Injectable composite material for bone repair, and preparation method thereof

Abstract

An injectable composite material for bone repair comprises a biological tissue material and bioceramics in order to serve as a three-dimensional scaffold for bone regeneration. The biological tissue material consists of microfibers having a naturally cross-linked structure without additional physical or chemical cross-linking, has superior biological compatibility, and can be slowly and completely degraded in vivo. The bioceramics in the composite material serves as a reinforcing phase. When combining the biological tissue material with the bioceramics, the composite material provides a template for bone tissue regeneration to effectively induce bone growth. The injectable composite material for bone repair can be used to fill bone defects, particularly critical-sized bone defects, and can be combined with a biological agent such as bone marrow to improve its biological activity. Therefore, the composite material can be widely used to repair bone defects caused by trauma, tumor resection, osteonecrosis, and infection.

Claims

1. An injectable composite material for bone repair, comprising the following raw materials in parts by weight: 1-7 parts of biological tissue matrix material, 1-9 parts of bioceramics, or minerals containing strontium, zinc, magnesium or silicon, or salts containing strontium, zinc, magnesium or silicon as a material for reinforcing phase, and 2-8 parts of physiological saline or other isotonic aqueous solution, wherein said biological tissue matrix material is an extracellular matrix maintaining its natural cross-linked structure, which is prepared from soft tissue of mammals, and consists essentially of collagen fibers, and wherein said biological tissue matrix material has a microfibrillar shape; the biological tissue matrix material with the microfibrillar shape has a diameter of 1 to 1500 micrometers; and the aspect ratio of the biological tissue matrix material with the microfibrillar shape is in the range of 0.05-0.95, and wherein said bioceramics or minerals containing strontium, zinc, magnesium or silicon, or salts containing strontium, zinc, magnesium or silicon are distributed in a form of microparticles within said biological tissue matrix material; and bioceramic, or minerals containing strontium, zinc, magnesium or silicon, or salts containing strontium, zinc, magnesium or silicon microparticles form a three-dimensional network structure in the biological tissue matrix material, wherein the bioceramic particles have a particle size of 1 to 500 micrometers, and wherein the biological tissue matrix material is obtained by micronizing tissue.

2. The injectable composite material for bone repair according to claim 1, wherein said soft tissue of mammals is a soft tissue from a pig, a soft tissue from a bovine, or a soft tissue from human body; said soft tissue is selected from the group consisting of skin, dermis, blood vessel, diaphragm, muscle tendon, ligament, large intestine, small intestine, and nerve tissue.

3. The injectable composite material for bone repair according to claim 1, wherein said bioceramics are hydroxyapatite, α-tricalcium phosphate, β-tricalcium phosphate, calcium hydrophosphate, calcium hydrophosphate dihydrate, calcium dihydrophosphate, tetracalcium phosphate, octacalcium phosphate, calcium sulfate, or calcium carbonate.

4. A method for preparing the injectable composite material for bone repair according to claim 1, comprising the following steps: 1) preparing a microfibrillar biological tissue matrix material, comprising the following steps: 1.1) collecting raw tissue material, cleaning blood off of the raw tissue material, cutting the raw tissue into strips having dimensions of 0.5-2 centimeters in width and 4-8 centimeters in length, and preserving the strips at −20° C.; 1.2) disinfecting and sterilizing the raw tissue material by: sterilizing the raw tissue material by using ammonia aqueous solution with a weight percentage of 0.1%; soaking the raw tissue material in the solution and slowly shaking for 6-36 hours; washing the raw tissue material with sterile deionized water; and rinsing the raw tissue material with sterile physiological saline to produce a disinfected raw tissue material; 1.3) micronizing the disinfected raw tissue by homogenizing the disinfected raw tissue material with a grinder; 1.4) decellularizing the tissue with a decellularization solution, followed by removing remaining deoxyribonucleic acid with deoxyribonuclease solution, and removing α-galactoside with a solution for α-galactoside removal; inactivating one or more viruses with a mixture solution of hydrogen peroxide, acetic acid and peroxyacetic acid; 1.5) using physiological saline with a weight percent of 0.9% to wash the material resulting from step 1.4) three or more times to remove any residues resulting from step 1.4); 1.6) washing the material resulting from step 1.5) three or more times with sterile deionized water; 1.7) terminally sterilizing the material resulting from step 1.6) by Co60 gamma rays, X-rays or electron beams to form biological tissue matrix microfibers, sealing and storing the biological tissue matrix microfibers in a closed container, and preserving the biological tissue matrix microfibers in a buffer with neutral pH, said buffer comprising physiological saline and a phosphate buffer; 2) preparing bioceramic microparticles, comprising the following steps: 2.1) obtaining bioceramic microparticles with a particle size of 0.1-500 microns from a bioceramic by a process comprising mechanical pulverization, high-rate ball-milling, and sieving, and then disinfecting and sterilizing the bioceramic microparticles to inactivate one or more viruses located theron; 2.2) mixing the bioceramic microparticles with sterile physiological saline in a ratio of 1:1 to 1:5, and stirring and vibrating the mixture of bioceramic particles and sterile physiological saline to obtain a homogeneous suspension; 3) suspending the biological tissue matrix microfibers obtained in step 1.7) in an amount of physiological saline having a weight 1-5 times that of the microfibers, and shaking the suspension until the suspension is homogeneous; 4) mixing 1-9 parts of the bioceramic particles obtained from step 2.2) with 1-7 parts of the biological tissue matrix microfibers obtained from step3); 5) vortex mixing the resulting mixture in a container for one hour until the bioceramic particles are completely adsorbed onto the biological tissue matrix microfibers; 6) removing excess water from the vortexed mixture by centrifugation at 200 to 10000 rpm, and allowing the biological tissue matrix microfibers with bioceramic particles adsorbed thereon to be deposited on the bottom of the container in solid state; 7) adding an amount of physiological saline, blood, bone marrow, or platelet plasma having a weight 1-5 times that of the biological tissue matrix microfibers with bioceramic particles adsorbed thereon to form a fluid mixture, and vortex mixing the fluid mixture to form a homogeneous fluid mixture, wherein the moisture content of the homogeneous fluid mixture in mass percent is 20-80%; 8) sealing and storing the homogeneous fluid mixture in a closed container, and sterilizing the homogeneous fluid mixture with gamma rays, X-rays or electron beams.

5. The method for preparing the injectable composite material for bone repair according to claim 4, wherein said decellularization solution in step 1.4) contains 1 to 10% of sodium deoxycholate, 2 to 15 mM of ethylenediaminetetraacetic acid, and 10 to 50 mM of 4-hydroxyethyl piperazine ethanesulfonic acid per liter; the pH of said decellularization solution is 6.8 to 7.2; said deoxyribonuclease solution contains 10 to 50 mM of 4-hydroxyethyl piperazine ethanesulfonic acid, 1 to 20 mM of calcium chloride, and 1 to 20 mM of magnesium chloride and 0.5 to 5 mg of deoxyribonuclease per liter; said solution for α-galactoside removal contains 0.2 to 10 mg of α-galactosidase, and 2 to 40 mM of 4-hydroxyethyl piperazine ethanesulfonic acid per liter; the pH of said solution for α-galactoside removal is 5.0 to 7.5; and the solution used for inactivating one or more viruses is a mixture solution of 0.10% of hydrogen peroxide, 0.50% of acetic acid, and 0.50% of peroxyacetic acid.

6. The method for preparing the injectable composite material for bone repair according to claim 4, wherein the fluid mixture of step 7) is formed with blood, bone marrow, or platelet plasma.

7. The method for preparing the injectable composite material for bone repair according to claim 4, wherein the fluid mixture of step 7) is formed with physiological saline.

8. A method for delivering stem cells to a bone defect, comprising: forming a cell suspension from bone marrow mesenchymal stem cells, adipose-derived stem cells or stem cells extracted from blood; mixing the cell suspension with the injectable composite material for bone repair according to claim 1 to form a mixture; and delivering the mixture to the bone defect.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a diagram of differential scanning calorimetry of the biological tissue microfibers;

(2) FIG. 2 shows the injectability of the biological tissue matrix/calcium hydrophosphate composite material;

(3) FIG. 3A is a scanning electron microscope image of the biological tissue matrix/calcium hydrophosphate composite material, showing typical morphology of the composite material;

(4) FIG. 3B is a scanning electron microscope image of the biological tissue matrix/calcium hydrophosphate composite material, showing distribution of the calcium hydrophosphate microparticles;

(5) FIG. 3C is a scanning electron microscope image of the biological tissue matrix/calcium hydrophosphate composite material, showing distribution of the calcium hydrophosphate microparticles;

(6) FIG. 3D is a scanning electron microscope image of the biological tissue matrix/calcium hydrophosphate composite material, showing triple-helix structure of collagen microfibers;

(7) FIG. 4A shows an implantation experiment of calvarial defect repair using the biological tissue matrix/calcium hydrophosphate composite material, showing a gross view of the mouse calvarial defect after repair;

(8) FIG. 4B shows an implantation experiment of calvarial defect repair using the biological tissue matrix/calcium hydrophosphate composite material, showing x-ray observations of the mouse calvarial defect after repair;

(9) FIG. 4C shows an implantation experiment of calvarial defect repair using the biological tissue matrix/calcium hydrophosphate composite material, showing histological staining of the mouse calvarial defect after repair;

(10) FIG. 5A shows an implantation experiment for repairing the mouse calvarial defect after combining the biological tissue matrix/calcium hydrophosphate composite material with fresh bone marrow, showing x-ray observations of the skull of the mouse after repair;

(11) FIG. 5B shows an implantation experiment for repairing the mouse calvarial defect after combining the biological tissue matrix/calcium hydrophosphate composite material with fresh bone marrow, showing micro-CT Scanning of the skull of the mouse after repair;

(12) FIG. 5C shows an implantation experiment for repairing the mouse calvarial defect after combining the biological tissue matrix/calcium hydrophosphate composite material with fresh bone marrow, showing quantitative analysis of the micro-CT Scanning;

(13) FIG. 6A shows the histological observation after repairing the skull of the mouse after combining the biological tissue matrix/calcium hydrophosphate composite material with fresh bone marrow using Von-Kassa staining (bone calcium deposition);

(14) FIG. 6B shows the histological observation after repairing the skull of the mouse after combining the biological tissue matrix/calcium hydrophosphate composite material with fresh bone marrow using the MASSON trichrome staining method; and

(15) FIG. 6C shows the histological observation after repairing the skull of the mouse after combining the biological tissue matrix/calcium hydrophosphate composite material with fresh bone marrow using the hematoxylin-eosin staining method.

DETAILS OF THE INVENTION

(16) The present invention will be described in detail with reference to the examples, which are intended to illustrate the invention rather than limit the invention.

Example 1: The Preparation and Characterization of Biological Tissue Matrix/Calcium Hydrophosphate Composite Material

(17) 1) A microfibrillar biological tissue matrix material was weighted, which was obtained from decellularized and antigen-extracted porcine dermis and sterilized by Co60 gamma rays. The material was suspended in 0.9% of physiological saline in a concentration of 10 mg/mL.

(18) 2) The material was washed 3 or more times with sterile deionized water. The parameters of the prepared decellularized matrix microfiber were shown in FIG. 1. It has an average aspect ratio of 0.05-0.95, and a particle size distribution of 40-1000 μm.

(19) 3) Calcium hydrophosphate microparticles were weighted and sterile deionized water was added to prepare a 10 mg/mL of homogeneous suspension.

(20) 4) The calcium hydrophosphate microparticles were rapidly passed through a filter membrane with a pore diameter of 40 micron and the oversized agglomerated particles therein were removed.

(21) 5) The suspension was centrifuged at 1200 rpm for 2 minutes, and the supernatant was removed.

(22) 6) 0.5 milliliter of sterile deionized water was added to 1 g of calcium hydrophosphate microparticles to prepare a paste by repeatedly vortex oscillation and stirring.

(23) 7) The calcium hydrophosphate microparticles were added into the decellularized microfiber matrix in a mass ratio of decellularized microfiber matrix to calcium hydrophosphate microparticle of 4:6.

(24) 8) The calcium hydrophosphate microparticles and the decellularized microfiber matrix were placed on a vortex mixer and fully mixed for one hour so that they were mixed homogeneously;

(25) 9) The mixture was centrifuged at 1200 rpm for 2 minutes, and the supernatant was removed.

(26) 10) 2 parts by weight of sterile physiological saline was added into 4 parts by weight of the decellularized matrix microfiber and 6 parts by weight of the calcium hydrophosphate microparticle.

(27) 11) An injectable composite material was obtained after sufficient agitation. As shown in FIG. 2, this material can be applied to the patient's wound with a standard syringe;

(28) 12) The composite material was stored as paste, and the microstructure thereof was shown in FIGS. 3A-D. The scanning electron microscope image shows that the calcium hydrophosphate microparticles are evenly distributed around the decellularized matrix microfibers without obvious agglomeration. In addition, the image under high magnification shows that the decellularized matrix consists of a large amount of collagen fibers, and the collagen fiber completely maintains its natural triple helix structure and is distributed within the composite material as micelle.

Example 2: Bone Defect Repair Experiment of the Injectable Biological Tissue Matrix/Calcium Hydrophosphate Composite Material

(29) The injectable biological tissue matrix/calcium hydrophosphate composite material prepared according to the present application was used to evaluate the bone forming ability of this material with a mouse calvarial defect model of nude mouse without thymus. The mice (6 cases) were anesthetized and then the head furs thereof were shaved off A 1 centimeter skin incision was cut along the sagittal suture of the mouse head. Circular defect with a diameter of 3.5 millimeter was made on the skull with a dental drill after putting the skin apart. The bone defect was filled with the material prepared according to the present invention, and then the wound was sutured. Six weeks later, the mice were sacrificed humanely. The skull after repairing was observed grossly. It is found that the material and the surrounding bone tissue integrated well. X-ray observation shows that a large amount of bones are formed at the defects. Histological observation also showed that new bone has successfully formed within the defect. It is found that the bone defects are filled with cells. The bone calcium staining shows that a large amount of bone calcium is deposited at the defects. The bone defects are completely bridged, as shown in FIGS. 4A-C. This example is the first which achieves repair of large area bone defect only by biological material, without adding any biological activity factor or stem cells.

Example 3: Bone Defect Repair Experiment of the Injectable Biological Tissue Matrix/Calcium Hydrophosphate Composite Material as a Carrier for Fresh Bone Marrow

(30) The injectable biological tissue matrix/calcium hydrophosphate composite material prepared in Example 1 was mixed with fresh mouse bone marrow aspirate to prepare a paste-like material. The mouse model used in Example 2 was used. The bone defects were filled with the composite material loaded with fresh bone marrow aspirate, and then the wounds were sutured. Six weeks later, X-ray observations revealed that the entire bone defect was filled with newly formed bone tissue. No tissue gap was found at the edge of the bone defects. Micro-CT scanning showed the same results that the entire bone defect had been filled with newly formed bone tissues, and a seamless connection was formed between the new bone and the implant. Quantitative analysis of bone formation showed that the material prepared in Example 1 could form much more bones than Healos® (J&J product), and the bone density thereof was closer to the autologous bone tissue (FIGS. 5A-C). Histological observations showed that the entire bone defect had been bridged by mineralized bone tissue. A large amount of bone trabeculas had been formed. Masson trichrome staining indicated that a large amount of collagens were deposited at the bone defect site and osteoblasts were found in the new bone, demonstrating that they participated in the formation of new bones. The result of the hematoxylin-eosin staining method was consistent with the results obtained in the previous two staining methods. No fibrous tissue is found around the bone defects, and bone union was achieved, such that the repair method described in this example is a viable clinical strategy.

(31) The foregoing is only an illustration of several specific embodiments of the present invention, but is not to be construed as limiting the scope of protection of the present invention. Equivalent changes or modifications in accordance the spirit of the invention are considered to fall within the protection scope of the present invention.