CN109172863B

CN109172863B - Method for modifying nano decalcification bone matrix particle coating of polycaprolactone-tricalcium phosphate bone tissue engineering scaffold

Info

Publication number: CN109172863B
Application number: CN201810948498.2A
Authority: CN
Inventors: 陈雄生; 王智巍; 苑博; 周盛源; 唐一钒; 许国峰
Original assignee: Second Affiliated Hospital Army Medical University
Current assignee: Second Affiliated Hospital Army Medical University
Priority date: 2018-08-20
Filing date: 2018-08-20
Publication date: 2021-04-27
Anticipated expiration: 2038-08-20
Also published as: CN109172863A

Abstract

The invention discloses a method for modifying a nano decalcified bone matrix particle coating on a polycaprolactone-tricalcium phosphate bone tissue engineering scaffold, which comprises the following steps: mixing the allogeneic nano decalcified bone matrix material with 75% ethanol, soaking the polycaprolactone-tricalcium phosphate bone tissue engineering scaffold in the diluted nano decalcified bone matrix slurry, and placing in liquid nitrogen to finish preliminary freezing; and (3) putting the obtained frozen scaffold into a vacuum freeze dryer, freeze-drying for at least 48 hours to finish surface coating modification, and obtaining the polycaprolactone-tricalcium phosphate bone tissue engineering scaffold modified by the nano decalcified bone matrix particle coating. The invention adopts a low-temperature freeze-drying method, thereby effectively ensuring the activity of the nanometer DBM biomaterial; on the basis of reserving the pores of the original stent, the osteogenic capacity of the stent is increased. The invention provides a certain theoretical basis for the development of bone graft substitutes and bone defect repair materials.

Description

Method for modifying nano decalcification bone matrix particle coating of polycaprolactone-tricalcium phosphate bone tissue engineering scaffold

Technical Field

The invention relates to the technical field of bone tissue engineering, in particular to a method for modifying a nano decalcified bone matrix particle coating on a polycaprolactone-tricalcium phosphate bone tissue engineering scaffold.

Background

The repair of bone defects has been a difficult and hot spot in the orthopedic field. At present, autologous bone, allogeneic bone or tissue engineering bone is generally adopted for filling treatment for treating bone defects. Autologous bone, as a gold standard for bone defect repair, has ideal properties of inducing osteogenesis, conducting osteogenesis and osteogenesis, however, its source is limited and there are complications such as infection of supply area, chronic pain, etc. Although the source of the allogeneic bone is wide and has good osteogenesis inducing and bone formation conducting capabilities, the source control is limited, and meanwhile, the allogeneic bone has risks of pathogen transmission, immunological rejection and the like. More researchers are focusing on tissue engineered bone.

Bone tissue engineering studies have shown that natural bone is, at the microscopic level, a multi-layered, structurally complex nanocomposite material ([1] Li X, Wang L, Fan Y, Feng Q, Cui FZ, Watari f. nanostructured scans for bone tissue engineering. j biomedical materials Res a.2013.101(8):2424-35.), inorganic nano calcium phosphate and hydroxyapatite crystals deposited between extracellular matrices, together with organic nanostructured collagen fibers and some protein molecules that promote bone formation promote cell growth and osteogenic differentiation ([2] sources CD, Jansen JA, Leeuwenburgh sc. synthesis and application of nanostructured calcium phosphate ceramics gene generation. j biomedical materials applicator. B. application.6.2318.23126). The nature of the organic polymer itself, which is easily modified, makes it common to use as the organic portion of the scaffold, mainly nanofibers ([3] polyplane H, Lebourg M, Ripalda P, et al. biomedical hydroxyl coating on pores improprovides oil extraction of poly (L-lactic acid) scaffold. J Biomed. Mater Res B Appl. biometer.2013.101 (1): 173-86.). Recent researches show that the proportioning structure of the nanofiber scaffold is extremely similar to the structural characteristics of I-type collagen fibers in natural bones, and can effectively promote the adhesion growth of cells so as to promote the regeneration and reconstruction of bone tissues (4 Gupte MJ, Ma PX. Nanofibrous scaffold for denal and cardiovascular applications. J Dent Res.2012.91(3): 227-34). BMSCs are extremely sensitive to nanoscale materials, and the nano structure can effectively increase the sensing capability of filopodia, promote cell diffusion and adhesion, and enable the filopodia to rapidly proliferate in a 3D micro-environment formed by nano fibers, and the main reason is that the special surface structure of the nano material can obviously increase the specific surface area of protein adsorption and cell adhesion ([5] Stevens MM, George JH.expansion and engineering the cell surface interface.science.2005.310(5751): 1135-8.).

At present, there are many reports on organic-inorganic composite nanomaterials, and the hybrid scaffold material shows better biocompatibility and osteogenic induction characteristics than inorganic materials such as hydroxyapatite alone ([6] Yan LP, Silva-Correia J, Correia C, et al.Bioactive macro/micro porous with porous materials for bone-tissue-engineering applications. Nanomedicine (Lond) 2013.8(3):359-78.[7] Yan LP, Oliveira JM, Oliveira AL, Caridade SG, Mano JF, Reis.Macro/porous fibrous tissue strain with porous tissue strain, and [ 6.6 ] calcium oxide, molecular tissue strain, and strain E.12.12.12.12.8. molecular tissue strain, tissue strain E.12.12.12.12.8.12.12.12.12.12.12.12.12.12.12.12.12.12.12.12.12.12.12.12.12.12.12.12.12.12.12.12.12.12.12.12.12.12.12.12.12.12.12.12.12.12.12.12.12.1.12.12.12.12.12.12.1.12.12.12.12.1.12.12.12.12.12.12.12.12.12.12.12.12.12.1.12.12.12.12.12.12.12.12.12.1.12.12.12.12.12.12.12.12.1.12.12.12.12.12.12.12.12.12.12.12.: [9] Kim H, Che L, Ha Y, Ryu W.mechanical-repaired electronic composite site cellulose nanoparticles Mater Sci Eng C Mater Biol appl.2014.40: 324-35). Chae et al ([10] Chae T, Yang H, Leung V, Ko F, Troczynski T. novel biological hydrogel/alginate nanocomposite fibers for bone tissue regeneration. J. Mater Sci Mater Med.2013.24(8):1885-94.) uniformly deposit nHAP particles along the direction of alginate nanofibers, avoid inorganic salt aggregation caused by conventional mechanical mixing or electrostatic spinning methods, and effectively promote the adhesion of osteoblasts on the surface of nHAP/alginate scaffold. Qian et al ([11] Zhou H, Touny AH, Bhaduri SB.Fabrication of novel PLA/CDHA bionical porous fibers for tissue engineering Via electrospanning.J. Mater Sci Mater. Meer Med.2011.22(5):1183-93.) prepared a nHAP/PLGA composite scaffold (lactic acid-polyglycolic acid copolymer and nano-hydroxyapatite), which has significantly enhanced biocompatibility compared with pure nHAP and can effectively promote adhesion, proliferation and differentiation of MC3T3-E1 cells. Ganesh N ([12] Ganesh N, Ashokan A, Rajeshkannan R, Chennanazhi K, Koyakutty M, Nair SV.magnetic resistance functional nano-no-hydro-tissue in-doped poly (calcoacetane) complex scaffolds for in situ monitoring of bone tissue regeneration by MRI.tissue Eng Part A.2014.20(19-20):2783-94.) A composite scaffold was designed, in vitro cell experiments showed that expression of nHAP/PCL scaffold alkaline phosphatase (ALP) was increased by about 43% compared to a pure PCL scaffold, and that expression of Runx-2 gene on nHAP/PCL scaffold was also earlier than that of a pure PCL scaffold, indicating that the composite scaffold had better osteogenic induction than that of a scaffold with a single nanostructure.

Although more and more synthetic nanocomposite scaffold materials exhibit good biocompatibility and osteogenesis inducing properties, studies on the processing of natural bone tissues into nanoscale materials and their application to scaffold surface modification have rarely been reported. A scaffold material suitable for bone tissue regeneration should also have a porous structure with a pore diameter of at least 100 μm to allow for absorption and transmission of nutrients and oxygen (13 Rouwkema J, Rivron NC, van Blittterswijk CA. Vasculation in tissue engineering. trends Biotechnol.2008.26(8): 434-41.). Scaffolds with pore sizes of 200-. Pores of unequal size are more favorable for cell proliferation and differentiation ([15] Woodard JR, Hilldore AJ, Lan SK, et al. the mechanical properties and osteo-conductivity of hydro-xapatite bone scans with multi-scale porosity. biomaterials.2007.28(1): 45-54.). The PCL-TCP scaffold prepared in the early stage of the experimental group has the pore size of 350-450 mu m and the porosity of 50 percent, and meets the optimal requirement of bone tissue growth.

An ideal bone graft substitute should of course mimic as much as possible the real bone growth environment. The introduction of tissue engineering provides a new idea for the repair of bone defects. The scaffold, the seed cells and the active growth factors form three elements of bone tissue engineering, wherein the scaffold is the most important. The ideal bone tissue engineering scaffold should have the following conditions that 1) the internal interconnected pore structure has both macropores (the pore diameter is more than 100 mu m) and micropores (the pore diameter is less than 20 mu m), which is beneficial to the growth of tissues, the material transfer and the vascularization; 2) the material is made of biodegradable or absorbable material, has enough biomechanical strength and controllable degradation dynamics at the same time, and can transmit load to surrounding tissues; 3) good interface affinity, and is beneficial to the adhesion, proliferation and differentiation of cells; 4) is easy to prepare into various forms and sizes; 5) has the ability of controllably releasing active biological factors.

Polycaprolactone (PCL) is an artificially synthesized macromolecular organic compound and has good biocompatibility, biodegradability and plasticity. But the material has no bioactivity, poor mechanical strength, too slow degradation speed, smooth surface and strong hydrophobicity, is not suitable for osteoblast adhesion and bone tissue regeneration, and is often used in combination with one or more other biological materials to enhance the induced osteogenesis property or the biomechanical strength. The composite material has obviously enhanced mechanical strength after being compounded with Tricalcium phosphate (TCP), belongs to ceramic calcium inorganic biological materials, has chemical components and crystal structures similar to those of natural bone mineral substances, has good osteogenesis inducing and conductive properties and sufficient mechanical strength, can promote osteoblast adhesion and bone tissue deposition, but has high brittleness and poor biological absorbability. The advantages of the two can be complemented by 3D printing technology. The Decalcified Bone Matrix (DBM) mainly comprises 93% of collagen fibers (surface active substances for conducting osteogenesis), 5% of soluble proteins (BMPs for inducing osteogenesis, cooperative proteins such as transforming growth factors and insulin-like growth factors) and 2% of residual mineralized Matrix, has a microstructure very similar to that of natural Bone, and is a commercial biological material with good osteogenesis inducing and Bone formation conducting properties. The organic combination of the three has not been reported in the literature.

CN201410023751.5 discloses a moldable bone repair material for bone repair, which is prepared from the following raw materials: 20-90 parts of alpha-calcium sulfate hemihydrate and hydroxyapatite, 20-40 parts of bioactive mineral powder, 10-80 parts of autologous bone powder particles or DBM particles, and hydrogel with the total dosage of the raw materials being 1: 0.5-1: 15. The plastic repairing material for bone repair is suitable for bone repair by in-situ curing and in-vitro rapid curing after wound injection, wherein all components are proved to be degradable materials with good biocompatibility.

CN200510027391.7 discloses a method for preparing a nano decalcified bone matrix material (DBM) for bone regeneration, comprising the following steps: 1) removing soft tissue from fresh healthy adult bone to obtain bone pieces of different sizes, washing with flowing water for 2-6hr, dehydrating with anhydrous ethanol for 1-3hr, defatting with diethyl ether for 0.5-2hr, drying in ventilated place, freeze drying at-10 deg.C to-100 deg.C, and pulverizing into 0.5-2.5mm granule; 2) soaking in anhydrous ethanol for 1-3hr, soaking in diethyl ether for 1-3hr, washing with distilled water for 2-5 times, soaking in hydrochloric acid for 2-8hr, soaking in diethyl ether for 0.5-2hr, drying in ventilated place to obtain demineralized bone matrix DBM, and freeze-drying at-10 deg.C to-100 deg.C in refrigerator; 3) grinding DBM to 1-300nm nanometer particles by a grinder, and then sterilizing by gamma rays or ethylene oxide to obtain the nanometer bone regeneration material. The nano DBM prepared by the method can obviously improve the performance of the DBM in the following three aspects: 1) the internal particle structure of the DBM is constructed by using nano, the internal porosity of the whole graft can be artificially regulated, and the bone conduction function of the DBM can be reasonably guided so as to meet the requirements of various bone grafts. 2) After the DBM is subjected to nano treatment, the hydrophobicity of the DBM is enhanced, the DBM is more stable after being implanted in a body, the antigenicity of the DBM can be reduced, and the osteogenesis inducing activity is more stable. 3) The nanometer technology is applied to construct the DBM, so that the biomechanical reduction effect caused by the decalcification of the DBM can be reduced as much as possible, and the DBM is more suitable for the bone supporting effect during bone grafting fusion.

CN200810202524.3 discloses an allogeneic bone graft substitute material nano DBM, which is characterized in that the diameter of the nanoparticle is 50-100nm, and the preparation method is as follows: 1) preparing a DBM bone block from fresh allogeneic bone by adopting an improved Urist method according to a conventional method; 2) placing the DBM bone blocks into a liquid nitrogen freezing pulverizer to be pre-pulverized to obtain DBM powder; 3) preparing nano DBM: placing DBM powder into an ultrafine grinding pulverizer, adding a proper amount of distilled water, keeping the temperature at 17-25 ℃, increasing the rotating speed from 800r/min to 1200r/min, fully grinding, performing high-speed centrifugation on homogenate, removing supernatant to obtain nano DBM, drying, subpackaging, sealing and sterilizing. The invention takes the allogeneic bone as the raw material, and the material is easy to obtain; the nanometer DBM can be shaped randomly; because of the nano structure, the nano-structure can be used as a bone graft substitute material, has good biocompatibility, can be used as a good carrier of exogenous growth factors for promoting bone regeneration such as bone morphogenetic protein and the like, and has good capability of promoting bone regeneration.

Disclosure of Invention

The invention aims to provide a method for modifying a nanometer decalcified bone matrix particle coating on a polycaprolactone-tricalcium phosphate bone tissue engineering scaffold, and particularly provides a theoretical basis for designing a novel bone defect repairing material by coating nanometer DBM particles on the surface of a PCL-TCP bone tissue engineering scaffold by a freeze-drying method.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

the invention provides a method for modifying a nano decalcified bone matrix particle coating on a polycaprolactone-tricalcium phosphate bone tissue engineering scaffold, which comprises the following steps:

mixing an allogeneic nano Decalcified Bone Matrix (DBM) material with 75% ethanol, soaking a polycaprolactone-tricalcium phosphate bone tissue engineering scaffold (PCL-TCP bone tissue engineering scaffold) in diluted nano Decalcified Bone Matrix (DBM) slurry, and placing the nano Decalcified Bone Matrix (DBM) slurry in liquid nitrogen to finish preliminary freezing; and (3) putting the obtained frozen scaffold into a vacuum freeze dryer, freeze-drying for at least 48 hours to finish surface coating modification, and obtaining the polycaprolactone-tricalcium phosphate bone tissue engineering scaffold modified by the nano decalcified bone matrix particle coating.

The volume ratio of the allogeneic nano Decalcified Bone Matrix (DBM) material to 75% ethanol is 1 (5-10), preferably 1: 5; specifically, the ratio can be 1:5, 1:6, 1:7, 1:8, 1:9 and 1: 10.

The polycaprolactone-tricalcium phosphate bone tissue engineering scaffold (PCL-TCP bone tissue engineering scaffold) is constructed by a 3D printing technology; the 3D printing technique is a fused deposition modeling technique.

The preparation method of the polycaprolactone-tricalcium phosphate bone tissue engineering scaffold (PCL-TCP bone tissue engineering scaffold) comprises the following steps:

mixing Polycaprolactone (PCL) particles and Tricalcium phosphate (TCP) powder according to the ratio of (3-6): 1, then fully and uniformly mixing the materials by a torque rheometer, and preparing the materials into granular mixed materials suitable for 3D printing by a granulator;

firstly, CAD software matched with a 3D printer is utilized to design a PCL-TCP bone tissue engineering scaffold model, the fiber diameter is 0.5mm, the layer height is 0.3mm, and the fiber superposition angle of each layer is 0/90 degrees; and then placing the prepared granular mixed material into a melting tank, setting the feeding temperature of the melting tank to be 120 ℃, the discharging temperature to be 110 ℃, and keeping the temperature of the granular mixed material in the melting tank for more than 120 minutes to ensure the granular mixed material to be fully melted, extruding fibers from the melted material through a motor-assisted micro-injector type fine nozzle, and superposing layer by layer to form a polycaprolactone-tricalcium phosphate bone tissue engineering bracket (PCL-TCP bone tissue engineering bracket).

The preparation method of the allogeneic nano decalcified bone matrix material comprises the following steps:

first, preparing a nano Decalcified Bone Matrix (DBM) bone block: removing soft tissues from fresh allogeneic bone, and preparing a massive nano Decalcified Bone Matrix (DBM) bone block by adopting an improved Urist method;

second, preparing nano Decalcified Bone Matrix (DBM) powder: placing the massive nano decalcified bone matrix bone blocks obtained in the first step into a zirconia nano grinding tank, placing zirconia grinding balls, screwing down the tank opening, placing the grinding tank into a full-automatic liquid nitrogen freezing grinder for pre-crushing to obtain nano Decalcified Bone Matrix (DBM) powder with the particle size of 5-10 microns;

thirdly, preparing an allogeneic nano Decalcified Bone Matrix (DBM) material: and (3) putting the nano Decalcified Bone Matrix (DBM) powder obtained in the second step into a zirconia nano grinding tank, adding zirconia grinding balls, adding 75% ethanol until the grinding balls are completely covered, putting the zirconia nano grinding tank into a full-automatic low-temperature high-energy ball mill for ball milling, and obtaining the allogeneic nano Decalcified Bone Matrix (DBM) material with the particle size of 20-50 nm.

The preparation of the massive nanometer Decalcified Bone Matrix (DBM) bone block by the improved Urist method comprises the following steps:

removing soft tissue from fresh allogeneic bone, processing the allogeneic bone into bone blocks with size of about 1cm × 1cm, precisely weighing, and freezing at-80 deg.C;

placing the cryopreserved allogeneic bone blocks into a beaker, and soaking in absolute ethyl alcohol for 2 hours for dehydration; pouring off the absolute ethyl alcohol, adding ether for degreasing, and soaking for 12 hours; removing diethyl ether, washing with large amount of sterile distilled water for 3 times; adding 0.6mmol/L hydrochloric acid for decalcification, soaking for 72 hr, changing hydrochloric acid every 12 hr during soaking, stirring with precise power-increasing electric stirrer, washing with sterile distilled water for 5 times, and soaking overnight; soaking with anhydrous ethanol for 2 h; removing ethanol, adding ether, and soaking for 1 hr; removing the ether, and placing the bone blocks on a ventilation experiment table to volatilize overnight; freeze-drying in a vacuum freeze dryer, packaging and sealing for later use to obtain the massive nanometer Decalcified Bone Matrix (DBM) bone block.

In the second step, zirconia grinding balls with the diameter of 1.5cm are used in the zirconia nano grinding tank.

In the second step, the pre-crushing setting parameters are 30 times/second, and the grinding time is 5 min.

In the third step, zirconia grinding balls with the diameter of 1mm are used in the zirconia nano grinding tank.

And step three, setting the temperature to be 25-35 ℃, the rotating speed to be 1500r/min and the grinding time to be 20min in a full-automatic low-temperature high-energy ball mill, automatically reducing the rotating speed of the ball mill to 500r/s when the temperature in the grinding tank exceeds 35 ℃, and automatically grinding the ball mill again at a high speed when the temperature of the grinding tank is reduced to 30 ℃ by a water cooler.

The second aspect of the invention provides a polycaprolactone-tricalcium phosphate bone tissue engineering scaffold modified by a nano decalcified bone matrix particle coating, which is prepared by the preparation method.

The third aspect of the invention provides an application of the polycaprolactone-tricalcium phosphate bone tissue engineering scaffold modified by the nano decalcified bone matrix particle coating prepared by the preparation method in preparing bone defect repair materials or bone graft substitutes.

Due to the adoption of the technical scheme, the invention has the following advantages and beneficial effects:

in the method for carrying out nano decalcification bone matrix particle coating modification on the polycaprolactone-tricalcium phosphate bone tissue engineering scaffold, the allogeneic bone is adopted as the allogeneic bone, so that the material is easy to obtain, the source is wide, and the clinical requirements can be met; the whole process adopts controllable low-temperature grinding, so that the damage of heat generated inside the grinding tank to collagen and active protein components in the DBM in the grinding process is avoided, and the characteristics of inducing osteogenesis and conducting osteogenesis of the DBM are better kept; the plasticity is good, and the material can be used alone or as a mixed or coating material; the nano-scale DBM has good biocompatibility and is not easy to cause adverse reactions such as organism rejection and the like.

In the method for modifying the nano decalcified bone matrix particle coating by using the polycaprolactone-tricalcium phosphate bone tissue engineering scaffold provided by the invention, the activity of the nano DBM biomaterial is effectively ensured by adopting a low-temperature freeze-drying method; on the basis of reserving the pores of the original stent, the osteogenic capacity of the stent is increased. The invention provides a certain theoretical basis for the development of bone graft substitutes and bone defect repair materials.

In the method for modifying the nano decalcified bone matrix particle coating by the polycaprolactone-tricalcium phosphate bone tissue engineering scaffold provided by the invention, the nano DBM is a bioactive material and cannot be added into 3D printing in a high-temperature process for direct mixed printing, the nano DBM is coated on the surface layer of the scaffold, and the method is more favorable for accelerating the adhesion, proliferation, migration and differentiation of osteoblasts in the early post-operation stage.

Drawings

Fig. 1 is a scanning electron microscope image of a polycaprolactone-tricalcium phosphate bone tissue engineering scaffold before freeze-drying a coating in a method for modifying a nanometer decalcified bone matrix particle coating by using the polycaprolactone-tricalcium phosphate bone tissue engineering scaffold provided by the embodiment of the present invention.

FIG. 2 shows a comparative example of the present invention based on polycaprolactone: and (3) a scanning electron microscope image of freeze-drying the tricalcium phosphate 8:2 printed bone tissue engineering scaffold and nano DBM particle slurry diluted by distilled water or normal saline after coating is shown, and as can be seen from the image, the DBM is unevenly covered on the surface of the scaffold and blocks the pores of the part of the scaffold.

Fig. 3 is a scanning electron microscope image of a polycaprolactone-tricalcium phosphate bone tissue engineering scaffold subjected to nano decalcified bone matrix particle coating modification in the method for performing nano decalcified bone matrix particle coating modification on the polycaprolactone-tricalcium phosphate bone tissue engineering scaffold provided by the embodiment of the invention, wherein the polycaprolactone-tricalcium phosphate bone tissue engineering scaffold and nano DBM particle slurry diluted by 75% ethanol are subjected to freeze-drying coating.

Fig. 4 is an enlarged scanning electron microscope image of the polycaprolactone-tricalcium phosphate bone tissue engineering scaffold shown in fig. 3 after being coated with a freeze-dried slurry of nano DBM particles diluted with 75% ethanol.

Fig. 5 is an enlarged scanning electron microscope image of the polycaprolactone-tricalcium phosphate bone tissue engineering scaffold shown in fig. 4 after being coated with a freeze-dried slurry of nano DBM particles diluted with 75% ethanol.

Fig. 6 is a scanning electron microscope image of an allogeneic nano-decalcified bone matrix material in the method for performing nano-decalcified bone matrix particle coating modification on the polycaprolactone-tricalcium phosphate bone tissue engineering scaffold provided by the embodiment of the present invention.

Fig. 7 is an enlarged view of the scanning electron microscope image of the allogeneic nano-decalcified bone matrix material shown in fig. 6.

Fig. 8 is an enlarged view of the scanning electron microscope image of the allogeneic nano-decalcified bone matrix material shown in fig. 7.

Detailed Description

In order to more clearly illustrate the invention, the invention is further described below in connection with preferred embodiments. It is to be understood by persons skilled in the art that the following detailed description is illustrative and not restrictive, and is not to be taken as limiting the scope of the invention.

The main reagents and instruments used in the invention are as follows:

the relative molecular mass of polycaprolactone, 65000, was purchased from Sigma-Aldirch, usa; tricalcium phosphate, having an average particle size of 500nm, was purchased from Nanjing Epimeri nanometer, Inc., China; normal saline was purchased from chenxin pharmaceutical industry, inc; 75% ethanol was purchased from national pharmaceutical group, china; torque rheometer Rheomix 600Haake germany; the 3D printer was Motor Assisted Microsyringe, purchased from fuqifan electromechanical corporation, china; christ vacuum freeze dryer, shanghai zhiyao instruments ltd, china; high resolution thermal field emission scanning electron microscope, Sigma300, available from ZEISS Merlin, germany.

The PCL-TCP bone tissue engineering scaffold is successfully constructed in the early stage test of the invention, and the nano DBM particle slurry is prepared. The key point is how to effectively attach the nano DBM particle slurry on the surface of the PCL-TCP bone tissue engineering scaffold. The freeze-drying method can effectively coat the DBM particle slurry on the surface of the stent in a low-temperature environment and ensure the activity of the DBM biomaterial; meanwhile, physicochemical and biological denaturation of the material in the drying process is prevented to the maximum extent, growth of microorganisms and enzyme action cannot be performed, drying is performed under a vacuum condition, and easily-oxidized substances are protected, so that the original properties can be maintained, the biological material stored in a freeze-dried manner can be immediately dissolved after being added with water, the stability is good, the chance of pollution is reduced, and the biological material can be stored for a longer time.

According to the invention, the nanometer decalcified bone matrix is finally compounded with the PCL-TCP 3D printing support in a vacuum freeze-drying mode after multiple attempts, so that a novel organic-inorganic nanometer composite support is constructed. After several attempts, the ndma/PCL-TCP composite scaffold is finally prepared in a vacuum freeze-drying manner, because the DBM is thickened, but the liquid nano-slurry has poor adhesion when being combined with the scaffold and cannot be directly combined. In the invention, the nDBM composite stent is constructed in a mode of electrostatic or air spraying on the surface of the stent, but the spraying effect is poor, and the stent and the nDBM can not be effectively combined; then, the stent and the diluted nDBM slurry are soaked and then put into a 37 ℃ incubator for natural drying, but the nDBM is completely deposited at the bottom of the stent after drying, and no obvious nano slurry is attached to the surface and in the pores of the stent.

The PCL-TCP scaffold is firstly soaked in nDBM diluent by adopting a vacuum freeze-drying method, after the scaffold is frozen in a refrigerator at the temperature of minus 80 ℃, the nDBM can be fixed in the state of being soaked with the scaffold, and then the solid liquid is directly sublimated by a vacuum freeze-dryer, so that the nDBM is fixed on the surface of the scaffold fiber. The freeze-drying method can prevent the physicochemical and biological denaturation of the material in the drying process to the maximum extent, the growth of microorganisms and the action of enzymes cannot be carried out, the drying is carried out under the vacuum condition, the easily oxidized substances are protected, the original properties can be kept, the biological material stored in the freeze-drying method can be immediately dissolved after being added with water, the stability is good, and the pollution chance is reduced, so the biological material can be stored for a longer time ([15] Woodard JR, Hilldore AJ, Lan SK, et al.the mechanical properties and the biocompatibility with multi-scale porosity. biomaterials.2007.28(1): 45-54.).

Proper dispersant is needed for dispersing the nano material in the liquid phase, the dispersion (dispersant) of the nano decalcified bone matrix is not reported, and in order to avoid the damage of the biological activity of the nDBM, the solution selected by dilution is sterile distilled water, normal saline and ethanol used in the decalcification of allogeneic bone. In the dilution process, after the distilled water and the normal saline are mixed with the nDBM slurry, the nDBM is agglomerated and solidified, which indicates that the nDBM is not easy to dissolve in the distilled water and the normal saline; after adding 75% ethanol with the ratio of 1:5 into the nDBM slurry, the nDBM is uniformly dispersed without solidification and precipitation; when the diluted concentration of the nDBM and the ethanol is 1:1 and 1:2, the nDBM is unevenly dispersed on the surface of the stent and partially blocks pores of the stent, and when the diluted concentration is 1:10, the concentration of the nDBM is too low, and partial surfaces of fibers of the stent are not adhered with the nDBM, which is also the reason for finally selecting the ethanol with the ratio of 1:5 as the diluent of the nDBM.

Example 1

A method for modifying a nanometer decalcified bone matrix particle coating of a polycaprolactone-tricalcium phosphate bone tissue engineering scaffold comprises the following steps:

mixing allogeneic nano Decalcified Bone Matrix (DBM) material with 75% ethanol, and diluting the nano slurry

An allogeneic nano Decalcified Bone Matrix (DBM) material (prepared in example 3) is mixed with 75% ethanol according to the volume ratio of 1:5, and the mixture is fully vibrated to ensure that nano DBM particles are uniformly distributed in the ethanol.

The mixing conditions were as follows: 20-25 ℃ at room temperature, instrument: the temperature control range of the table type constant temperature shaking table NHWY-100B is 5-60 ℃ (30 ℃ constant temperature is adopted), the rotating speed is 40-300 rmp, the amplitude range is phi 26mm, and the time is 5 min.

Second, preliminary freezing

Soaking the PCL-TCP bone tissue engineering scaffold (20-25 ℃ at room temperature for 2 minutes) in diluted nano Decalcified Bone Matrix (DBM) slurry, placing the slurry in a 20ml plastic test tube, fully oscillating the test tube again, immediately placing the plastic test tube in liquid nitrogen, and completing preliminary freezing (instruments used for preliminary freezing have no special requirements, and both a liquid nitrogen test vessel and a common plastic test tube can be normally placed). The polycaprolactone-tricalcium phosphate bone tissue engineering scaffold (PCL-TCP bone tissue engineering scaffold) is constructed by a 3D printing technology; the 3D printing technique is a fused deposition modeling technique.

mixing Polycaprolactone (PCL) granules with Tricalcium phosphate (TCP) powder according to a ratio of 8:2, then fully and uniformly mixing the mixture by a torque rheometer, and preparing the mixture into a granular mixed material suitable for 3D printing by a granulator.

Firstly, CAD software (HTS slice software purchased from Fuqifan company) matched with a 3D printer is utilized to design a PCL-TCP bone tissue engineering scaffold model, the fiber diameter is 0.5mm, the layer height is 0.3mm, and the stacking angle of each layer of fiber is 0/90 degrees; and then placing the prepared granular mixed material into a melting tank, setting the feeding temperature of the melting tank to be 120 ℃, the discharging temperature to be 110 ℃, and keeping the temperature of the granular mixed material in the melting tank for more than 120 minutes to ensure the granular mixed material to be fully melted, extruding fibers from the melted material through a motor-assisted micro-injector type fine nozzle, and superposing layer by layer to form a polycaprolactone-tricalcium phosphate bone tissue engineering bracket (PCL-TCP bone tissue engineering bracket).

Thirdly, finishing coating modification by freeze-drying

And (3) putting the preliminarily frozen scaffold into a vacuum freeze dryer, and freeze-drying for 48 hours to finish surface coating modification to obtain the polycaprolactone-tricalcium phosphate bone tissue engineering scaffold modified by the nano decalcified bone matrix particle coating.

A vacuum freeze dryer: labconco, Freezone lyophilizer, Cold trap temperature-84 ℃, vacuum: <1.5mbar, lyophilization time 48 hours.

Fourth, observation by scanning electron microscope

Spraying gold on the polycaprolactone-tricalcium phosphate bone tissue engineering scaffold modified by the nano decalcified bone matrix particle coating obtained in the third step to prepare a sample, and observing the shape change of the surface of the scaffold under a scanning electron microscope. As shown in fig. 1 and 3 to 5, fig. 1 is a scanning electron microscope image of a polycaprolactone-tricalcium phosphate bone tissue engineering scaffold before freeze-drying a coating in a method for modifying a nanometer decalcified bone matrix particle coating by using the polycaprolactone-tricalcium phosphate bone tissue engineering scaffold provided by the embodiment of the present invention. Fig. 3 is a scanning electron microscope image of a polycaprolactone-tricalcium phosphate bone tissue engineering scaffold subjected to nano decalcified bone matrix particle coating modification in the method for performing nano decalcified bone matrix particle coating modification on the polycaprolactone-tricalcium phosphate bone tissue engineering scaffold provided by the embodiment of the invention, wherein the polycaprolactone-tricalcium phosphate bone tissue engineering scaffold and nano DBM particle slurry diluted by 75% ethanol are subjected to freeze-drying coating. Fig. 4 is an enlarged scanning electron microscope image of the polycaprolactone-tricalcium phosphate bone tissue engineering scaffold shown in fig. 3 after being coated with a freeze-dried slurry of nano DBM particles diluted with 75% ethanol. Fig. 5 is an enlarged scanning electron microscope image of the polycaprolactone-tricalcium phosphate bone tissue engineering scaffold shown in fig. 4 after being coated with a freeze-dried slurry of nano DBM particles diluted with 75% ethanol. As can be seen from fig. 1, 3, 4 and 5, the surface of each fiber of the scaffold is uniformly covered with the nano DBM, the bottom layer is dense, the combination with the scaffold is good, nano DBM particles uniformly distributed on the surface layer of the scaffold are shown, the nano DBM is partially agglomerated, and the surface has rich nano grooves (fig. 3, 4 and 5).

The results show that: the method of the invention is used for finishing the nano DBM particle coating modification of the PCL-TCP bone tissue engineering scaffold, and the nano DBM particles are uniformly distributed.

Example 2

An allogeneic nano Decalcified Bone Matrix (DBM) material (prepared in example 3) is mixed with 75% ethanol according to the volume ratio of 1:10, and the mixture is fully vibrated to ensure that nano DBM particles are uniformly distributed in the ethanol.

The remaining coating steps were the same as in example 1.

Example 3

The preparation method of the adult allogeneic nano decalcified bone matrix material comprises the following steps:

firstly, after soft tissues of fresh adult allogeneic bone are removed, a massive nanometer Decalcified Bone Matrix (DBM) bone block is prepared by adopting an improved Urist method

(1) Source of bone tissue

Allogeneic bone used for bone tissue decalcification is derived from hip joint of joint surgery in 2015 to 2016 of joint surgery and femoral head, tibial plateau and femoral condyle parts cut by knee replacement patients in the surgery of subsidiary long-term hospital of naval military medical university, approved by medical ethics committee of Shanghai long-term hospital and approved for clinical research. The donors selected all met the following specifications of the american tissue bank association: no history of acute and chronic infectious diseases; no toxic substance contact and drug absorption history; no history of tumor, sexually transmitted diseases, hepatitis and the like; no history of application of chemoradiotherapy drugs or long-term large-dose hormones; three routine tests before the operation check the normal. The allograft bone 1400.9g was collected altogether.

(2) Allogeneic bone treatment

Removing soft tissues, periosteum and marrow in marrow cavity attached to the allogeneic bone, removing articular cartilage as much as possible from bone tissues around the joint, washing with sterile distilled water, processing the allogeneic bone into bone blocks with size of about 1cm × 1cm by using tools such as rongeur, weighing by using a precision electronic balance instrument, and freezing and storing the allogeneic bone blocks in a refrigerator at-80 ℃.

(3) Preparation of massive nanometer Decalcified Bone Matrix (DBM) bone block by improved Urist method

Putting the allogeneic bone blocks frozen in the step (2) into a 2000ml beaker, and soaking in absolute ethyl alcohol for 2h for dehydration; pouring off the absolute ethyl alcohol, adding ether for degreasing, and soaking for 12 hours; removing diethyl ether, washing with large amount of sterile distilled water for 3 times; adding prepared 0.6mmol/L hydrochloric acid for decalcification, soaking for 72h, replacing hydrochloric acid every 12h in the soaking process, and continuously stirring with a precise force-increasing electric stirrer to ensure that the bone blocks are fully contacted with hydrochloric acid and decalcification; rinsing with sterile distilled water for 5 times and soaking overnight to ensure sufficient removal of hydrochloric acid; soaking with anhydrous ethanol for 2 h; removing ethanol, adding ether, and soaking for 1 hr; removing the ether, and placing the bone blocks on a ventilation experiment table to volatilize overnight; freeze-drying in a vacuum freeze dryer, packaging and sealing for later use to obtain 257g of blocky nano Decalcified Bone Matrix (DBM) bone blocks.

Secondly, preparing nano Decalcified Bone Matrix (DBM) powder

A proper amount of freeze-dried massive nano Decalcified Bone Matrix (DBM) bone blocks are put into a zirconia grinding tank with the capacity of 30ml, zirconia grinding balls with the diameter of 1.5cm are put into the zirconia grinding tank, after the tank opening is screwed down, the grinding tank is put into a CryoMill full-automatic liquid nitrogen freeze grinder (Leichi company, Germany) for pre-crushing, the parameters are set for 30 times/second, and the grinding time is 5min, so that nano Decalcified Bone Matrix (DBM) powder with the particle size of 5-10 mu m is obtained. In the whole grinding process, the addition and supplementation of liquid nitrogen and the precooling of the instrument are automatically controlled by a program.

Thirdly, preparing an allogeneic nano Decalcified Bone Matrix (DBM) material:

putting the nano Decalcified Bone Matrix (DBM) powder into a zirconia nano grinding tank with the capacity of 50ml, adding zirconia grinding balls with the diameter of 1mm, uniformly mixing with the DBM, and adding 75% ethanol until the grinding balls are completely covered. Then the nano grinding tank is placed into an E-Max full-automatic low-temperature high-energy ball mill (E-Max, Leichi company, Germany), and the parameters are set as follows: 1500r/s for 20min, setting the temperature threshold value to be 25-35 ℃, automatically reducing the rotating speed of the ball mill to 500r/s when the temperature in the grinding tank exceeds 35 ℃, and automatically grinding the ball mill again at high speed when the temperature of the grinding tank is reduced to 30 ℃ by the water cooler; obtaining the allogeneic nano Decalcified Bone Matrix (DBM) material with the grain diameter of 20-50nm, sucking out the material by a suction pipe and putting the material into a 20ml plastic test tube.

Observation by scanning electron microscope

And (3) spraying gold on the allogeneic nano Decalcified Bone Matrix (DBM) material obtained in the third step to prepare a sample, and observing the surface appearance under a scanning electron microscope. As shown in fig. 6 to 8, fig. 6 is a scanning electron microscope image of an allogeneic nano-decalcified bone matrix material in a method for performing nano-decalcified bone matrix particle coating modification on a polycaprolactone-tricalcium phosphate bone tissue engineering scaffold provided by an embodiment of the present invention, fig. 7 is an enlarged view of the scanning electron microscope image of the allogeneic nano-decalcified bone matrix material shown in fig. 6, and fig. 8 is an enlarged view of the scanning electron microscope image of the allogeneic nano-decalcified bone matrix material shown in fig. 7; as can be seen from fig. 6 to 8, the DBM bottom layer is compact and is in fibrous interconnection, the fiber has different thicknesses and diameters of 40 to 80nm, the surface is filled with irregular nanoparticles, the particle size is not uniform, the diameter is 20nm to 50nm, the nanoparticles are mutually agglomerated, nano-level grooves are densely distributed on the surface, the nano-structure fibers are interconnected, a plurality of micron-level pore structures are formed inside the fibers, and the pores have different sizes and are communicated with one another.

The results show that: after the DBM prepared by the improved Urist method is physically ground at low temperature, the microstructure of the prepared nano DBM accords with the category of nano biological materials.

Example 4

The preparation method of the rabbit allogenic nano decalcified bone matrix material comprises the following steps:

100g of fresh bone was obtained from New Zealand rabbit (provided by the animal experiment center of the university of naval military medical science), and the preparation process of the other allogeneic nano-decalcified bone matrix material was the same as that of example 3.

Example 5

An allogeneic nano Decalcified Bone Matrix (DBM) material (prepared in example 3) is mixed with 75% ethanol according to the volume ratio of 1:7.5, and the mixture is fully vibrated to ensure that nano DBM particles are uniformly distributed in the ethanol.

The remaining coating steps were the same as in example 1.

Comparative example 1

Mixing allogeneic nano Decalcified Bone Matrix (DBM) material with distilled water or normal saline, diluting the nano-slurry

The allogeneic nano Decalcified Bone Matrix (DBM) material (prepared in example 3) is mixed with distilled water or normal saline according to the volume ratio of 1:5, and the mixture is fully vibrated to ensure that nano DBM particles are uniformly distributed in the distilled water or the normal saline.

Second, preliminary freezing

And (3) soaking the PCL-TCP bone tissue engineering scaffold in diluted nano Decalcified Bone Matrix (DBM) slurry, placing the nano Decalcified Bone Matrix (DBM) slurry in a 20ml plastic test tube, fully oscillating the nano decalcified bone matrix again, and immediately placing the plastic test tube in liquid nitrogen to finish preliminary freezing. The polycaprolactone-tricalcium phosphate bone tissue engineering scaffold (PCL-TCP bone tissue engineering scaffold) is constructed by a 3D printing technology; the 3D printing technique is a fused deposition modeling technique.

Thirdly, finishing coating modification by freeze-drying

And (3) putting the preliminarily frozen stent into a vacuum freeze dryer, and freeze-drying for 48 hours to finish surface coating modification. The observation of the scanning electron microscope is shown in figure 2, and figure 2 shows that the polycaprolactone is used as a comparative example of the invention: and (3) a scanning electron microscope image of freeze-drying the tricalcium phosphate 8:2 printed bone tissue engineering scaffold and nano DBM particle slurry diluted by distilled water or normal saline after coating is shown, and as can be seen from the image, the DBM is unevenly covered on the surface of the scaffold and blocks the pores of the part of the scaffold. After the stent is soaked in nano DBM particle slurry diluted by distilled water or normal saline, the nano DBM is unevenly covered on the surface of the stent and blocks the pores of a part of the stent, nano-level DBM particles and grooves are not obvious, the nano-level DBM particles and the grooves are compact and flaky, and the part of the nano-level DBM particles and the grooves are loose and meshed.

Comparative example 2

In the invention, the nDBM composite scaffold is constructed by electrostatic or air spraying on the surface of the scaffold, but the spraying effect is poor, the scaffold and the nDBM cannot be effectively combined, and the result of visual observation is as follows: the nanopaste cannot adhere or adhere to the surface of the stent.

Firstly, air spraying: diluting and uniformly mixing nDBM slurry with 75% ethanol at room temperature of 20-25 ℃, placing the nDBM slurry into a Finex air spray gun liquid supply bottle, aligning a gun mouth to the surface of a PCL-TCP support for spraying, and observing by naked eyes: the nDBM can not be effectively adhered to the stent, the nDBM is easy to block a gun mouth in the spraying process, and obvious nano-slurry adhesion does not exist on the surface of the stent and in pores.

Secondly, electrostatic spraying: diluting and uniformly mixing nDBM slurry with 75% ethanol at room temperature of 20-25 ℃, placing the nDBM slurry into a Graco Pro Xp air-assisted electrostatic spray gun liquid supply bottle, spraying with a gun mouth aligned to the surface of a PCL-TCP support, and observing with naked eyes: the nDBM can not be effectively adhered to the stent, the nDBM is easy to block a gun mouth in the spraying process, and obvious nano-slurry adhesion does not exist on the surface of the stent and in pores.

Comparative example 3

Soaking the polycaprolactone-tricalcium phosphate bone tissue engineering scaffold and the diluted nDBM slurry, putting the immersed nDBM slurry into a 37 ℃ incubator for natural drying, wherein the nDBM is completely deposited at the bottom of the scaffold after drying, and no obvious nano slurry is attached to the surface and in pores of the scaffold.

The method comprises the following specific steps: soaking the polycaprolactone-tricalcium phosphate bone tissue engineering scaffold and the diluted nDBM slurry at the room temperature of 20-25 ℃, putting the immersed nDBM slurry into a 37 ℃ incubator, drying the immersed nDBM slurry for 20min, taking out the immersed nDBM slurry, and observing the immersed nDBM slurry by naked eyes: the nDBM is completely deposited at the bottom of the stent, no obvious nano-slurry is attached to the surface of the stent and in pores, and the nano-slurry cannot be adhered or attached to the surface of the stent.

Comparative example 4

Preparing an allogeneic nano Decalcified Bone Matrix (DBM) material:

The method is characterized in that ethanol grinding is adopted in the preparation process (namely grinding process) of the allogeneic nano Decalcified Bone Matrix (DBM) material, the principle is consistent with the dilution principle, effective dispersion of particles in liquid is required to be ensured in the grinding process, the DBM can be fully ground to a nano level, namely, ethanol is adopted as a solvent for grinding, the grinding effect is good, and the DBM can be effectively ground to a nano level; and water is adopted as a solvent for grinding, DBM is not uniformly dispersed, particles are still coarse after grinding for 20 minutes, the particles cannot be effectively ground into nanometer DBM slurry, and even if the grinding time is prolonged, the improvement is not obvious.

The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are given by way of illustration of the principles of the present invention, and that various changes and modifications may be made without departing from the spirit and scope of the invention as defined by the appended claims. The scope of the invention is defined by the appended claims and equivalents thereof.

Claims

1. A method for modifying a nanometer decalcified bone matrix particle coating on a polycaprolactone-tricalcium phosphate bone tissue engineering scaffold is characterized by comprising the following steps: the method comprises the following steps:

mixing the allogeneic nano decalcified bone matrix material with 75% ethanol, soaking the polycaprolactone-tricalcium phosphate bone tissue engineering scaffold in the diluted nano decalcified bone matrix slurry, and placing in liquid nitrogen to finish preliminary freezing; putting the obtained frozen scaffold into a vacuum freeze dryer, freeze-drying for at least 48 hours to finish surface coating modification, and obtaining the polycaprolactone-tricalcium phosphate bone tissue engineering scaffold modified by the nano decalcified bone matrix particle coating;

the volume ratio of the allogeneic nano decalcified bone matrix material to 75% ethanol is 1 (5-10);

the polycaprolactone-tricalcium phosphate bone tissue engineering scaffold is constructed by a 3D printing technology; the 3D printing technology is a fused deposition modeling technology;

the preparation method of the polycaprolactone-tricalcium phosphate bone tissue engineering scaffold comprises the following steps:

mixing polycaprolactone particles and tricalcium phosphate powder according to the ratio of (3-6): 1, then fully and uniformly mixing the materials by a torque rheometer, and preparing the materials into granular mixed materials suitable for 3D printing by a granulator;

firstly, CAD software matched with a 3D printer is utilized to design a polycaprolactone-tricalcium phosphate bone tissue engineering scaffold model with the fiber diameter of 0.5mm, the layer height of 0.3mm and the fiber superposition angle of each layer of 0/90 degrees; placing the prepared granular mixed material into a melting tank, setting the feeding temperature of the melting tank to be 120 ℃, the discharging temperature to be 110 ℃, and keeping the temperature of the granular mixed material in the melting tank for more than 120 minutes to ensure sufficient melting, extruding fibers from the melted material through a motor-assisted micro-injector type fine nozzle, and superposing layer by layer to form a polycaprolactone-tricalcium phosphate bone tissue engineering support;

firstly, preparing a nano decalcified bone matrix bone block: removing soft tissues from fresh allogeneic bone, and preparing a block-shaped nanometer decalcified bone matrix bone block by adopting an improved Urist method;

secondly, preparing nano decalcified bone matrix powder: placing the blocky nano decalcified bone matrix bone blocks obtained in the first step into a zirconia nano grinding tank, placing a zirconia grinding ball, screwing down the tank opening, placing the grinding tank into a full-automatic liquid nitrogen freezing grinder for pre-crushing to obtain nano decalcified bone matrix powder with the particle size of 5-10 microns;

step three, preparing an allogeneic nano decalcified bone matrix material: placing the nano decalcified bone matrix powder obtained in the second step into a zirconia nano grinding tank, adding zirconia grinding balls, adding 75% ethanol until the grinding balls are completely covered, placing the zirconia nano grinding tank into a full-automatic low-temperature high-energy ball mill for ball milling to obtain an allogeneic nano decalcified bone matrix material with the particle size of 20-50 nm;

the preparation of the massive nanometer decalcified bone matrix bone block by the improved Urist method comprises the following steps:

placing the cryopreserved allogeneic bone blocks into a beaker, and soaking in absolute ethyl alcohol for 2 hours for dehydration; pouring off the absolute ethyl alcohol, adding ether for degreasing, and soaking for 12 hours; removing diethyl ether, washing with large amount of sterile distilled water for 3 times; adding 0.6mmol/L hydrochloric acid for decalcification, soaking for 72 hr, changing hydrochloric acid every 12 hr during soaking, stirring with precise power-increasing electric stirrer, washing with sterile distilled water for 5 times, and soaking overnight; soaking with anhydrous ethanol for 2 h; removing ethanol, adding ether, and soaking for 1 hr; removing the ether, and placing the bone blocks on a ventilation experiment table to volatilize overnight; freeze-drying in a vacuum freeze dryer, packaging and sealing for later use to obtain massive nanometer decalcified bone matrix bone blocks;

2. The method for coating and modifying the bone matrix particles of the polycaprolactone-tricalcium phosphate bone tissue engineering scaffold according to the claim 1, wherein the coating and modifying step comprises the following steps: the volume ratio of the allogeneic nano decalcified bone matrix material to 75% ethanol is 1: 5.

3. The method for coating and modifying the bone matrix particles of the polycaprolactone-tricalcium phosphate bone tissue engineering scaffold according to the claim 1, wherein the coating and modifying step comprises the following steps: in the second step, zirconia grinding balls with the diameter of 1.5cm are used in the zirconia nano grinding tank;

4. The method for coating and modifying the bone matrix particles of the polycaprolactone-tricalcium phosphate bone tissue engineering scaffold according to the claim 1, wherein the coating and modifying step comprises the following steps: in the third step, zirconia grinding balls with the diameter of 1mm are used in the zirconia nano grinding tank.

5. A polycaprolactone-tricalcium phosphate bone tissue engineering scaffold modified by the coating of nano-decalcified bone matrix particles prepared by the preparation method of any one of claims 1 to 4.

6. The use of the polycaprolactone-tricalcium phosphate bone tissue engineering scaffold modified by the nano decalcified bone matrix particle coating prepared by the preparation method of any one of claims 1 to 4 in the preparation of bone defect repair materials or bone graft substitutes.