INJECTABLE COMPOSIT 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, characterized in comprising the following raw materials in parts by weight: 1-7 parts of biological tissue matrix material, 1-9 parts of bioceramics and 2-8 parts of physiological saline or other isotonic aqueous solution.

2. The injectable composite material for bone repair according to claim 1, characterized in that said biological tissue matrix material is an extracellular matrix maintaining its natural cross-linked structure, which is prepared from soft tissue of mammals, and is mainly comprised of collagen fibers.

3. The injectable composite material for bone repair according to claim 1, characterized in that 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 comprises skin, dermis, blood vessel, diaphragm, muscle tendon, ligament, large intestine, small intestine, and nerve tissue.

4. The injectable composite material for bone repair according to claim 1, characterized in that said biological tissue matrix material has a microfibrillar shape; the microfibrillar biological tissue matrix material has a diameter of 1 to 1500 micrometers; and the aspect ratio of the microfibrillar biological tissue matrix material is in the range of 0.05-0.95.

5. The injectable composite material for bone repair according to claim 1, characterized in that said bioceramics are replaced with bioglass, or minerals containing strontium, zinc, magnesium or silicon, or salts containing strontium, zinc, magnesium or silicon as a material for reinforcing phase; and said bioceramics are hydroxyapatite, -tricalcium phosphate, -tricalcium phosphate, calcium hydrophosphate, calcium hydrophosphate dihydrate, calcium dihydrophosphate, tetracalcium phosphate, octacalcium phosphate, calcium sulfate, or calcium carbonate.

6. The injectable composite material for bone repair according to claim 5, characterized in that said bioceramics are distributed in a form of particles within said biological tissue matrix material; and bioceramic particles 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.

7. A method for preparing the injectable composite material for bone repair according to claims 1, characterized in comprising the following steps: 1) preparing a microfibrillar biological tissue matrix material, comprising the following steps: 1.1) collecting raw tissue material, cleaning off blood, cutting into long strips of 0.5-2 centimeter wide and 4-8 centimeter long, and preserving them at 20 C.; 1.2) disinfecting and sterilizing: 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 sufficiently with sterile deionized water, and then rinsing with sterile physiological saline; 11.3) micronizing the tissue: homogenizing the disinfected raw tissue material by a grinder till micro size; 1.4) decellularizing the tissue with decellularized solution, followed by removing the remaining deoxyribonucleic acid with deoxyribonuclease solution; and removing -galactoside with a solution for -galactoside removal; inactivating virus by a mixture solution of hydrogen peroxide, acetic acid and peroxyacetic acid; 1.5) washing the tissue matrix: using physiological saline with a weight percent of 0.9% to wash the products in step 1.5) three or more times so as to remove the residues resulted from the treatment in step 1.4); 1.6) washing: washing the products in step 1.5) three or more times with sterile deionized water; 1.7) terminally sterilizing: after sterilizing by Co60 gamma rays, X-rays or electron beams, sealing and storing the resulting biological tissue matrix microfibers in a closed container, the biological tissue matrix materials are preserved in a buffer with neutral pH, said buffer comprises physiological saline and phosphate buffer; 2) preparing bioceramic microparticles, comprising the following steps: 2.1) obtaining bioceramic microparticles with a particle size of 0.1-500 micron after the steps of mechanical pulverization, high-rate ball-milling, and sieving of the bioceramics according to specific application, and then disinfecting and sterilizing the bioceramic microparticles under high temperature and high pressure to inactivate virus; 2.2) mixing the bioceramic microparticles with sterile physiological saline in a ratio of 1:1 to 1:5, sufficiently stirring and vibrating to obtain a homogeneous suspension; 3) suspending the biological tissue matrix microfibers obtained in step 1.7) into physiological saline of a weight 1-5 times of that of the microfibers, and shaking sufficiently to obtain a homogeneous suspension of biological tissue matrix microfiber; 4) mixing the bioceramic particles with the biological tissue matrix microfibers in the ratio of raw materials as defined in claim 1; 5) placing the mixture on a vortex mixer and mixing sufficiently for one hour until the bioceramic particles are completely adsorbed onto the biological tissue matrix microfibers; 6) removing excess water by centrifugation at 200 to 10000 rpm, and allowing the mixture to be deposited on the bottom in solid state; 7) adding physiological saline of a weight 1-5 times of that of the mixture, placing them on the vortex mixer to form a homogeneous fluid mixture, wherein the moisture content in mass percent is 20-80%; 8) terminally sterilizing: sealing and storing the resulting fluid mixture in a closed container, and sterilizing with gamma rays, X-rays or electron beams.

8. The method for preparing the injectable composite material for bone repair according to claim 7, characterized in that said accellular 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 accellular 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 -galactoside removal contains 0.2 to 10 mg of -galactosidase, and 2 to 40 mM of 4-hydroxyethyl piperazine ethanesulfonic acid per liter; the pH of said solution for -galactoside removal is 5.0 to 7.5; and the solution used for inactivating virus is a mixture solution of 0.10% of hydrogen peroxide, 0.50% of acetic acid, and 0.50% of peroxyacetic acid.

9. The method for preparing the injectable composite material for bone repair according to claim 7, characterized in that the solution for dispersing the bioceramic particles and the biological tissue matrix material, i.e., the physiological saline from step 7), is replaced by blood, bone marrow, or high concentration of platelet plasma.

10. A method for delivering stem cells to a bone defect, characterized in forming cell suspensions from bone marrow mesenchymal stem cells, adipose-derived stem cells or stem cells extracted from blood, and then mixing with the composite material for bone repair obtained according to claim 7, using as means for delivering stem cells to the bone defect.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0040] FIG. 1 is a diagram of differential scanning calorimetry of the biological tissue microfibers;

[0041] FIG. 2 shows the injectability of the biological tissue matrix /calcium hydrophosphate composite material;

[0042] FIG. 3 is a scanning electron microscope image of the biological tissue matrix /calcium hydrophosphate composite material:

[0043] A. typical morphology of the composite material;

[0044] B & C. distribution of the calcium hydrophosphate microparticles;

[0045] D. Triple-helix structure ofcollagen microfibers;

[0046] FIG. 4 shows an implantation experiment of calvarial defect repair using the biological tissue matrix/calcium hydrophosphate composite material:

[0047] A. Gross view of themouse calvarial defect after repair;

[0048] B. X-ray observations of the mouse calvarial defect after repair;

[0049] C. Histological staining of the mouse calvarial defects after repair;

[0050] FIG. 5 shows an implantation experiment for repairing the mouse calvarial defect after combining the biological tissue matrix/calcium hydrophosphate composite material with fresh bone marrow:

[0051] A. X-ray observations of the skull of the mouse after repair;

[0052] B. micro-CT Scanning of the skull of the mouse after repair;

[0053] C. quantitative analysis of the micro-CT Scanning;

[0054] FIG. 6 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:

[0055] A. Von-Kassa staining (bone calcium deposition);

[0056] B. MASSON trichrome staining method;

[0057] C. hematoxylin-eosin staining method.

DETAILS OF THE INVENTION

[0058] 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

[0059] 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.

[0060] 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 m.

[0061] 3) Calcium hydrophosphate microparticles were weighted and sterile deionized water was added to prepare a 10 mg/mL of homogeneous suspension.

[0062] 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.

[0063] 5) The suspension was centrifuged at 1200 rpm for 2 minutes, and the supernatant was removed.

[0064] 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.

[0065] 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.

[0066] 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;

[0067] 9) The mixture was centrifuged at 1200 rpm for 2 minutes, and the supernatant was removed.

[0068] 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.

[0069] 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;

[0070] 12) The composite material was stored as paste, and the microstructure thereof was shown in FIG. 3. 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

[0071] 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 FIG. 4. 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

[0072] 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 (FIG. 5). 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.

[0073] 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.