implanted or granular material is loaded into the affected area.

To ensure bone formation, it is desirable to implant or fill such material in the affected area without an opening (space). However, it is not easy to process a dense or porous material in order to adjust to the dimensions of the affected area comfortably. In addition, a granular material, which is loaded into the affected area, often moves out of the affected area after surgery (implantation). These techniques are therefore susceptible to improvement.

Independently, the guided bone regeneration technique that uses a masking membrane to cover a bone defect is also known. The guided bone regeneration technique does not involve loading a material into the affected area. Instead, this technique uses a masking membrane, which has the functions of preventing the invasion of cells and tissues that are not involved in the formation of bone within the bone defect, allowing the self-healing ability of the bone to expose, and help to rebuild the bone. This technique is intended to heal the bone defect using the healing ability that a living body inherently has. For example,

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Publication of Unexamined Japanese Patent Application (JPA) number 2009-61109 discloses a guided bone regeneration membrane and a method of producing it, characterized by the fact that guided bone regeneration membrane has a double layer structure, including a first layer layer of non-woven fabric and a second layer of non-woven fabric, characterized by the fact that the first layer of non-woven fabric contains a silicon release from calcium carbonate and a biodegradable resin as main components, and the second layer of non-woven fabric woven contains a biodegradable resin as a main component. The use of this membrane has been reported to provide satisfactory proliferation of osteoblast-like cells in mice (MC3T3-E1 cells), and when a bone defect formed in a rabbit's skull bone is covered by such a membrane, satisfactory bone formation (osteogenesis ) is observed (see T. Wakita, A. and T. Obata Kasuga New Fabrication Process of Layered Membranes Based on Poly (Lactic Acid) Fibers for Guided Bone Regeneration, Materials Transactions, 50 [7], 1737-1741 (2009)) . This membrane, however, is not used as a material for filling bone defects, because it has a small thickness of 230 to 300 pm. In addition, the apparent density of such membranes, estimated to be about 0.4 g / cm ³ or more, is generally too high to be used as a material for filling bone defects.

SUMMARY OF THE INVENTION

Therefore, an object of the present invention is to provide a fiber fill formed by a

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- * resorbable material for filling bone defects, the material may have a prolonged release system with such a chemical composition in order to effectively guide the bone reconstruction capacity and has a three-dimensional structure presenting a flexibility such as to fit in an area affected satisfactorily.

In one aspect, the present invention relates to a fiber filling for filling bone defects, 10 characterized by the fact that the fiber filling has a three-dimensional flocculation structure, including several fibers. The fibers contain a biodegradable resin as a main component and contain or carry a siloxane.

In one aspect, the fibers in a fiber fill of the present invention can be coated with hydroxyapatite on their surfaces. The biodegradable resin can be poly (lactic acid) or a copolymer thereof. The fibers may contain siloxane dispersed in microparticles of calcium carbonate.

In one aspect, the present invention relates to a fiber filler for filling bone defects which comprises several fibers consisting of a biodegradable resin and vaterite phase 25 calcium carbonate microparticles, characterized by the fact that the various fibers are tangled with each other, three-dimensionally to form a fiber fill such that the fiber density by volume is from 0.005 to 0.3 g / cm ³ .

In one aspect, the present invention relates to a

8/33 fiber filling to fill bone defects which comprises several biodegradable fibers containing several microparticles of calcium carbonate and siloxane. Biodegradable fibers are entangled with each other, three-dimensionally, to create three-dimensional spaces of about several tenths of a micrometer or more through the fiber filling structure, such that the fiber filling presents sufficient flexibility in all three-dimensional directions in the formation of a flat mat structure. The bulk density of the fiber filler as measured according to JIS L 1097 is from about 0.005 to about 0.3 g / cm ³ .

In one aspect, the present invention relates to a fiber filler for filling bone defects that comprises several biodegradable fibers containing several microparticles of calcium carbonate and siloxane. Biodegradable fibers are entangled with each other, three-dimensionally, to create three-dimensional spaces of about several tenths of a micrometer or more through the fiber filling structure, such that the fiber filling presents sufficient flexibility in all three-dimensional directions in the formation of a flat mat structure. The bulk density of the fiber filler as measured according to JIS L 1097 is from about 0.005 to about 0.3 g / cm ³ .

In one aspect, the present invention relates to a fiber filling for filling bone defects that comprises several fibers that contain a biodegradable resin, vaterite phase calcium carbonate microparticles and siloxane. The various fibers are tangled

9/33 to each other, in the three dimensional directions, to create a fiber filling structure, such that the fiber filling presents substantially the same flexibility in all three dimensional directions. The volume density of the fiber filler is 0.005 to 0.3 g / cm ³ , the compressibility of the fiber filler as measured, according to JIS L 1097, is about 10% to about 55% and the recovery of Compression of the fiber fill, as measured according to JIS L 1097, is about 5% to about 76%.

BRIEF DESCRIPTION OF THE DRAWINGS

Other objectives, characteristics and advantages of the present invention will be more fully understood from the detailed description that follows made with reference to the accompanying drawings.

In the drawings:

Figure 1 shows an explanatory view of a common electrostatic deposition technique;

Figure 2 shows an explanatory view of an electrostatic deposition technique according to an embodiment of the present invention;

Figure 3 illustrates the appearance of a three-dimensional structure of a fiber fill, prepared in Example 1, with the indication of 10 square millimeters;

Figure 4 shows a scanning electron micrograph (SEM) of fibers that make up the three-dimensional structure of a fiber fill, prepared in Example 1;

Figure 5 is a graph showing the amount of silicon ions released from a structure

10/33 three-dimensional Si-PLAi ₅ , prepared in Example 1, in a cell culture medium;

Figure 6 is a graph showing the amount of silicon ions released from a three-dimensional structure of Si-PLA _{50 /} prepared in Example 1, in a cell culture medium;

Figure 7 shows a scanning electron micrograph (SEM) of fibers that constitute a three-dimensional structure of Si-CaCO3 / PLA prepared in Example 2;

Figure 8 shows a scanning electron micrograph (SEM) of fibers that constitute a three-dimensional SiCaCO ₃ / PLA structure obtained after immersion in 1.5 SBF, the three-dimensional Si-CaCO ₃ / PLA structure prepared in Example 2;

Figure 9 illustrates an X-ray diffraction pattern of the three-dimensional Si-CaCO3 / PLA structure prepared in Example 2, before and after immersion in SBF 1.5;

Figure 10 is a graph showing the results in the cell proliferation test of a three-dimensional structure Si-CaCO3 / PLA coated with hydroxyapatite, prepared in Example 2 and a comparison sample.

Figure 11 shows a SEM photograph of silicon microparticles released by calcium carbonate obtained by the carbonation process.

Figure 12 shows an explanatory view of an electrostatic deposition technique according to an embodiment of the present invention;

Figure 13 (A) shows an explanatory view of the density measurement in fiber fill volume, prepared in Example 3. Figure 13 (B) shows an explanatory view of

11/33 measurement of compressibility of the fiber fill, prepared in Example 3, characterized by the fact that the weight is placed on the cover. Figure 13 (C) is an explanatory view of the compression recovery measurement of the fiber fill, prepared in Example 3, characterized by the fact that the weight is removed from the cover.

DETAILED DESCRIPTION

The present invention will be further described with reference to the various embodiments of the drawings. The modalities of the present invention refer to bioactive materials for filling bone defects. The materials comprise fiber filler. Such a material has a flocculating three-dimensional structure of a fibrous substance comprising a biodegradable resin, as a main component. The fibrous substance contains or carries a siloxane.

As used in this document, a three-dimensional (3D) structure is a soft and loose 3D structure. A biomaterial of the invention showing such a soft and loose 3D structure can be defined by several physical / chemical parameters, such as its weight, density in volume, compressibility, the recoverability of compression, as described in the present document. These physical / chemical parameters can be determined using known standard methods, such as the Japanese Industrial Standard (JIS) methods, in particular the JIS L 1097 method.

A siloxane is any chemical compound consisting of units of the form (R ₂ SiO) _n / characterized by the fact that R is a hydrogen atom or a hydrocarbon group

12/33 (for example, methyl, ethyl, propyl and butyl). The siloxane has a backbone that alternately comprises Si and O. A siloxane suitable for use with the embodiments of the invention can be any siloxane that can release Si in vivo. See, for example, Wakita et al., Preparation of electrospun siloxane-poly (lactic acid) -vaterite hybrid fibrous membranes for guided bone regeneration, Compos. Sci. Technol., 2010; 70: 1889-1893.

Bioactive materials of the present invention can be prepared using electrostatic deposition techniques. Electrostatic deposition uses an electrical charge to drag very thin fibers (typically on the micro or nano scale) from a liquid or paste. When a sufficiently high voltage is applied to a drop of liquid, the body of the liquid becomes charged. Electrostatic repulsion in the drop could counteract surface tension and the droplet would be stretched. When the repulsion force exceeds the surface tension, a stream of liquid erupts from the surface. This eruption point is known as a Taylor cone. If the molecular cohesion of the liquid is high enough, the current does not break and a jet of charged liquid is formed. After the jet dries in flight, the flow current changes from ohmic to convective as the load migrates to the

surface of the fiber. 0 jet is then stretched by a process of beat caused per repulsion electrostatic started with small folds at fiber, until it is finally deposited on one collector grounded. 0

fiber elongation and thinning resulting from this bending instability leads to the formation of fibers

13/33 uniform with diameters in nanometer scale.

While the voltage is normally applied to the solution or suspension in a regular electrostatic deposition process, according to the modalities of the present invention, the voltage is applied to the collector, and not to the polymeric solution (or suspension), and therefore the polymeric solution is grounded. The polymeric solution or paste is sprayed into the fibers while applying the tension in this way, and the fibers are interwoven to form a three-dimensional structure.

Alternatively, a bone defect filler material that has a three-dimensional structure and is satisfactorily flexible can also be obtained by performing the improved electrostatic deposition technique, followed by immersing the electrorotated material in a buffer solution that is supersaturated with hydroxyapatite.

Examples of biodegradable resins usable with the embodiments of the invention include synthetic polymers, such as polyethylene glycols (PEGs), polycaprolactones (PCLs), poly (lactic acid) (PLAs), polyglycolic acids (PGAs) and PEG and PCL copolymers, in addition to natural polymers, such as fibrin, collagen, alginic acid, hyaluronic acid, chitin and chitosan. Preferred examples of the biodegradable resin include a (lactic acid), poly (PLA), and a copolymer of a poly (lactic acid) and a poly (glycolic acid) (PGA) (i.e., lactic acid glycolic acid copolymer).

According to modalities of the invention, a bone defect filling material can be produced from

14/33 as follows. Initially, a solution is prepared by dissolving a biodegradable resin (eg, PLA), in a suitable solvent, eg, chloroform (CHCl ₃ ) and / or dichloromethane. An aqueous solution of aminopropyltriethoxysilane (APTES) is added to the solution. In this procedure, the weight ratio of PLA: APTES (PLA to APTES) is possibly 1: 0.01 to 1: 0.5, but is preferably 1: 0.01 to 1: 0.05 (by weight ). This is because most APTES, if added in an excessively large amount, dissolve in the initial phase of immersion in the aqueous solution and, therefore, are not as effective. The PLA has a molecular weight of about 20 x 10 ⁴ to about 30 x 10 ⁴ kDa. The concentration of PLA in the solution is preferably 4 to 12 weight percent for satisfactory spinning. In order to maintain satisfactory spinning conditions, dimethylformamide and / or methanol can be added to the solution, in a proportion of about 50 weight percent or less with respect to chloroform and / or dichloromethane.

A liquid having a relative dielectric constant greater than that of biodegradable resin can be added to the resulting solution to produce a spinning solution for the preparation of a three-dimensional structure. Typically, when the biodegradable resin is a poly (lactic acid), a liquid having a relative dielectric constant greater than that of lactic acid can be added. Examples of liquids with a relative dielectric constant greater than that of lactic acid (relative dielectric constant: 22.0) include methanol (relative dielectric constant: 32.6), ethanol (dielectric constant

Relative 15/33: 24.6), ethylene glycol (relative dielectric constant: 37.7), 1,2-propanediol (relative dielectric constant: 32.0), 2,3-butanediol, glycerol (relative dielectric constant: 42 , 5), acetonitrile (relative dielectric constant: 37.5), propionitrile (relative dielectric constant: 29.7), benzonitrile (relative dielectric constant: 25.2), sulfolane (relative dielectric constant: 43.3), and nitromethane (relative dielectric constant: 35.9). Any of these is effective, but more advantageously water (relative dielectric constant: 70 to 80) can be used. However, water is immiscible and completely separated from chloroform and / or dichloromethane used as a solvent for PLA. To avoid this problem, an amphiphilic liquid, such as methanol, ethanol, propanol and / or acetone, preferably coexists with the solvent and water. Such amphiphilic liquids for use in the invention are not limited in their relative dielectric constants, as they are amphiphilic and satisfactorily both miscible with the solvent (such as chloroform and / or dichloromethane) and water. As an example, 0.5 to 5 g of the amphiphilic liquid (such as methanol, ethanol, propanol and / or acetone) and 0.5 to 3 g of water can be added per 1 g of PLA.

The spinning solution can be further combined with a calcium carbonate to form a spinning paste. This helps to streamline (accelerate) the immersion step of the article that has undergone electrostatic deposition in a buffer solution that is supersaturated with hydroxyapatite to form the corresponding absorbable hydroxyapatite. Absorbable hydroxyapatite contributes to greater cell adhesion

Initial 16/33. The amount of calcium carbonate is possibly 60 percent by weight or less, because calcium carbonate, if added in an amount of more than 60 percent by weight, can be difficult to mix with the solution to obtain a homogeneous paste. However, calcium carbonate, if added in an amount of less than 10 percent by weight, cannot have its noticeable beneficial effects. The solution or suspension can also include one or more inorganic substances that can be used in vivo without problems. Examples of such inorganic substances include hydroxyapatite, tricalcium phosphate, calcium sulfate, chloride, sodium phosphate, sodium hydrogen phosphate, calcium hydrogen phosphate, octaccal phosphate, tetracalcium phosphate, calcium pyrophosphate and calcium chloride.

According to embodiments of the invention, a bone defect-filling material can also be a substance that contains a biodegradable resin, such as a main component and additionally or contains a siloxane. This substance can be prepared by preparing calcium carbonate containing siloxane microparticles dispersed in it (Si-CaCO ₃ ), typically by the method described in Unexamined Japanese Patent Application Publication (JP-A) number 2008-100878, and mixture of 60 weight percent or less of Si-CaCO ₃ microparticles with PLA. The amount of Si-CaCO ₃ microparticles is preferably 10 to 60 weight percent, relative to PLA, as in calcium carbonate. In order to disperse the microparticles evenly, the substance is preferably prepared

17/33 by kneading the PLA and Si-CaCO ₃ microparticles in predetermined proportions of a heating kneader to obtain a composite. The composite is then dissolved in the solvent to obtain a spinning solution.

According to a common electrostatic deposition technique, as shown in figure 1, a charge is applied by a voltage source 1 to a tip of a syringe 2. In this way, a positive charge is applied to a spinning solution, and the solution it is slowly extruded from the tip of the nozzle.

When the effect of the electric field is greater than the surface tension, the solution will be stretched into fibers and will move in relation to the collector 3, which is connected to an earth electrode. On the way to the collector 3, the solvent in the solution evaporates, thus forming a thin layer of non-woven fiber fabric. This technique, however, does not produce a three-dimensional structure, even if the spinning conditions change (such as the concentration of the spinning solution, the type of solvent contained in the solution, the speed of solution feeding, the spinning time, the applied tension and the distance between the nozzle and the collector. This is due to the fact that the residual solution and the resin deposited on the collector 3 are properly loaded and repel. Repulsion prevents deposition in the direction of the thickness. In this respect, the fibrous resin derived from the solution deposits in the collector 3 would have more solvent evaporated, however, a trace amount of the solution is deposited intact (that is, containing the solvent) on the collector 3.

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In contrast, according to the modalities of the present invention, as illustrated in figure 2, the three-dimensional fiber filling structure can be formed by performing electrostatic deposition, while grounding the syringe nozzle 2 (that is, without the application of a charge to it). At the same time, a positive charge is applied to the collector 3. According to this technique, if a normal spinning solution is slowly extruded from the tip of the nozzle, the spinning solution would fall into droplets as the solution is not loaded. However, when the spinning solution additionally contains a liquid, such as water, with a relative dielectric constant greater than that of biodegradable resin, the liquid may be affected by the electric field, and the spinning solution may be attracted to the collector by polarizing action. In this case, the wiring solution is not properly charged and easily forms three-dimensional deposits in the collector 3, without undergoing electrostatic repulsion. In this process, the liquid (solution) can be divided into two or more threads and dragged from the syringe nozzle 2 to the direction of the collector 3. These threads are intertwined to form a three-dimensional flocculant structure in the collector 3.

In order to allow this phenomenon to occur, however, the spinning solution must have a slightly low viscosity. If the wiring solution has an excessively high viscosity, it will not be able to reach the collector 3 even when affected by the electric field. Therefore, the diameter of the fibrous substance that constitutes the three-dimensional structure prepared according to the modalities of the invention can be substantially controlled by

19/33 viscosity of the spinning solution. When the spinning solution has an especially low viscosity, the fibrous substance can settle more easily to form a three-dimensional structure and the fibrous substance is more likely to have smaller fiber diameters. Typically, when a spinning solution is prepared by dissolving PLA in chloroform, to obtain a solution followed by the addition of ethanol and water, the resulting fibrous substance will have a fiber diameter in the range of about 0.05 pm to about 10 pm. It is acceptable not to apply a positive charge, but a negative charge to the collector 3, while the wiring solution may be attracted to the collector by polarization.

The three-dimensional structure resulting from a fiber filling can be cut into a piece of a desired size, and the cut piece can be soaked in a buffer solution, which contains calcium ions and phosphate ions and being saturated with respect to hydroxyapatite, to coat the surface of the fibrous main structure with hydroxyapatite. Examples of the buffer solution for use in the present invention include a Tris buffer solution (pH 7.2 to 7.4) containing ions, at a concentration substantially equal to the concentration of inorganic ion in human plasma (simulated body fluid, or SBF) and a solution (1.5 SBF), containing ions in concentrations 1.5 times that of the SBF. SBF 1.5 is more advantageous because the fibrous substance can be coated with hydroxyapatite more quickly.

According to the present modality, a flexible material is provided to fill bone defects,

20/33 the material having a three-dimensional structure of a fiber fill, including a fibrous substance, where the fibrous substance contains a biodegradable resin, represented by poly (lactic acid) (PLA), as a main component, and additionally contains or port siloxane. A filling material for bone repair is also provided, where the surface of the fibrous substance that makes up the three-dimensional structure is coated with hydroxyapatite. Such material, which includes the communication space for the entrance of the cells, presenting an improved fit in the affected area, can be easily prepared when adopting the technique for the production of a non-woven fabric through electrostatic deposition for the production of the three-dimensional structure. In addition, coating with an absorbable hydroxyapatite can be easily accomplished by immersing the article that has undergone electrostatic deposition in a buffer solution supersaturated with hydroxyapatite and the coated absorbable hydroxyapatite helps to provide greater initial cell adhesion.

The fiber filling, thus obtained, for filling bone defects has satisfactory flexibility in all three dimensional directions, in order to adjust to a bone defect derived from the three-dimensional structure constituted by the fibrous substance. The flexibility of fiber filling is substantially the same in all three dimensional directions. The fiber filling does not tend to form a flat mat structure in which the fibers are interwoven and extended in two dimensional directions. Such fiber filling shows the

21/33 high cell proliferation in the cell affinity assay using osteoblast-like cells (MC3T3El cells) and excellent bone reconstruction capacity. The bulk density of the fiber filling in this modality is much less than the masking membrane of the prior art, as disclosed, for example, JP-A 2009-61109.

According to some embodiments of the present invention, the fibers of the fiber filler are formed of a silicon compound, calcium carbonate-releasing silicon and a biodegradable polymer.

Similar to the exemplary modality discussed above, calcium carbonate microparticles vaterite phase can be used as calcium carbonate-releasing silicon, the Si content of which may be, for example, 2% by weight (in this document below) referred to as 2SiV). As described in detail in Unexamined Japanese Patent Application Publication (JP-A) number 2008-100878, calcium carbonate-release silicon can be obtained using the carbonation process, in which the carbonate gas is sprayed into a suspension of a mixture of methanol, quenched lime and organic silicone compound. Figure 11 shows a SEM photograph of calcium carbonate microparticles, which is revealed in (JP-A) number 2008100878. Although the diameters of the microparticles vary and can be changed by adjusting the production conditions, the preferred range of the diameter of the microparticles is approximately 0.5 to 1.5 pm.

A compound of silicone and calcium carbonate released from the biodegradable polymer can be formed by

22/33 heating / kneading the mixture of silicon powders vaterite and polylactic acid or a copolymer of polylactic acid and polyglycolic acid. The molecular weights of polylactic acid or a copolymer of polylactic acid and polyglycolic acid are preferably 150,000 to 300,000. In order to obtain a desirable elasticity of the fiber filling, the content of 2SiV of the compound is preferably from 30% to 40% by weight. This compound can be dissolved with a solvent (for example, CHCl3) to obtain a spinning solution. The spinning solution thus obtained can be transformed into a fiber fill using electrostatic deposition techniques.

In order to process the spinning solution in the fiber fill using electrostatic deposition, as illustrated in figure 12, a positive voltage is applied to the spinning solution in a state in which a collector container filled with ethanol is grounded. The spinning solution is then made into fibers and the fibers undergo electrostatic deposition being attracted and traveling to the collector container, while the solvent of the spinning solution is evaporated in the electric field during the process. These fibers attracted to the collector vessel are accumulated in the vessel's ethanol to form a cotton-like structure. When changing the wiring conditions (such as the density of the wiring solution, the type of solvent, the delivery speed, electrostatic deposition time period, the applied voltage, the distance between the nozzle and the ethanol in the collector vessel) , desired morphology of the fiber fill can be obtained.

The outer diameter of the fiber filling of this

The embodiment is preferably between about 0.05 pm to about 30 pm, preferably from about 0.1 pm to about 20 pm, and more preferably from about 10 pm to about 20 pm. The bulk density of the fiber filler, as measured according to JIS L 1097, is from about 0.005 g / cm ³ to about 0.3 g / cm ³ , preferably from about 0.01 g / cm ³ to about 0.1 g / cm ³ , more preferably about 0.014 g / cm ³ to about 0.021 g / cm ³ . The compressibility of the fiber fill in this embodiment is from about 10% to about 55%, preferably from about 20% to about 55%, more preferably from 29% to 55%. The compression recovery of the fiber fill in this embodiment is from about 5% to about 76%, preferably from about 7% to about 76%, more preferably from about 44% to about 58%.

Advantage of the Invention

As the fiber filler of the present invention is formed by Si releasable calcium carbonate and a biodegradable polymer, it shows high cell proliferation and has excellent bone reconstruction capacity. In comparison to the masking membrane, the volume density of a fiber filler of the present invention is very low. In addition, a fiber filler of the present invention is flexible and has excellent elasticity. Therefore, it can be easily filled in bone defects during surgery and can be implanted in bone defects without gap (spacing).

Example 1 and Example 2

The present invention will be illustrated in more detail with reference to the various examples below, which are

24/33 refer to the methods of producing three-dimensional structures. It should be noted, however, that these examples are illustrated for a better understanding of the present invention and are not intended to limit the scope of the present invention in any way. One skilled in the art will appreciate that various modifications are possible without thereby departing from the scope of the invention.

Raw materials used in Example 1 and Example 2

Poly (lactic acid) (PLA): PLPURASORB PL PolÍ (Llactide), with a molecular weight of 20 x 10 ⁴ to 30 x 10 ⁴ obtained from PURAC Biochem (a division of CSM; Netherlands). Chloroform (CHC1 ₃ ): analytical grade reagent, with a purity of 99.0% or more, obtained from Kishida Chemical Co., Ltd., Japan, γ-aminopropyltriethoxysilane (APTES): (TSL8331, with a purity of 98% or more, obtained from GE Toshiba Silicones Co., Ltd., Japan).

Calcium carbonate doped with siloxane (Si-CaCO ₃ ): Vaterita containing a siloxane in terms of a silicon ion content of 2.9 percent by weight, and prepared using the extinct lime (Microstar T; with a degree of purity 96% or more; Yabashi Industries Co., Ltd., Japan), methanol (analytical grade reagent, with a purity of 99.8% or more; Kishida Chemical Co., Ltd., Japan), APTES, and gas carbon dioxide (greater purity of liquefied carbon dioxide, with a purity of 99.9%; Taiyo Kagaku Kogyo KK, Japan).

Electrostatic deposition conditions in Example 1 and Example 2

Spinning solution feed rate: 0.1 mL / minute,

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Applied voltage: A voltage was applied to the plate collector at [25 kV, while the nozzle is grounded,

Distance between the nozzle and the plate collector: 100 mm,

Spinning time: about 60 minutes

Example 1

APTES (1 g) was added to ultrapure water (0.5 g) with stirring to provide a solution. The solution was added dropwise to an 8 wt% PLA solution in CHCl ₃ to provide an APTES content of 0.015 g and 0.050 g, respectively, followed by stirring. During this procedure, APTES was condensed to provide a siloxane. The resulting mixtures received the addition of 1.5 g of ethanol and 1 g of ultrapure water in order to provide spinning solutions. These spinning solutions were subjected to electrostatic deposition and thus yielded three-dimensional structures, each including a fibrous substance containing a biodegradable resin, as a main component and containing or presenting a siloxane (hereinafter these steric structures are referred to as SÍ-PLA15 and Si- PLA ⁵⁰ , respectively).

Figure 3 shows the appearance of the resulting three-dimensional structure (SÍ-PLA15). Figure 4 shows a scanning electron micrograph (SEM) of this steric structure, demonstrating that the structure is a steric structure and is a flocculent structure including fibers with diameters from several tens of nanometers to eight micrometers. Structures under this condition have a weight of 40 mg. The structures do not lose their flexibility and elasticity, even after being embedded in a cell culture medium and recovered from it.

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Each of the steric structures prepared above was cut into a 10 mm wide, 10 mm long and 1 mm thick piece, soaked in 4 ml of a-MEM cell culture medium, maintained at a temperature of 37 ° C in an incubator, in a 5% carbon dioxide gas atmosphere, and the cell culture medium was changed to fresh medium on day 1, day 3, and day 5. Figure 5 and figure 6 show the amounts of release of silicon ions, when steric structures were embedded in cell culture medium, as measured by inductively coupled plasma emission spectrometry. The data demonstrate that both samples (steric structures) released a large amount of silicon ions on day 1, and thereafter released silicon ions in a significantly decreased amount, but continued to release silicon ions until at least day 7 SiPLA ₅ released approximately 6.5 ppm of silicon ions on day 1, but released 1 ppm or less of silicon ions from day 6 to day 7, showing only a small difference from that in SÍ-PLA15.

Example 2

A three-dimensional Si-CaCO ₃ / PLA structure was prepared by kneading PLA and Si-CaCO ₃ in a kneader with heating at 200 ° C for 15 minutes in order to provide a Si-CaCO ₃ / PLA compound containing 40 weight percent Si-CaCO ₃ ; 1.67 g of the mixture of the compound SiCaCO ₃ / PLA with 8.33 g of CHC1 ₃ in order to provide a solution, adding 1.5 g of ethanol and 1 g of ultrapure water to the solution to obtain a spinning solution, and subjecting the wiring solution to electrostatic deposition

27/33 under the conditions mentioned above.

The prepared three-dimensional structure had a flocculent appearance substantially the same as shown in figure 3 and had superior flexibility and elasticity. Figure 7 is a scanning electron micrograph (SEM) of the three-dimensional Si-CaCO ₃ / PLA structure, which demonstrates that this structure is a steric structure, including fine fibers with diameters from about 0.1 to about 3 μτη and spherical particles of calcium carbonate having diameters of about 1 pm incorporated between the fibers. The fibers have small diameters and the gaps (spacing) between the fibers are large enough to be several tens of micrometers or more, in order to give enough space to allow the cells to enter them. The amount of silicon ion release from this steric structure was determined by the procedure described in Example 1, to find that the steric structure released silicon ions in amounts of 5.3 ppm on Day 1, 0.8 ppm from day 2 on day 3, 0.4 ppm from Day 4 to day 5, and 0.4 ppm from Day 6 to Day 7, indicating that the release of trace amount of silicon ion continued.

The steric structure was cut into a sample piece 10 mm wide, 10 mm long and 10 mm thick, soaked in 40 mL of 1.5 SBF, and kept at 37 ° C for one day. The sample piece was then recovered from 1.5 SBF and observed under a scanning electron microscope (SEM), to find that many aggregated particles, as shown in figure 8, were precipitated and that spaces of about

28/33 several tens of micrometers, in order to allow the cells to enter. Figure 9 shows the X-ray diffraction pattern of the sample piece before and after immersion in 1.5 SBF, demonstrating that the peaks from hydroxyapatite were observed in the sample piece after immersion. These results demonstrate that the fiber surfaces that make up the three-dimensional Si-CaCO ₃ / PLA steric structure can be easily coated with a hydroxyapatite just by immersing the steric structure in 1.5 SBF.

Figure 10 shows how cell numbers (in terms of cell number per 1 cm ² ) vary after inoculation of cells similar to murine osteoblasts (MC3T3-E1 cells) on the steric structure coated with hydroxyapatite and on a comparative sample ( Thermanox: plastic disk for cell culture). The comparative sample of Thermanox was treated on its surface to improve cell proliferation and for use in cell culture. The data in figure 10 demonstrate that the stereochemical structure provides a much higher growth capacity of the cells than that of the comparative sample treated on the surface and is expected to be a material that stands out in the capacity of bone reconstruction.

Conditions for Cell Culture Experiment

Cultivation:

Cultivation using 24-well plate,

Cell type: cells similar to murine osteoblasts (MC3T3-E1 cells; Riken Institute of Physical and Chemical Research, Japan).

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Cell inoculation number: 1 x 10 ⁴ cells / well.

Medium: a-MEM (containing 10% fetal bovine serum).

Exchange Medium: the day after the inoculation, then every other day

Sample piece: The sample a steric 3D structure was cut into a piece 10 mm long, 10 mm wide and 10 mm thick (that is, a 10 mm cube).

Cell counting method: The measurement was performed using the Cell Counting Kit-8 (cell growth / cell toxicity analytical reagent; Dojindo

Laboratories, Japan) , according to the protocols attachments to reagent. Example 3 Carbonate calcium from stage (18 g) presenting a silicon content 2% by weight (2SiV) and polylactic acid classification medical (42 g) were

mixed and heated / kneaded at 200 ° C for 45 minutes. They were then cooled a composite (SiPVH composite), where the content of 2SiV was 30% by weight.

g of SiPVH thus obtained is dissolved in chloroform (9.3 g) and stirred to obtain a spinning solution. This spinning solution is loaded on an electrostatic deposition machine and undergoes electrostatic deposition to obtain the fiber filling, under the following conditions.

Electrostatic deposition conditions

Feeding speed of the spinning solution: 0.2 mL / minute, Voltage applied: 17 kV, and a distance between the nozzle and collector (filled with ethanol): about 20 cm, Nozzles: Syringe: 18G.

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As shown in figure 12, the fibers accumulated in ethanol in the collector are recovered from the collector (figure 12). Samples 1 to 4 were prepared from the filling of fibers recovered from the collectors and analyzed. Volume density, compressibility and compression recovery were measured according to JISL1097.

1) Diameter of fibers

The diameters of the fibers were measured using an 18-point laser microscope. It was found that, while the diameter of each fiber so measured varied significantly, the diameter distribution range was around 10 μτη ~ 21 pm.

2) Bulk density

Each of samples 1 to 4 (average weight: 0.055 g) was placed in a glass cylinder with an internal diameter of 22 mm. The circular glass cover with approximately the same diameter (weight: 1.148 g) was placed over the fiber filling in the glass cylinder. The height of the fiber filling (h ₀ ) in the glass cylinder was measured with the lid placed on it. The volume density of the fiber filler was calculated based on the volume of the fiber fill so measured and its weight, (as shown in figure 13 (A)).

The result was that, although it varied significantly between samples, the volume densities of each sample were as follows: Sample 1: 0.015 g / cm ³ ; Sample 2: 0.014 g / cm ³ ; Sample 3: 0.018 g / cm ³ ; and Sample 4: 0.021 g / cm ³ (mean: 0.017 g / cm ³ ).

3) Compressibility and compression recovery

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As shown in figure 13 (B), a weight of 9.914 g is placed on the glass cover arranged inside the glass cylinder. After 30 minutes had passed, the height of the fiber filling (h _x ) in the glass cylinder, in that state was measured. From the change before and after placing the weight on the cap, the compressibility of the fiber fill was calculated using the following formula.

Compressibility (%) = (h ₀ -hi) / h ₀ x 100

The compressibility of the fiber filling of samples 1 to 4 was: Sample 1: 45.56%, Sample 2: 34.90%, Sample 3: 29.01%, Sample 4: 37.80% (36.82% in average).

Then, as shown in figure 13 (C), the weight is removed from the glass cover. After 30 minutes, the height of the fiber filling (h ₂ ) in the glass cylinder in that state was measured. From the change before and after placing the weight on the cover and removing the weight from the cover, the compression recovery of the fiber fill was calculated using the following formula: Compressibility recovery (%) = (h ₂ -h _x ) / (h ₀ -hi) x 100

The compression compression of the fiber filling of Sample 1 to 4 was as follows: Sample 1: 58.47%, Sample 2: 44.02%, Sample 3: 56.14%, Sample 4: 57.20% ( average 53.96%).

JIS Test Method L1097 - 1982 (confirmed in 2008)

The Japanese Industrial Standard (JIS) published the L 1097 standard method for the synthetic fiber filling test in 1982, which was confirmed in 2008. The test was conducted in a room with class 2 standard temperature and humidity (temperature 20 + 2 ° C, relative humidity 65 ± 2%) of

32/33 according to JIS Z8703 (standard test room condition).

Sample Preparation

Remove filling pieces from an unpackaged product and leave each piece in the room for more than 8 hours. Then, randomly choose samples for testing. The test samples of 20 cm x 20 cm in size are stacked until their weight becomes about 40 g. The parts of the test samples needed to conduct the test are left in that state for about an hour.

The tests could include the color test, specific volume (in volume), compressibility (compression, recovery), fiber length and quality.

Test Method

Specific Volume (Volume Density)

The specific volume is measured by measuring the weight of the test samples prepared as described above. A thick plate (a flat plate measuring 20 x 20 cm in size and 0.5 g / cm ² ) is placed on a test piece and a weight of A (2 kg) is placed on it for 30 minutes. Then, the weight is removed and a test sample is left for 30 minutes in that state. This procedure is repeated three times. After the weight To be removed and left for thirty minutes in that state, the heights of the sample in the four corners are measured and an average height is obtained. The specific volume is calculated using the following formula. The test must be performed with three samples and an average of three samples is obtained.

Specific volume (cm ³ / g) - (20 x 20 xh ₀ ) / 10) / W where h ₀ is an average height value in the four corners

33/33 of the test sample (mm), and W is the weight of the test sample (g). The specific volume (cm ³ / g) can be readily converted to density values (g / cm ³ ), if desired.

____ elasticity of ____ compression ____ (compressibility, compression, recovery)

The compression elasticity is measured by measuring the height of the four corners of a test piece described above. A B mass (4 kg) is placed on it for 30 minutes. The heights at the four corners are measured. After that, weight B is removed, and the test sample is left for 3 minutes. The heights at the four corners are measured. Their average values are obtained. Compressibility and compression recovery are calculated according to the following formula. The test is conducted on three samples, and an average of the three samples is obtained.

Compressibility (%) = (h ₀ -hi) / h ₀ ) x 100

Compressibility recovery (%) = (h ₂ -hi) / (h ₀ -hi) x 100

h ₀ : average height in the four corners, before placement weight: B over them (mm). h _i: average height in four corners with weight B placed about the same (mm) h ₂ : average height of four corners B after weight B is removed (mm).

While the above description is one of the preferred embodiments of the present invention, it will be appreciated by one skilled in the art that the invention can be modified or altered without departing from the scope and meaning of the claims that follow.

Claims (12)

1. Filling of fibers to fill bone defects characterized by the fact that it comprises: a plurality of biodegradable fibers containing a plurality of microparticles of calcium carbonate and siloxane, and being produced by the use of an electrostatic deposition apparatus having a collector filled with ethanol, in which fibers which have undergone electrostatic deposition emitted from an apparatus nozzle electrostatic deposition enters the collector's ethanol, and the fibers sink into the ethanol and are deposited in the collector, where the plurality of biodegradable fibers are entangled with each other to form a flocculating three-dimensional structure having a volume density of about 0.01 g / cm ³ to about 0.1 g / cm ³ , as measured according to JIS L 1097, wherein the plurality of biodegradable fibers comprises a biodegradable resin and a releasable calcium compound Si. 2. Filling of fibers according to claim 1, characterized by the fact that the releasable calcium compound Si comprises calcium phosphate 3. Filling of fibers according to claim 1, characterized by the fact that the outer diameter of the biodegradable fibers is from about 0.05 pm to about 30 pm. 4. Filling of fibers according to claim 1, characterized by the fact that the biodegradable resin comprises poly (lactic acid) or a copolymer thereof. 5. Filling of fibers according to the claim Petition 870200035420, of 03/16/2020, p. 13/15 2/3 1, characterized by the fact that the compressibility of the fiber filler, as measured according to JIS L 1097, is from about 10% to about 55%. 6. Filling of fibers, according to claim 1, characterized by the fact that the compressibility of the fiber filling, as measured according to JIS L 1097 is from about 20% to about 55%. 7. Filling of fibers according to the claim 1, characterized by the fact that the compressibility of the fiber filler, as measured according to JIS L 1097 is from about 29% to about 55%. 8. Filling of fibers, according to claim 1, characterized by the fact that the compression recovery of the fiber filling, as measured according to JIS L 1097 is from about 5% to about 76%. 9. Filling of fibers according to claim 1, characterized by the fact that the compression recovery of the fiber filling, as measured according to JIS L 1097 is from about 7% to about 76%. 10. Filling of fibers, according to claim 1, characterized by the fact that the compression recovery of the fiber filling, as measured according to JIS L 1097 is from 44% to about 58%. 11. Filling of fibers to fill bone defects characterized by the fact that it comprises: a plurality of biodegradable fibers produced by the use of an electrostatic deposition apparatus having a collector filled with ethanol, in which fibers that have undergone electrostatic deposition emitted from a nozzle of the Petition 870200035420, of 03/16/2020, p. 14/15 3/3 device in electrostatic deposition come in in ethanol of collector, and the fibers sink into ethanol and they are deposited at the collector, in that the plurality of fibers biodegradable comprises a biodegradable resin and an element in formation in bone, and wherein the plurality of biodegradable fibers are entangled with each other to form a three-dimensional flocculant structure having a volume density of about 0.01 g / cm ³ to about 0.1 g / cm ³ , compressibility of about 10 % to about 5% compression to according to JIS L 1097. 12. Filling of 55% and recovery of about 76%, as measured fibers, according to claim 11, characterized in that the bone-forming element comprises a releasable calcium compound Si.