Preparation method of gradient mineralized cancellous bone matrix material

#The present invention relates to a technical field of bone tissue repair and regeneration, and more particularly to a natural-tissue-derived gradient mineralized cancellous bone matrix material and a preparation method thereof.

#Bone regeneration is a long process, and most of the serious bone defects are difficult to repair by themselves. Conventionally, bone graft materials have been considered as an alternative treatment than can be widely used. Among them, natural bone tissue, especially natural cancellous bone matrix, is rich in collagen, growth factors and other substances that promote cell growth and bone tissue repair. Furthermore, it also contains a lot of minerals (including Ca2+, PO4−3, Mg2+ and other ions). In recent years, more and more studies have shown that mineral ions (Ca2+, PO4−3, Mg2+, etc.) have extremely important regulating and promoting effects on the repair of new bone tissue, osteogenesis-related angiogenesis, collagen mineralization, etc. In addition, the mineral content also affects material properties such as three-dimensional structure, porosity and microscopic biomechanical. Characterization of the above materials has a significant regulatory effect on bone repair and regeneration. For example, studies have shown that the hardness and elastic modulus of the material can significantly promote osteoblast behavior induction and bone repair ability, and biological collagen mineralization is also believed by more and more scholars to significantly promote the osteogenesis and bone tissue regeneration process.

#In recent years, biomaterials derived from natural bone matrix have been gradually prepared and initially used in clinic. However, due to lack of early research on the theoretical mechanism of bone repair, the conventional bone matrix materials have certain detects. For example, it was reported that cancellous bone ECM (Extracellular matrix) scaffold material was prepared by completely removing the cellular components of the natural bone matrix, thereby reducing immunogenicity and promoting bone repair. However, bone repair needs to go through a hematoma organizing period, a callus formation period and a callus shaping period, and the application of biological materials is commonly before and after the hematoma organizing period, so the mature, dense and fully-mineralized bone ECM material cannot be well fused with new bone to promote regeneration. There are also reports in the literature that fully demineralized bone matrix materials were used for bone tissue repair and regeneration. Conventional treatment process uses strong acid and long-term EDTA-2Na soaked decalcification, which often causes irreversible damage to the natural bone ECM scaffold material and leads to the decline of biomechanical properties, the loss of minerals and growth active factors, and the change of three-dimensional microstructure. As a result, biological regeneration repair activity of the scaffold is seriously decreased.

#FIG. 1A and FIG. 1B are general appearance images of gradient mineralized decellularized cancellous bone matrix materials; FIG. 1C is a histogram illustrating that DNA content of the decellularized material is significantly reduced, and almost contains no cellular components and immunogenic substances; FIG. 1D is a histogram illustrating materials with mineralization degrees of 100%, 90%, 60% and 0% after gradient demineralization; FIG. 1E is EDS analysis diagram indicating that contents of calcium and phosphorus are decreased significantly after the material is demineralized; and FIG. 1F is Masson dyeing images indicating that exposure of immaturely mineralized collagen fiber is increased after the mineralization degree of the material is reduced, and all groups of cells are completely removed without obvious immunogenic substances after decellularization:

#FIG. 2A is scanning electron micrographs indicating that under 500 times magnification observation, surfaces of a demineralized bone ECM material are smoother and calcium nodule distribution is reduced, and under 5000 times magnification observation, exposure of mineralized collagen fiber is increased after the mineralization degree of the material is reduced; and FIG. 2B is immunohistochemical staining images indicating that BMP-2 exposure in the demineralized material is gradually increased;

#FIG. 3A and FIG. 3B are histograms indicating that porosity of the demineralized bone ECM material is increased, and stiffness of deformation resistance index is decreased; FIG. 3C is images indicating frontal force difference of the bone ECM materials with different mineralization degrees (scale bar of 1 μm); and FIG. 3D is histograms of micromechanics property changes of the hone ECM material, wherein as the mineralization degree is decreased, Young's modulus on material surfaces is decreased, deformation is increased, and adhesion force is decreased.

#The present invention provides a natural-tissue-derived gradient mineralized cancellous bone matrix material and a preparation method thereof.

#1) selecting fresh porcine scapula and washing for 4 times with sterile saline, removing cancellous bone with a 6 mm drill, and cutting the cancellous bone into cylindrical bone blocks about 2 mm high with a scalpel;

#2) then rinsing the bone blocks with sterile physiological saline for 2 hours before sending to an irradiation center for sterilizing by irradiation, wherein an irradiation dose is 25 w: