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KR-102963763-B1 - A BONE REGENERATION MATERIAL HAVING A COTTON-WOOL LIKE STRUCTURE FORMED OF A PLURALITY OF ELECTROSPUN FIBERS

KR102963763B1KR 102963763 B1KR102963763 B1KR 102963763B1KR-102963763-B1

Abstract

A bone regeneration material having a cotton-like structure formed from a plurality of electrospun fibers in which BMP-2 is bound through a β-TCP binding peptide is disclosed. The electrospun fibers contain 25-65 vol% of β-TCP particles dispersed within the fibers, so that a portion of the β-TCP particles is exposed on the surface of the electrospun fibers, while the remainder of the β-TCP particles is embedded within the fibers without being exposed to the outside. The β-TCP binding peptide fused with BMP-2 is bound to the β-TCP particles so that BMP-2 is connected to the β-TCP particles on the surface of the fibers. The bone regeneration material is embedded in a bone defect site in the human body, and BMP-2 connected to the β-TCP particles on the surface of the bone regeneration material promotes cell proliferation and differentiation at the bone defect site.

Inventors

  • 타이라 히로유키
  • 알바레즈 루이스

Assignees

  • 오쏘리버스 가부시키가이샤

Dates

Publication Date
20260512
Application Date
20191225
Priority Date
20181225

Claims (10)

  1. A bone-inductive bone regeneration material comprising a target-type bone morphogenetic protein-2 (tBMP-2) and a cotton-shaped scaffold material formed of a plurality of field-spun biodegradable fibers, The biodegradable polymer included in the above-mentioned field-spun biodegradable fiber is a lactic acid-glycolic acid copolymer (PLGA), and The above field-spun biodegradable fibers have the above-mentioned bone-inducing bone regeneration material having mechanical strength, and furthermore, have a space between the fibers of the plurality of field-spun biodegradable fibers for body fluids to penetrate through the bone regeneration material, and The field-spun biodegradable fiber contains 40 to 60 vol% of β-TCP particles, the diameter of the β-TCP particles is 1 to 5 μm, and a portion of the β-TCP particles is exposed on the surface of the field-spun biodegradable fiber, while the remainder of the particles is embedded within the field-spun biodegradable fiber. By contacting the above cotton-shaped scaffold material with the above tBMP-2 contained in an acetic acid buffer for a predetermined time, the tBMP-2 is specifically bound to the β-TCP particles exposed on the surface of the above field-spun biodegradable fiber, and The above tBMP-2 comprises a β-TCP binding peptide fused to BMP-2, and the β-TCP binding peptide comprises LLADTTHHRPWT(SEQ ID NO: 1), GQVLPTTTPSSP(SEQ ID NO: 2), VPQHPYPVPSHK(SEQ ID NO: 3), HNMAPATLHPLP(SEQ ID NO: 4), QSFASLTNPRVL(SEQ ID NO: 5), HTTPTTTYAAPP(SEQ ID NO: 6), QYGVVSHLTHTP(SEQ ID NO: 7), TMSNPITSLISV(SEQ ID NO: 8), IGRISTHAPLHP(SEQ ID NO: 9), MNDPSPWLRSPR(SEQ ID NO: 10), QSLGSMFQEGHR(SEQ ID NO: 11), KPLFTRYGDVAI(SEQ ID NO: 12), MPFGARILSLPN(SEQ ID It has an amino acid sequence selected from the group consisting of NO: 13), QLQLSNSMSSLS(SEQ ID NO: 14), TMNMPAKIFAAM(SEQ ID NO: 15), EPTKEYTTSYHR(SEQ ID NO: 16), DLNELYLRSLRA(SEQ ID NO: 17), DYDSTHGAVFRL(SEQ ID NO: 18), SKHERYPQSPEM(SEQ ID NO: 19), HTHSSDGSLLGN(SEQ ID NO: 20), NYDSMSEPRSHG(SEQ ID NO: 21), and ANPIISVQTAMD(SEQ ID NO: 22), and When the above-described osteoinductive bone regeneration material is implanted into a patient's bone defect, a body fluid containing mesenchymal stem cells penetrates into the gaps between a plurality of fibers forming the cotton-shaped scaffold material and comes into contact with the tBMP-2 combined with the β-TCP particles, thereby inducing bone formation in the bone defect.
  2. A bone-inducing bone regeneration material according to claim 1, wherein the diameter of the field-spun biodegradable fiber is 10 to 100 μm.
  3. The field-spun biodegradable fiber according to claim 1 or 2 is a bone-inductive bone regeneration material further comprising silicon-doped vaterite-phase calcium carbonate (SiV).
  4. A method for manufacturing a cotton-shaped osteo-inductive bone regeneration material formed from field-spun biodegradable fibers, wherein the method A cotton-shaped scaffold material formed from biodegradable fibers spun using an electrospinning method is produced, wherein the electrospun biodegradable fibers contain 25 to 65 vol% of β-TCP particles, the diameter of the β-TCP particles is 1 to 5 μm, a portion of the particles is exposed on the surface of the biodegradable fibers, the remainder of the particles is embedded within the electrospun biodegradable fibers, and a space is formed between the fibers for body fluids to penetrate. By contacting the above cotton-shaped scaffold material with target-type bone morphogenetic protein-2 (tBMP-2) contained in an acetic acid buffer for a predetermined time, the tBMP-2 is specifically bound to the β-TCP particles exposed on the surface of the field-spun biodegradable fiber, wherein the tBMP-2 comprises a β-TCP binding peptide fused to BMP-2, and the β-TCP binding peptide is LLADTTHHRPWT(SEQ ID NO: 1), GQVLPTTTPSSP(SEQ ID NO: 2), VPQHPYPVPSHK(SEQ ID NO: 3), HNMAPATLHPLP(SEQ ID NO: 4), QSFASLTNPRVL(SEQ ID NO: 5), HTTPTTTYAAPP(SEQ ID NO: 6), QYGVVSHLTHTP(SEQ ID NO: 7), TMSNPITSLISV(SEQ ID NO: 8), From the group consisting of IGRISTHAPLHP(SEQ ID NO: 9), MNDPSPWLRSPR(SEQ ID NO: 10), QSLGSMFQEGHR(SEQ ID NO: 11), KPLFTRYGDVAI(SEQ ID NO: 12), MPFGARILSLPN(SEQ ID NO: 13), QLQLSNSMSSLS(SEQ ID NO: 14), TMNMPAKIFAAM(SEQ ID NO: 15), EPTKEYTTSYHR(SEQ ID NO: 16), DLNELYLRSLRA(SEQ ID NO: 17), DYDSTHGAVFRL(SEQ ID NO: 18), SKHERYPQSPEM(SEQ ID NO: 19), HTHSSDGSLLGN(SEQ ID NO: 20), NYDSMSEPRSHG(SEQ ID NO: 21), and ANPIISVQTAMD(SEQ ID NO: 22) It has a selected amino acid sequence, and A method for manufacturing a cotton-shaped bone-inducing bone regeneration material formed from field-spun biodegradable fibers, comprising a process of cleaning the cotton-shaped scaffold material, in which the tBMP-2 is specifically bound to the β-TCP particles exposed on the surface of the field-spun biodegradable fibers, using a cleaning buffer solution.
  5. In paragraph 4, the cleaning buffer is PBS or a cleaning acetic acid buffer.
  6. A method according to claim 4 or 5, wherein the field-spun biodegradable fiber comprises PLGA.
  7. A method according to claim 4 or 5, wherein the field-spun biodegradable fiber further comprises silicon-doped vaterite-phase calcium carbonate (SiV).
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Description

A bone regeneration material having a cotton-wool-like structure formed of multiple electrospun fibers The present invention relates to a material for bone regeneration, and in particular to a material for bone regeneration formed from biodegradable fibers comprising β-tricalcium phosphate and bone-forming protein. Calcium phosphate (particularly β-tricalcium phosphate, β-TCP) is widely used for bone regeneration because it exhibits osteoconductivity. The release of calcium and phosphorus ions promotes bone regeneration by regulating the activation of osteoblasts and osteoclasts. Controlling the surface characteristics and porosity of calcium phosphate influences cell/protein adhesion and proliferation, thereby controlling the formation of bone minerals (Jeong Ji-woon et al., "Bioactive Calcium Phosphate Materials and Applications for Bone Regeneration," Biomater. Res. 23, 4(2019) doi:10.1186/s40824-018-0149-3). One of the applicants of this application developed a biodegradable fiber containing β-TCP using an electrospinning process, wherein a spinning solution is extruded from a nozzle as a microfiber and attracted by the electrostatic attraction of an electric field and deposited on a collector. The applicant of this application succeeded in preparing such biodegradable fibers into a cotton-wool-shaped structure containing β-TCP and a biodegradable resin using a novel electrospinning device (see U.S. Patents No. 8,853,298 and 10,092,650). The cotton-like structure is unique and provides several features, including (1) having a large space within the tissue to allow fluid to easily penetrate the structure of the bone graft material, (2) providing a large surface area to enable the release of calcium and phosphorus from β-TCP into the fluid, (3) having a flexible structure that can be adapted to the shape of the bone repair site, and (4) providing a large surface area to support other bioactive or bone-forming factors such as BMP-2. As a result of evaluating the cotton-like composite material as a bone substitute in vivo and in vitro , it was demonstrated that it is advantageous for the repair of complex bone defects. Bone morphogenetic protein-2 (BMP-2) induces osteoinduction, making it possible to promote bone formation and regeneration. For example, Infuse™ Bone Graft (Medtronic) contains a manufactured bone graft material containing recombinant human BMP-2 (rhBMP-2) and has been approved by the U.S. Food and Drug Administration (FDA) for use as a bone graft material in maxillary sinus lifts and local alveolar ridge augmentations. BMP-2 is encapsulated in an infuse and transported to the bone defect site. BMP-2 is slowly released from the bone defect site to stimulate bone formation. The stimulation of bone formation by BMP is localized and persists there for several weeks. If BMP-2 leaks to a distant site, adverse effects occur. In fact, several side effects resulting from rhBMP-2 have been reported. These side effects include postoperative inflammation and associated adverse effects, ectopic ossification, bone resorption via osteoclasts, and inappropriate lipogenesis (Aaron W. James et al., "A review of the Clinical Side Effects of Bone Morphogenetic Protein-2," Tissue Eng. Part B Rev., 2016, 22(4):284-297). FIGS. 1A to 1F are electron microscope images of ReBOSSIS® fibers. FIG. 1A shows an image of several ReBOSSIS (85) fibers (PLGA 30 wt%, SiV 30 wt%, β-TCP 40 wt%) at 200x magnification, showing the inter-fiber space between the fibers with a cotton-like structure. FIG. 1B shows an image of one ReBOSSIS (85) fiber at 2000x magnification. Calcium particles on the fiber surface can be easily identified. FIG. 1C shows the same fiber magnified at 5000x, where the white arrows indicate β-TCP particles and the dark arrows indicate SiV particles. Figure 2 shows SDS-PAGE gel images illustrating the binding of tBMP-2 and ReBOSSIS®. Panel A shows a gel image obtained using an acidic buffer (acetic acid buffer) as a washing buffer, and Panel B shows a gel image obtained using a neutral buffer (PBS) as a washing buffer. In each gel image, the four lanes on the right show the analysis results of tBMP-2, and the four lanes on the left show the analysis results of BSA. FIG. 3 is a gel image of the binding of tBMP-2 with a plurality of calcium-containing materials. tBMP2 binds well to materials containing β-TCP and/or SiV (SiV70, ReBOSSIS (85), and ORB-03). FIG. 4 is a gel image of the combination of rhBMP-2 and a material containing a plurality of calcium. rhBMP-2 is retained only on the material containing β-TCP (ReBOSSIS (85) and ORB-03) and not on the material containing SiV. Figure 5 shows a schematic diagram of a Chronic Caprine Critical Defect (CCTD) model in goats. Prior to treatment, a 5 cm segment critical defect is created in a skeletally mature female goat. A polymethyl methacrylate (PMMA) spacer measuring 5 cm in length × 2 cm in diameter is placed in the defect to induce biomembrane formation. After 4