CN106110395B - Bone repair support with self-repairing and antibacterial effects and manufacturing method thereof - Google Patents

Bone repair support with self-repairing and antibacterial effects and manufacturing method thereof Download PDF

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CN106110395B
CN106110395B CN201610685779.4A CN201610685779A CN106110395B CN 106110395 B CN106110395 B CN 106110395B CN 201610685779 A CN201610685779 A CN 201610685779A CN 106110395 B CN106110395 B CN 106110395B
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composite material
support body
patient
antibacterial
porous nano
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CN106110395A (en
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陈卫
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Shaanxi Dongwang Technology Trade Co ltd
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Shaanxi Dongwang Technology Trade Co ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
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    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
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    • A—HUMAN NECESSITIES
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    • A61L27/3843—Connective tissue
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    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
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Abstract

The invention discloses a bone repair bracket with self-repairing and antibacterial effects, which can shorten the recovery time of a patient. The invention also discloses a manufacturing method of the bone repair bracket with self-repairing and antibacterial effects, which comprises the following steps: step 1, manufacturing a mesh-shaped hollow support body with connecting blocks and fixing holes, and culturing stem cells of a patient; step 2, preparing an antibacterial porous nano hydroxyapatite composite material; step 3, filling the antibacterial porous nano hydroxyapatite composite material into the hollow part of the mesh hollow support body; and 4, uniformly inoculating the cultured stem cells of the patient on the antibacterial porous nano-hydroxyapatite composite material, and then carrying out aseptic packaging to obtain the bone repair scaffold with self-repairing and antibacterial effects. The bone repair scaffold prepared by the method can repair itself in a patient body and resist bacterial infection.

Description

Bone repair support with self-repairing and antibacterial effects and manufacturing method thereof
Technical Field
The invention belongs to the technical field of biomedical materials, and particularly relates to a bone repair support with self-repairing and antibacterial effects, and a manufacturing method of the support.
Background
The clinical findings show that bacteria are easy to be planted on the surface of the implanted material after the bone repair material is implanted into an organism, which often causes bacterial infection related to the implanted material, influences the benign interaction between cells and tissues around the material and the material, even causes bone loss, and leads to the failure of bone repair. Once infection occurs, patients have difficulty in effective treatment even with oral or injection of large doses of antibiotics, and abuse of antibiotics is increasing bacterial resistance. Therefore, the development of bone repair materials with antibacterial and anti-infectious properties has become one of the research hotspots in the field of biological materials. Common bone repair techniques include mainly autologous and allogeneic bone grafting techniques, which are limited by the limitations of autologous donors and antigenicity. Other repair materials, such as metal, ceramic or other materials, although meeting the treatment requirements after being used, have poor long-term curative effect and higher incidence of complications after treatment. With the rapid development of medical technology, the research of analytic biology is also deepened continuously, and the rapid development of tissue engineering is added, so that the emerging composite material is widely used in bone defect repair.
The human bone can be approximately regarded as a composite material which is formed by taking collagen as a matrix material and taking hydroxyapatite as a reinforcing material, so that the composite material which takes the hydroxyapatite as the reinforcing material and takes a polymer, particularly a biodegradable polymer as the matrix has similar components and structures with the human bone, can make up the defects of metal and ceramic materials, and is expected to become an ideal artificial bone substitute material. However, the strength of the nano-hydroxyapatite-polymer composite bone repair material currently in development and test stages is still lower than that of cancellous bone, and the application of the nano-hydroxyapatite-polymer composite bone repair material is limited to a great extent.
Disclosure of Invention
The invention aims to provide a bone repair bracket with self-repairing and antibacterial effects, and the bone repair bracket can shorten the recovery time of a patient.
The invention also aims to provide a method for manufacturing the bone repair scaffold with self-repairing and antibacterial effects, and the bone repair scaffold manufactured by the method can be self-repaired in a patient body and resist bacterial infection.
The technical scheme adopted by the invention is as follows: the utility model provides a bone repair support with selfreparing, antibiotic efficiency, includes netted fretwork support body, and the both ends of netted fretwork support body all are provided with the connecting block, all are provided with the fixed orifices on every connecting block, and it has the filler to fill on the netted fretwork support body, and the filler is located the fretwork position of netted fretwork support body.
The present invention is also characterized in that,
the filler is a porous nano hydroxyapatite composite material, and stem cells of a patient are inoculated on the porous nano hydroxyapatite composite material.
Two fixing holes are arranged on each connecting block.
The net-shaped hollow bracket body is made of titanium alloy material.
The other technical scheme adopted by the invention is as follows: a manufacturing method of a bone repair bracket with self-repairing and antibacterial effects comprises the following steps:
step 1, manufacturing a mesh-shaped hollow support body with connecting blocks and fixing holes, and culturing stem cells of a patient;
step 2, preparing an antibacterial porous nano hydroxyapatite composite material;
step 3, filling the antibacterial porous nano hydroxyapatite composite material into the hollow part of the mesh hollow support body;
and 4, uniformly inoculating the cultured stem cells of the patient on the antibacterial porous nano-hydroxyapatite composite material, and then carrying out aseptic packaging to obtain the bone repair scaffold with self-repairing and antibacterial effects.
The present invention is also characterized in that,
the step 1 of culturing the stem cells of the patient comprises the following steps: taking bone marrow of a patient, separating stem cells of the patient, and performing sterile culture on the stem cells at 36-37 ℃;
step 1 preparation has netted fretwork support body of connecting block and fixed orifices specifically does:
the skeleton model of the pathological change part of the patient is obtained according to medical image data, a netted hollow support body model which accords with the size of the lesion part of the patient is manufactured, connecting blocks are added on the netted hollow support body model, fixing holes are added on the connecting blocks, then a netted hollow support body with the connecting blocks and the fixing holes is printed out through a metal 3D printer, a mold which can be filled in the netted hollow support body and is attached to the netted hollow support body is manufactured, and the metal 3D printer selects a medical-grade titanium alloy material for printing.
The step 2 specifically comprises the following steps:
step 2.1, mixing collagen and deionized water solution containing 3% silver phosphate by mass percent in a mass volume ratio of 5-6:10, stirring for 4-5 hours at normal temperature, and preparing into 0.6mg/ml collagen solution;
step 2.2, taking a calcium chloride solution with the concentration of 0.1mol/L, mixing the calcium chloride solution with the weight ratio of 7-7.5: adding 50 volume ratio into prepared collagen solution, mixing and stirring for 15-30min at normal temperature, slowly adding 0.1mol/L sodium dihydrogen phosphate solution, adjusting pH value with 0.1mol/L sodium hydroxide to 7.0-8.0 to start precipitation of compound A, and keeping pH value of the solution containing compound A at normal temperature to 7.0-8.0 for 23-24 h;
step 2.3, centrifuging the solution containing the compound A by using a centrifuge, wherein the rotating speed of the centrifuge is 6000rpm, removing supernatant after the centrifuge rotates for 4-5min, repeatedly washing the solution by using deionized water for 1-1.5h to remove salt ions to obtain a sample of the mixture A, freezing the sample in a refrigerator at-70 to-60 ℃ for 23-24h, taking out the sample, and drying the sample for 23-24h to obtain a yellow powdery silver phosphate-nano hydroxyapatite-collagen composite material;
and 2.4, mixing the obtained silver phosphate-nano hydroxyapatite-collagen composite material powder and medical polycaprolactone powder according to a mass ratio of 8-7:2-1, then placing the mixture into a torque rheometer, stirring the mixture for 4-5min at a normal temperature at a rotating speed of 80-85r/min to fully mix the mixture, and then melting and banburying the mixture for 8-10min in the torque rheometer at a rotating speed of 110-100r/min and a temperature of 95-105 ℃ until the torque is balanced to obtain the silver phosphate-nano hydroxyapatite-collagen-polycaprolactone composite material, namely the antibacterial porous nano hydroxyapatite composite material.
The step 2 specifically comprises the following steps:
step 2.1, dissolving collagen in deionized water, and stirring for 4-5h at normal temperature to prepare a collagen solution of 0.5 mg/ml;
step 2.2, adding a calcium chloride solution with the concentration of 0.1mol/L into the prepared collagen solution according to the volume ratio of 7-7.5:50, mixing and stirring for 25-30min at normal temperature, then slowly adding a sodium dihydrogen phosphate solution with the concentration of 0.1mol/L, adjusting the pH value with 0.1mol/L sodium hydroxide to 7.0-8.0, beginning to precipitate a mixture A, and keeping the pH value of the mixture A at normal temperature to 7.0-8.0 for 23-24 h;
step 2.3, centrifuging the mixture A by using a centrifuge, wherein the rotating speed of the centrifuge is 6000rpm, removing supernatant after the centrifuge rotates for 4-5min, repeatedly washing the mixture A by using deionized water for 1-1.5h to remove salt ions, starting to obtain a sample separated from the mixture A, freezing the sample in a refrigerator at-70 to-60 ℃ for 23-24h, taking out the sample, and drying the sample for 23-24h to obtain a white powdery nano hydroxyapatite-collagen composite material;
step 2.4, mixing the obtained nano hydroxyapatite-collagen composite material powder with deionized water according to a mass-volume ratio of 1-1.5:25, stirring for 8-15min by using a magnetic stirrer to prepare a white turbid liquid B, mixing silver nitrate with the deionized water according to a mass-volume ratio of 1-1.5:250 to obtain a silver nitrate solution C, then dropping the silver nitrate solution C into the stirred liquid B at a constant speed to obtain a mixed solution D, continuously stirring the mixed solution D6-7h at the temperature of 36-38 ℃, centrifuging the mixed solution D by using a centrifuge at the rotating speed of 6000rpm for 4-5min, removing supernatant, repeatedly washing with deionized water for 1-1.5h to remove salt ions to obtain a sample of the mixed solution D, freezing the sample in a refrigerator at the temperature of-70 ℃ to-60 ℃ for 23-24h, and taking out, drying for 23-24h to obtain a yellow powdery silver phosphate-nano hydroxyapatite-collagen composite material;
mixing the obtained silver phosphate-nano hydroxyapatite-collagen composite material with medical polycaprolactone powder according to the mass ratio of 8-7:2-1, then placing the mixture into a torque rheometer, stirring the mixture for 4-5min at the normal temperature at the rotating speed of 80-85r/min to fully mix the mixed solution, and then melting and banburying the mixture for 8-10min in the torque rheometer at the rotating speed of 110-100r/min and at the temperature of 95-105 ℃ until the torque reaches balance to obtain the silver phosphate-nano hydroxyapatite-collagen-polycaprolactone composite material, namely the antibacterial porous nano hydroxyapatite composite material.
The step 3 specifically comprises the following steps:
heating the antibacterial porous nano-hydroxyapatite composite material prepared in the step 2.4 to 90-100 ℃ in a sterile environment, putting the mesh hollow-out support body with the connecting blocks and the fixing holes, which is 3D printed in the step 1, into the mold prepared in the step 1, heating to 90-100 ℃, then pressing the antibacterial porous nano-hydroxyapatite composite material with the temperature of 90-100 ℃ into the mould from one side of the mould under the pressure of 3-3.5Mpa, stopping filling the material after the antibacterial porous nano-hydroxyapatite composite material on the other side of the mould is pressed out from a flash port of the mould, maintaining at 90-100 deg.C and 0.9-1.1Mpa for 1-2 hr, cooling and solidifying, correcting to remove the redundant antibacterial porous nano hydroxyapatite composite material, then cleaning, freeze drying and sterilizing.
The step 4 specifically comprises the following steps:
extracting appropriate amount of bone marrow from patient, separating stem cells from patient, culturing, growing to fuse, preparing stem cells into cell suspension at 2 × 10 8 L -1 The cell density is 2mL, the cell suspension is evenly inoculated on the antibacterial porous nano-hydroxyapatite composite material, the cell suspension is subjected to proliferation culture for 23-24h in a carbon dioxide incubator with the volume fraction of 5% of carbon dioxide and the humidity of 95% and the constant temperature of 37-38 ℃, and then the cell suspension is subjected to aseptic packaging and storage at the temperature of 2-6 ℃ to obtain the bone repair scaffold with self-repairing and antibacterial effects.
The invention has the beneficial effects that: a bone repair support with self-repairing and antibacterial effects can shorten the recovery time of a patient. The bone repair bracket prepared by the method can perform self-repair in a patient body and resist bacterial infection. The silver phosphate-nano hydroxyapatite-collagen-polycaprolactone composite material is an antibacterial porous nano hydroxyapatite composite material, and the bone repair material of the material has high biocompatibility, can be absorbed and degraded by a human body, and can resist bacterial infection. The composite material can be self-repaired in vivo after being planted with stem cells, so that the biocompatibility of the bone repair material is improved, and the recovery time of a patient is shortened.
Drawings
FIG. 1 is a schematic structural view of a self-healing, antibacterial bone repair scaffold according to the present invention;
fig. 2 is an enlarged view of a portion a in fig. 1.
In the figure: 1. the net-shaped hollow support comprises a net-shaped hollow support body, 2 filler, 3 connecting blocks and 4 fixing holes.
Detailed Description
The invention is described in detail below with reference to the accompanying drawings and the detailed description;
the bone repair support with the self-repairing and antibacterial effects comprises a netted hollow support body 1, wherein connecting blocks 3 are arranged at two ends of the netted hollow support body 1, each connecting block 3 is provided with a fixing hole 4, the netted hollow support body 1 is filled with a filler 2, and the filler 2 is positioned at a hollow part of the netted hollow support body 1;
the filler 2 is a porous nano hydroxyapatite composite material, and stem cells of a patient are inoculated on the porous nano hydroxyapatite composite material;
two fixing holes 4 are arranged on each connecting block 3, and the connecting blocks 3 and the fixing holes 4 are used for fixing the support with the bone of a patient;
the reticular hollow bracket body 1 is made of titanium alloy material.
The invention relates to a manufacturing method of a bone repair bracket with self-repairing and antibacterial effects, which comprises the following steps:
step 1, manufacturing a reticular hollow support body 1 with a connecting block 3 and a fixing hole 4, and culturing stem cells of a patient;
the stem cells of the patient are cultured in the step 1 as follows: taking bone marrow of a patient, separating stem cells of the patient, and performing sterile culture on the stem cells at 36-37 ℃;
step 1 preparation has netted fretwork support body 1 of connecting block 3 and fixed orifices 4 specifically to be:
obtaining a skeleton model of a lesion part of a patient according to medical image data, manufacturing a mesh-shaped hollow support body 1 model which accords with the shape and size of the lesion part of the patient, adding a connecting block 3 on the mesh-shaped hollow support body 1 model and adding a fixing hole 4 on the connecting block 3, then printing the mesh-shaped hollow support body 1 with the connecting block 3 and the fixing hole 4 by a metal 3D printer, manufacturing a mold which can be arranged in the mesh-shaped hollow support body 1 and is attached to the mesh-shaped hollow support body 1, and selecting a medical grade titanium alloy material for printing by the metal 3D printer;
step 2, preparing an antibacterial porous nano hydroxyapatite composite material;
the step 2 specifically comprises the following steps:
step 2.1, mixing collagen and deionized water solution containing 3% silver phosphate by mass percent in a mass volume ratio of 5-6:10, stirring for 4-5 hours at normal temperature, and preparing into 0.6mg/ml collagen solution;
step 2.2, taking a calcium chloride solution with the concentration of 0.1mol/L, mixing the calcium chloride solution with the weight ratio of 7-7.5: adding 50 volume ratio into prepared collagen solution, mixing and stirring for 15-30min at normal temperature, slowly adding 0.1mol/L sodium dihydrogen phosphate solution, adjusting pH value with 0.1mol/L sodium hydroxide to 7.0-8.0 to start precipitation of compound A, and keeping pH value of the solution containing compound A at normal temperature to 7.0-8.0 for 23-24 h;
step 2.3, centrifuging the solution containing the compound A by using a centrifuge, wherein the rotating speed of the centrifuge is 6000rpm, removing supernatant after the centrifuge rotates for 4-5min, repeatedly washing the solution by using deionized water for 1-1.5h to remove salt ions to obtain a sample of the compound A, freezing the sample in a refrigerator at-70 to-60 ℃ for 23-24h, taking out the sample, and drying the sample for 23-24h to obtain a yellow powdery silver phosphate-nano hydroxyapatite-collagen composite material;
step 2.4, mixing the obtained silver phosphate-nano hydroxyapatite-collagen composite material powder and medical polycaprolactone powder according to a mass ratio of 8-7:2-1, then placing the mixture into a torque rheometer, stirring the mixture for 4-5min at a normal temperature at a rotating speed of 80-85r/min to fully mix the mixture, and then melting and banburying the mixture for 8-10min in the torque rheometer at a rotating speed of 110-100r/min and a temperature of 95-105 ℃ until the torque is balanced to obtain the silver phosphate-nano hydroxyapatite-collagen-polycaprolactone composite material, namely the antibacterial porous nano hydroxyapatite composite material;
step 3, filling the antibacterial porous nano hydroxyapatite composite material into the hollow part of the mesh hollow bracket body 1;
the step 3 specifically comprises the following steps:
heating the antibacterial porous nano-hydroxyapatite composite material prepared in the step 2.4 to 90-100 ℃ in a sterile environment, putting the mesh hollow-out support body 1 with the connecting blocks 3 and the fixing holes 4 printed in the step 1 in a 3D mode into a manufactured mould, heating to 90-100 ℃, then pressing the antibacterial porous nano hydroxyapatite composite material with the temperature of 90-100 ℃ into the mould from one side of the mould under the pressure of 3-3.5Mpa, stopping filling the material after the antibacterial porous nano hydroxyapatite composite material on the other side of the mould is pressed out from the flash port of the mould, maintaining at 90-100 deg.C and 0.9-1.1Mpa for 1-2 hr, cooling and solidifying, correcting to remove the redundant antibacterial porous nano hydroxyapatite composite material, then cleaning, and carrying out freeze drying and disinfection treatment;
step 4, uniformly inoculating the cultured stem cells of the patient on the antibacterial porous nano-hydroxyapatite composite material, and then carrying out aseptic packaging to obtain the bone repair scaffold with self-repairing and antibacterial effects;
the step 4 specifically comprises the following steps:
extracting appropriate amount of bone marrow from patient, separating stem cells from patient, culturing, growing to fuse, preparing stem cells into cell suspension at 2 × 10 8 L -1 The cell density is 2mL, the cell suspension is evenly inoculated on the antibacterial porous nano-hydroxyapatite composite material, the cell suspension is subjected to enrichment culture for 23-24h in a carbon dioxide incubator with the volume fraction of 5% of carbon dioxide and the humidity of 95% and the constant temperature of 37-38 ℃, and then the cell suspension is subjected to aseptic packaging and storage at the temperature of 2-6 ℃ to obtain the bone repair scaffold with self-repairing and antibacterial effects.
The bone repair scaffold which is customized by adopting a 3D printing technology and has self-repairing and antibacterial effects provides necessary strength support for the silver phosphate-nano hydroxyapatite-collagen-polycaprolactone composite material, namely the antibacterial porous nano hydroxyapatite composite material, and simultaneously, due to the uniqueness of the manufacturing process, the scaffold is more suitable for patients in aspects such as product morphological structure and the like. Meanwhile, the nano hydroxyapatite-collagen composite material has good biocompatibility although the strength is low, so that the silver phosphate-nano hydroxyapatite-collagen-polycaprolactone composite material, namely the antibacterial porous nano hydroxyapatite composite material, is ensured to have higher biocompatibility.
The combination of the netted hollowed-out support body 1 and the antibacterial porous nano-hydroxyapatite composite material is reliable, the contact area of the netted hollowed-out support body 1 and the antibacterial porous nano-hydroxyapatite composite material is increased, and the adhesiveness of the antibacterial porous nano-hydroxyapatite composite material to the netted hollowed-out support body 1 is improved.
The invention relates to a manufacturing method of a bone repair bracket with self-repairing and antibacterial effects, which comprises the following steps:
step 1, manufacturing a reticular hollow support body 1 with a connecting block 3 and a fixing hole 4, and culturing stem cells of a patient;
the stem cells of the patient are cultured in the step 1 as follows: taking bone marrow of a patient, separating stem cells of the patient, and performing sterile culture on the stem cells at 36-37 ℃;
step 1 preparation has netted fretwork support body 1 of connecting block 3 and fixed orifices 4 specifically to be:
obtaining a skeleton model of a lesion part of a patient according to medical image data, manufacturing a mesh-shaped hollow support body 1 model which accords with the shape and size of the lesion part of the patient, adding a connecting block 3 on the mesh-shaped hollow support body 1 model and adding a fixing hole 4 on the connecting block 3, then printing the mesh-shaped hollow support body 1 with the connecting block 3 and the fixing hole 4 by a metal 3D printer, manufacturing a mold which can be arranged in the mesh-shaped hollow support body 1 and is attached to the mesh-shaped hollow support body 1, and selecting a medical grade titanium alloy material for printing by the metal 3D printer;
step 2, preparing an antibacterial porous nano hydroxyapatite composite material;
the step 2 specifically comprises the following steps:
step 2.1, dissolving collagen in deionized water, and stirring for 4-5h at normal temperature to prepare a collagen solution of 0.5 mg/ml;
step 2.2, adding a calcium chloride solution with the concentration of 0.1mol/L into the prepared collagen solution according to the volume ratio of 7-7.5:50, mixing and stirring for 25-30min at normal temperature, slowly adding a sodium dihydrogen phosphate solution with the concentration of 0.1mol/L, adjusting the pH value with 0.1mol/L sodium hydroxide to 7.0-8.0, beginning to precipitate a compound A, and keeping the pH value of the solution of the compound A at the normal temperature for 23-24 h;
step 2.3, centrifuging the compound A solution by using a centrifuge, wherein the rotating speed of the centrifuge is 6000rpm, removing supernatant after the centrifuge rotates for 4-5min, repeatedly washing the sample for 1-1.5h by using deionized water to remove salt ions, beginning to obtain a sample separated from the mixture A, freezing the sample in a refrigerator at-70 to-60 ℃ for 23-24h, taking out the sample, and drying the sample for 23-24h to obtain a white powdery nano hydroxyapatite-collagen composite material;
step 2.4, mixing the obtained nano hydroxyapatite-collagen composite material with deionized water according to a mass-volume ratio of 1-1.5:25, stirring for 8-15min by using a magnetic stirrer to prepare a white turbid liquid B, mixing silver nitrate with the deionized water according to a mass-volume ratio of 1-1.5:250 to obtain a silver nitrate solution C, then dropping the silver nitrate solution C into the stirred liquid B at a constant speed to obtain a mixed solution D, continuously stirring the mixed solution D6-7h at the temperature of 36-38 ℃, centrifuging the mixed solution D by using a centrifuge at the rotating speed of 6000rpm for 4-5min, removing supernatant, repeatedly washing with deionized water for 1-1.5h to remove salt ions to obtain a sample of the mixed solution D, freezing the sample in a refrigerator at the temperature of-70 ℃ to-60 ℃ for 23-24h, and taking out, drying for 23-24h to obtain a yellow powdery silver phosphate-nano hydroxyapatite-collagen composite material;
mixing the obtained silver phosphate-nano hydroxyapatite-collagen composite material with medical polycaprolactone powder according to the mass ratio of 8-7:2-1, then placing the mixture into a torque rheometer, stirring the mixture for 4-5min at the normal temperature at the rotating speed of 80-85r/min to fully mix the mixed solution, and then melting and banburying the mixture for 8-10min in the torque rheometer at the rotating speed of 110-100r/min and at the temperature of 95-105 ℃ until the torque reaches balance to obtain the silver phosphate-nano hydroxyapatite-collagen-polycaprolactone composite material, namely the antibacterial porous nano hydroxyapatite composite material;
step 3, filling the antibacterial porous nano hydroxyapatite composite material into the hollow part of the mesh hollow bracket body 1;
the step 3 specifically comprises the following steps:
heating the antibacterial porous nano-hydroxyapatite composite material prepared in the step 2.4 to 90-100 ℃ in a sterile environment, putting the mesh hollow-out support body 1 with the connecting blocks 3 and the fixing holes 4 printed in the step 1 in a 3D mode into a manufactured mould, heating to 90-100 ℃, then pressing the antibacterial porous nano hydroxyapatite composite material with the temperature of 90-100 ℃ into the mould from one side of the mould under the pressure of 3-3.5Mpa, stopping filling the material after the antibacterial porous nano hydroxyapatite composite material on the other side of the mould is pressed out from the flash port of the mould, maintaining at 90-100 deg.C and 0.9-1.1Mpa for 1-2 hr, cooling and solidifying, correcting to remove the redundant antibacterial porous nano hydroxyapatite composite material, then cleaning, and carrying out freeze drying and disinfection treatment;
step 4, uniformly inoculating the cultured stem cells of the patient on the antibacterial porous nano-hydroxyapatite composite material, and then carrying out aseptic packaging to obtain the bone repair scaffold with self-repairing and antibacterial effects;
the step 4 specifically comprises the following steps:
extracting appropriate amount of bone marrow from patient, separating stem cells from patient, culturing, growing to fuse, preparing stem cells into cell suspension at 2 × 10 8 L -1 The cell density is 2mL, the cell suspension is evenly inoculated on the antibacterial porous nano-hydroxyapatite composite material, the cell suspension is subjected to enrichment culture for 23-24h in a carbon dioxide incubator with the volume fraction of 5% of carbon dioxide and the humidity of 95% and the constant temperature of 37-38 ℃, and then the cell suspension is subjected to aseptic packaging and storage at the temperature of 2-6 ℃ to obtain the bone repair scaffold with self-repairing and antibacterial effects.
The clinical findings show that bacteria are easy to be planted on the surface of the implanted material after the bone repair material is implanted into an organism, which often causes bacterial infection related to the implanted material, influences the benign interaction between cells and tissues around the material and the material, even causes bone loss, and leads to the failure of bone repair. Once infection occurs, patients have difficulty in effective treatment even with oral or injection of large doses of antibiotics, and abuse of antibiotics is increasing bacterial resistance. The antibacterial porous nano hydroxyapatite composite material has large specific surface area and strong adsorption capacity to drugs, the drugs exist in a molecular state, an amorphous state or a microcrystalline state in a carrier, and meanwhile, the antibacterial porous nano hydroxyapatite composite material has excellent biological activity and physiological compatibility, can be degraded and absorbed in vivo, and is very safe.
The antibacterial silver-based antibacterial agent, namely silver phosphate, is added in the manufacturing process of the antibacterial porous nano-hydroxyapatite composite material, so that the antibacterial porous nano-hydroxyapatite composite material has an antibacterial effect, and the risk of bacterial infection is reduced. The silver antibacterial agent has the advantages of wide antibacterial spectrum, difficult generation of drug resistance and the like, and has attracted extensive attention in the field of antibacterial bone repair materials. As silver ions in silver nitrate are released too fast, the silver ions serving as an antibacterial agent cannot meet the requirements of continuous and long-acting antibiosis; the silver phosphate is slightly soluble in water, and can slowly release silver ions with high antibacterial activity in the environment with water, so that the antibacterial effect is achieved.
The medical polycaprolactone has good biodegradability, biocompatibility and nontoxicity, is widely used as a medical biodegradable material and a drug controlled release system, can be used in tissue engineering as a drug slow release system, and is used as an adhesive of a silver phosphate-nano hydroxyapatite-collagen composite material due to low melting point (60 ℃) and good plasticity.
The nanometer hydroxyapatite has tissue and crystal structure similar to that of inorganic hydroxyapatite in human bone tissue, excellent biocompatibility and bone inductivity, and capacity of being combined directly with bone formation, and the three-dimensional porous netted structure in the composite material can ensure the composite material has great surface area, so as to facilitate the adhesion, growth, division, etc. of stem cell of patient, facilitate the transmission of nutrients, eliminate excrement, inoculate certain amount of stem cell of patient and induce culture. Effectively improves the clinical treatment effect, reduces the risk of rejection, improves the reliability of the operation and shortens the treatment period.
Example 1
A manufacturing method of a bone repair support with self-repairing and antibacterial effects comprises the following steps:
step 1, culturing the stem cells of the patient comprises the following steps: taking the bone marrow of a patient, separating out stem cells of the patient, and carrying out sterile culture on the stem cells at 36 ℃;
the preparation has netted fretwork support body 1 of connecting block 3 and fixed orifices 4:
obtaining a skeleton model of a diseased wrist part of a patient according to medical image data, manufacturing a mesh-shaped hollow support body 1 model which accords with the shape and size of a diseased part of the patient, adding a connecting block 3 on the mesh-shaped hollow support body 1 model and adding a fixing hole 4 on the connecting block 3, then printing the mesh-shaped hollow support body 1 with the connecting block 3 and the fixing hole 4 by a metal 3D printer, manufacturing a mold which can be arranged in the mesh-shaped hollow support body 1 and is attached to the mesh-shaped hollow support body 1, and selecting a medical grade titanium alloy material for printing by the metal 3D printer;
step 2, preparing the antibacterial porous nano hydroxyapatite composite material, which specifically comprises the following steps:
step 2.1, mixing collagen with a deionized water solution containing 3% silver phosphate by mass percent in a mass volume ratio of 5:10, and stirring for 4 hours at normal temperature to prepare a collagen solution of 0.6 mg/ml;
step 2.2, taking a calcium chloride solution with the concentration of 0.1mol/L, mixing the calcium chloride solution with the weight ratio of 7: adding 50 volume ratios of the components into the prepared collagen solution, mixing and stirring for 15min at normal temperature, slowly adding 0.1mol/L sodium dihydrogen phosphate solution, adjusting the pH value to 7.0 by using 0.1mol/L sodium hydroxide, separating out the compound A, and keeping the pH value of the solution containing the compound A at 7.0 at normal temperature for 23 h;
step 2.3, centrifuging the solution containing the compound A by using a centrifuge, wherein the rotating speed of the centrifuge is 6000rpm, removing supernatant after the centrifuge rotates for 4min, repeatedly washing the solution by using deionized water for 1h to remove salt ions to obtain a sample of the compound A, freezing the sample in a refrigerator at-70 ℃ for 23h, taking out the sample, and drying the sample for 23h to obtain a yellow powdery silver phosphate-nano hydroxyapatite-collagen composite material;
step 2.4, mixing the obtained silver phosphate-nano hydroxyapatite-collagen composite material powder and medical polycaprolactone powder according to a mass ratio of 8:2, then placing the mixture into a torque rheometer, stirring the mixture for 4min at a normal temperature at a rotating speed of 80r/min to fully mix the mixture, and then melting and banburying the mixture for 8min in the torque rheometer at a rotating speed of 110r/min and at a temperature of 95 ℃ until torque reaches balance to obtain the silver phosphate-nano hydroxyapatite-collagen-polycaprolactone composite material, namely the antibacterial porous nano hydroxyapatite composite material;
step 3, filling the antibacterial porous nano hydroxyapatite composite material into the hollow part of the mesh hollow bracket body 1, specifically:
heating the antibacterial porous nano-hydroxyapatite composite material prepared in the step 2.4 to 90 ℃ in an aseptic environment, putting the 3D-printed mesh-shaped hollow support body 1 with the connecting blocks 3 and the fixing holes 4 in the step 1 into a manufactured mould, heating to 90 ℃, pressing the antibacterial porous nano-hydroxyapatite composite material at 90 ℃ into the mould from one side of the mould under the pressure of 3Mpa, stopping filling the material after the antibacterial porous nano-hydroxyapatite composite material on the other side of the mould is pressed out from a flash port of the mould, maintaining the pressure at 0.9Mpa for 1h at the temperature of 90 ℃, correcting to remove the redundant antibacterial porous nano-hydroxyapatite composite material after cooling and solidification, cleaning, freeze-drying and sterilizing;
step 4, uniformly inoculating the cultured stem cells of the patient on the antibacterial porous nano-hydroxyapatite composite material, and then carrying out aseptic packaging to obtain the bone repair scaffold with self-repairing and antibacterial effects, which specifically comprises the following steps:
extracting appropriate amount of bone marrow from patient, separating stem cells from patient, culturing, growing to fuse, preparing stem cells into cell suspension at 2 × 10 8 L -1 The cell density of the composite material is 2mL, and the cell suspension is evenly inoculated on the antibacterial porous nano-hydroxyapatite composite materialAnd performing enrichment culture on the cell suspension in a carbon dioxide incubator with the volume fraction of 5% of carbon dioxide and the humidity of 95% and the constant temperature of 37 ℃ for 23 hours, then performing aseptic packaging and storing at the temperature of 2 ℃ to obtain the bone repair scaffold with self-repairing and antibacterial effects.
Example 2
A manufacturing method of a bone repair bracket with self-repairing and antibacterial effects comprises the following steps:
step 1, manufacturing a reticular hollow support body 1 with a connecting block 3 and a fixing hole 4, and culturing stem cells of a patient;
the stem cells of the patient are cultured in the step 1 as follows: taking the bone marrow of a patient, separating out stem cells of the patient, and carrying out sterile culture on the stem cells at 36.5 ℃;
step 1 preparation has netted fretwork support body 1 of connecting block 3 and fixed orifices 4 specifically to be:
obtaining a skeleton model of a diseased ankle part of a patient according to medical image data, manufacturing a mesh-shaped hollow support body 1 model which accords with the shape and size of the diseased ankle part of the patient, adding a connecting block 3 on the mesh-shaped hollow support body 1 model and adding a fixing hole 4 on the connecting block 3, then printing the mesh-shaped hollow support body 1 with the connecting block 3 and the fixing hole 4 by a metal 3D printer, manufacturing a mold which can be arranged in the mesh-shaped hollow support body 1 and is attached to the mesh-shaped hollow support body 1, and selecting a medical grade titanium alloy material for printing by the metal 3D printer;
step 2, preparing an antibacterial porous nano hydroxyapatite composite material;
the step 2 specifically comprises the following steps:
step 2.1, mixing collagen and deionized water solution containing 3% silver phosphate by mass according to a mass volume ratio of 5.5:10, stirring for 4.5 hours at normal temperature, and preparing into 0.6mg/ml collagen solution;
step 2.2, taking a calcium chloride solution with the concentration of 0.1mol/L, mixing the calcium chloride solution with the weight ratio of 7.25: adding 50 volume ratio into prepared collagen solution, mixing and stirring at normal temperature for 23min, slowly adding 0.1mol/L sodium dihydrogen phosphate solution, adjusting pH value with 0.1mol/L sodium hydroxide to 7.5 to precipitate compound A, and keeping pH value of the solution containing compound A at normal temperature to 7.5 for 23.5 h;
step 2.3, centrifuging the solution containing the compound A by using a centrifuge, wherein the rotating speed of the centrifuge is 6000rpm, removing supernatant after the centrifuge rotates for 4.5min, repeatedly washing the solution by using deionized water for 1.25h to remove salt ions to obtain a sample of the compound A, freezing the sample in a refrigerator at the temperature of-65 ℃ for 23.5h, taking out the sample, and drying the sample for 23.5h to obtain a yellow powdery silver phosphate-nano hydroxyapatite-collagen composite material;
step 2.4, mixing the obtained silver phosphate-nano hydroxyapatite-collagen composite material powder and medical polycaprolactone powder according to a mass ratio of 7.5:1.5, then placing the mixture into a torque rheometer, stirring the mixture for 4.5min at the normal temperature at the rotating speed of 83r/min, fully mixing the mixture, and then melting and banburying the mixture for 9min in the torque rheometer at the rotating speed of 105r/min and at the temperature of 100 ℃ until the torque reaches balance to obtain the silver phosphate-nano hydroxyapatite-collagen-polycaprolactone composite material, namely the antibacterial porous nano hydroxyapatite composite material;
step 3, filling the antibacterial porous nano hydroxyapatite composite material into the hollow part of the mesh hollow bracket body 1;
the step 3 specifically comprises the following steps:
heating the antibacterial porous nano-hydroxyapatite composite material prepared in the step 2.4 to 95 ℃ in an aseptic environment, putting the mesh-shaped hollow support body 1 with the connecting blocks 3 and the fixing holes 4 printed in the step 1 in a manufactured mould, heating to 95 ℃, pressing the antibacterial porous nano-hydroxyapatite composite material at 95 ℃ into the mould from one side of the mould under the pressure of 3.3Mpa, stopping filling the material after the antibacterial porous nano-hydroxyapatite composite material on the other side of the mould is extruded from a flash port of the mould, maintaining the pressure at 1Mpa for 1.5 hours at the temperature of 95 ℃, correcting to remove the redundant antibacterial porous nano-hydroxyapatite composite material after cooling and solidification, cleaning, freeze-drying and sterilizing;
step 4, uniformly inoculating the cultured stem cells of the patient on the antibacterial porous nano-hydroxyapatite composite material, and then carrying out aseptic packaging to obtain the bone repair scaffold with self-repairing and antibacterial effects;
the step 4 specifically comprises the following steps:
extracting appropriate amount of bone marrow from patient, separating stem cells from patient, culturing, growing to fuse, preparing stem cells into cell suspension at 2 × 10 8 L -1 The cell density is 2mL, the cell suspension is evenly inoculated on the antibacterial porous nano-hydroxyapatite composite material, the cell suspension is subjected to enrichment culture for 22h in a carbon dioxide incubator with the volume fraction of 5% of carbon dioxide and the humidity of 95% and the constant temperature of 37.5 ℃, and then the cell suspension is subjected to aseptic packaging and storage at the temperature of 4 ℃ to obtain the bone repair scaffold with self-repairing and antibacterial effects.
Example 3
A manufacturing method of a bone repair bracket with self-repairing and antibacterial effects comprises the following steps:
step 1, manufacturing a reticular hollow support body 1 with a connecting block 3 and a fixing hole 4, and culturing stem cells of a patient;
the stem cells of the patient are cultured in the step 1 as follows: taking the bone marrow of a patient, separating out the stem cells of the patient, and carrying out sterile culture on the stem cells at 37 ℃;
step 1 preparation has netted fretwork support body 1 of connecting block 3 and fixed orifices 4 specifically to be:
obtaining a skeleton model of a diseased thigh part of a patient according to medical image data, manufacturing a mesh-shaped hollow support body 1 model which accords with the shape and size of a diseased part of the patient, adding a connecting block 3 on the mesh-shaped hollow support body 1 model and a fixing hole 4 on the connecting block 3, then printing the mesh-shaped hollow support body 1 with the connecting block 3 and the fixing hole 4 by a metal 3D printer, manufacturing a mold which can be arranged in the mesh-shaped hollow support body 1 and is attached to the mesh-shaped hollow support body 1, and selecting a medical grade titanium alloy material for printing by the metal 3D printer;
step 2, preparing an antibacterial porous nano hydroxyapatite composite material;
the step 2 specifically comprises the following steps:
step 2.1, mixing collagen and deionized water solution containing 3% silver phosphate by mass percent in a mass volume ratio of 6:10, stirring for 5 hours at normal temperature, and preparing into 0.6mg/ml collagen solution;
step 2.2, taking a calcium chloride solution with the concentration of 0.1mol/L, mixing the calcium chloride solution with the weight ratio of 7.5: adding 50 volume ratios of the components into the prepared collagen solution, mixing and stirring for 30min at normal temperature, slowly adding 0.1mol/L sodium dihydrogen phosphate solution, adjusting the pH value to 8.0 by using 0.1mol/L sodium hydroxide, separating out the compound A, and keeping the pH value of the solution containing the compound A at 8.0 at normal temperature for 24 h;
step 2.3, centrifuging the solution containing the compound A by using a centrifuge, wherein the rotating speed of the centrifuge is 6000rpm, removing supernatant after the centrifuge rotates for 5min, repeatedly washing the solution by using deionized water for 1.5h to remove salt ions to obtain a sample of the compound A, freezing the sample in a refrigerator at the temperature of-60 ℃ for 24h, taking out the sample, and drying the sample for 24h to obtain a yellow powdery silver phosphate-nano hydroxyapatite-collagen composite material;
step 2.4, mixing the obtained silver phosphate-nano hydroxyapatite-collagen composite material powder and medical polycaprolactone powder according to a mass ratio of 7:1, then placing the mixture into a torque rheometer, stirring the mixture for 5min at a normal temperature at a rotating speed of 85r/min to fully mix the mixture, and then melting and banburying the mixture for 10min in the torque rheometer at a rotating speed of 100r/min and at a temperature of 105 ℃ until the torque reaches balance to obtain the silver phosphate-nano hydroxyapatite-collagen-polycaprolactone composite material, namely the antibacterial porous nano hydroxyapatite composite material;
step 3, filling the antibacterial porous nano hydroxyapatite composite material into the hollow part of the mesh hollow bracket body 1;
the step 3 specifically comprises the following steps:
heating the antibacterial porous nano-hydroxyapatite composite material prepared in the step 2.4 to 100 ℃ in an aseptic environment, putting the mesh-shaped hollow support body 1 with the connecting blocks 3 and the fixing holes 4 printed in the step 1 in a manufactured mould, heating to 100 ℃, pressing the antibacterial porous nano-hydroxyapatite composite material at 100 ℃ into the mould from one side of the mould under the pressure of 3.5Mpa, stopping filling the material after the antibacterial porous nano-hydroxyapatite composite material on the other side of the mould is extruded from a flash port of the mould, maintaining the pressure at 1.1 for 2 hours at the temperature of 100 ℃, correcting to remove the redundant antibacterial porous nano-hydroxyapatite composite material under the pressure of 100 Mpa after cooling and solidification, then cleaning, freeze-drying and sterilizing;
step 4, uniformly inoculating the cultured stem cells of the patient on the antibacterial porous nano-hydroxyapatite composite material, and then carrying out aseptic packaging to obtain the bone repair scaffold with self-repairing and antibacterial effects;
the step 4 specifically comprises the following steps:
extracting appropriate amount of bone marrow from patient, separating stem cells from patient, culturing, growing until they are fused, preparing stem cells into cell suspension at 2 × 10 8 L -1 The cell density is 2mL, the cell suspension is evenly inoculated on the antibacterial porous nano-hydroxyapatite composite material, and the cell suspension is subjected to multiplication culture for 24 hours in a carbon dioxide incubator with the volume fraction of 5% of carbon dioxide, the humidity of 95% and the constant temperature of 38 ℃, and then is subjected to aseptic packaging and storage at the temperature of 6 ℃ to obtain the bone repair scaffold with self-repairing and antibacterial effects.
Example 4
A manufacturing method of a bone repair bracket with self-repairing and antibacterial effects comprises the following steps:
step 1, culturing the stem cells of the patient comprises the following steps: taking the bone marrow of a patient, separating out the stem cells of the patient, and carrying out sterile culture on the stem cells at 36 ℃;
the preparation has netted fretwork support body 1 of connecting block 3 and fixed orifices 4:
obtaining a skeleton model of a diseased wrist part of a patient according to medical image data, manufacturing a mesh-shaped hollow support body 1 model which accords with the shape and size of a diseased part of the patient, adding a connecting block 3 on the mesh-shaped hollow support body 1 model and adding a fixing hole 4 on the connecting block 3, then printing the mesh-shaped hollow support body 1 with the connecting block 3 and the fixing hole 4 by a metal 3D printer, manufacturing a mold which can be arranged in the mesh-shaped hollow support body 1 and is attached to the mesh-shaped hollow support body 1, and selecting a medical grade titanium alloy material for printing by the metal 3D printer;
step 2, preparing the antibacterial porous nano hydroxyapatite composite material, which specifically comprises the following steps:
step 2.1, dissolving collagen in deionized water, and stirring for 4 hours at normal temperature to prepare a collagen solution of 0.5 mg/ml;
step 2.2, adding a calcium chloride solution with the concentration of 0.1mol/L into a prepared collagen solution according to the volume ratio of 7:50, mixing and stirring for 25min at normal temperature, then slowly adding a sodium dihydrogen phosphate solution with the concentration of 0.1mol/L, adjusting the pH value with 0.1mol/L sodium hydroxide to ensure that the pH value is 7.0, beginning to precipitate a compound A, keeping the pH value of the solution containing the compound A at the normal temperature to be 7.0, and keeping for 23 h;
step 2.3, centrifuging the compound A solution by using a centrifuge, wherein the rotating speed of the centrifuge is 6000rpm, removing supernatant after the centrifuge rotates for 4-5min, repeatedly washing the compound A solution by using deionized water for 1h to remove salt ions to obtain a compound A sample, freezing the compound A sample in a refrigerator at-70 ℃ for 23h, taking out the compound A sample, and drying the compound A sample for 23h to obtain a white powdery nano hydroxyapatite-collagen composite material;
step 2.4, mixing the obtained nano hydroxyapatite-collagen composite material powder with deionized water according to the mass-to-volume ratio of 1:25, stirring for 8min by using a magnetic stirrer to prepare white turbid liquid B, mixing silver nitrate with deionized water according to the mass-to-volume ratio of 1:250 to obtain silver nitrate solution C, then dropping the silver nitrate solution C into the liquid B under stirring at a constant speed to obtain a mixed solution D, continuously stirring the mixed solution D6h at the temperature of 36 ℃, centrifuging the mixed solution D by using a centrifuge, wherein the rotating speed of the centrifuge is 6000rpm, removing supernatant after the centrifuge rotates for 4min, repeatedly washing the mixed solution with deionized water for 1h to remove salt ions to obtain a sample of the mixed solution D, freezing the sample in a refrigerator at-70-DEG C for 23h, taking out the sample, and drying the sample for 23h to obtain a yellow powdery silver phosphate-nano hydroxyapatite-collagen composite material;
mixing the obtained silver phosphate-nano hydroxyapatite-collagen composite material with medical polycaprolactone powder according to the mass ratio of 8:2, then placing the mixture into a torque rheometer, stirring the mixture for 4min at the normal temperature at the rotating speed of 80r/min to fully mix the mixed solution, then melting and banburying the mixture for 8min in the torque rheometer at the rotating speed of 110r/min and at the temperature of 95 ℃ until the torque reaches balance, and obtaining the silver phosphate-nano hydroxyapatite-collagen-polycaprolactone composite material, namely the antibacterial porous nano hydroxyapatite composite material;
step 3, filling the antibacterial porous nano hydroxyapatite composite material into the hollow part of the mesh hollow bracket body 1, specifically:
heating the antibacterial porous nano-hydroxyapatite composite material prepared in the step 2.4 to 90 ℃ in an aseptic environment, putting the 3D-printed mesh-shaped hollow support body 1 with the connecting blocks 3 and the fixing holes 4 in the step 1 into a manufactured mould, heating to 90 ℃, pressing the antibacterial porous nano-hydroxyapatite composite material at 90 ℃ into the mould from one side of the mould under the pressure of 3Mpa, stopping filling the material after the antibacterial porous nano-hydroxyapatite composite material on the other side of the mould is pressed out from a flash port of the mould, maintaining the pressure at 0.9Mpa for 1h at the temperature of 90 ℃, correcting to remove the redundant antibacterial porous nano-hydroxyapatite composite material after cooling and solidification, cleaning, freeze-drying and sterilizing;
step 4, uniformly inoculating the cultured stem cells of the patient on the antibacterial porous nano-hydroxyapatite composite material, and then carrying out aseptic packaging to obtain the bone repair scaffold with self-repairing and antibacterial effects, which specifically comprises the following steps:
extracting appropriate amount of bone marrow from patient, separating stem cells from patient, culturing, growing to fuse, preparing stem cells into cell suspension at 2 × 10 8 L -1 The cell density is 2mL, the cell suspension is evenly inoculated on the antibacterial porous nano-hydroxyapatite composite material, the cell suspension is subjected to proliferation culture for 23 hours in a carbon dioxide incubator with the volume fraction of 5% of carbon dioxide and the humidity of 95% and the constant temperature of 37 ℃, and then the cell suspension is aseptically packaged and stored at the temperature of 2 ℃ to obtain the bone repair scaffold with self-repairing and antibacterial effects.
Example 5
A manufacturing method of a bone repair bracket with self-repairing and antibacterial effects comprises the following steps:
step 1, manufacturing a reticular hollow support body 1 with a connecting block 3 and a fixing hole 4, and culturing stem cells of a patient;
the stem cells of the patient are cultured in the step 1 as follows: taking the bone marrow of a patient, separating out stem cells of the patient, and carrying out sterile culture on the stem cells at 36.5 ℃;
step 1 preparation has netted fretwork support body 1 of connecting block 3 and fixed orifices 4 specifically to be:
obtaining a skeleton model of a diseased ankle part of a patient according to medical image data, manufacturing a mesh-shaped hollow support body 1 model which accords with the shape and size of the diseased ankle part of the patient, adding a connecting block 3 on the mesh-shaped hollow support body 1 model and adding a fixing hole 4 on the connecting block 3, then printing the mesh-shaped hollow support body 1 with the connecting block 3 and the fixing hole 4 by a metal 3D printer, manufacturing a mold which can be arranged in the mesh-shaped hollow support body 1 and is attached to the mesh-shaped hollow support body 1, and selecting a medical grade titanium alloy material for printing by the metal 3D printer;
step 2, preparing an antibacterial porous nano hydroxyapatite composite material;
the step 2 specifically comprises the following steps:
step 2.1, dissolving collagen in deionized water, and stirring for 4.5 hours at normal temperature to prepare a collagen solution of 0.5 mg/ml;
step 2.2, adding a calcium chloride solution with the concentration of 0.1mol/L into the prepared collagen solution according to the volume ratio of 7.25:50, mixing and stirring for 27min at normal temperature, then slowly adding a sodium dihydrogen phosphate solution with the concentration of 0.1mol/L, adjusting the pH value with 0.1mol/L sodium hydroxide to 7.5, beginning to precipitate a compound A, keeping the pH value of the solution containing the compound A at the normal temperature to 7.5, and keeping the solution for 23.5 h;
step 2.3, centrifuging the solution containing the compound A by using a centrifuge, wherein the rotating speed of the centrifuge is 6000rpm, removing supernatant after the centrifuge rotates for 4.5min, repeatedly washing the solution by using deionized water for 1.3h to remove salt ions to obtain a sample of the compound A, freezing the sample in a refrigerator at the temperature of-65 ℃ for 23.5h, taking out the sample, and drying the sample for 23.5h to obtain a white powdery nano hydroxyapatite-collagen composite material;
step 2.4, mixing the obtained nano hydroxyapatite-collagen composite material with deionized water according to the mass-to-volume ratio of 1.3:25, stirring for 12min by using a magnetic stirrer to prepare white turbid liquid B, mixing silver nitrate with the deionized water according to the mass-to-volume ratio of 1.3:250 to obtain silver nitrate solution C, then dropping the silver nitrate solution C into the liquid B under stirring at a constant speed to obtain a mixed solution D, continuously stirring the mixed solution at the temperature of 37 ℃ for 6.5h, centrifuging the mixed solution D by using a centrifuge at the rotating speed of 6000rpm, removing supernatant after the centrifuge rotates for 4.5min, repeatedly washing the mixed solution for 1.3h by using deionized water to remove salt ions to obtain a sample of the mixed solution D, freezing the sample in a refrigerator at the temperature of-65 ℃ for 23.5h, taking out the sample, and drying the sample for 23.5h to obtain a yellow powdery silver phosphate-nano hydroxyapatite-collagen composite material;
mixing the obtained silver phosphate-nano hydroxyapatite-collagen composite material with medical polycaprolactone powder according to the mass ratio of 7.5:1.5, then placing the mixture into a torque rheometer, stirring the mixture for 4.5min at the normal temperature at the rotating speed of 83r/min to fully mix the mixed solution, then melting and banburying the mixture for 8min in the torque rheometer at the rotating speed of 105r/min and at the temperature of 100 ℃ until the torque reaches balance, and obtaining the silver phosphate-nano hydroxyapatite-collagen-polycaprolactone composite material, namely the antibacterial porous nano hydroxyapatite composite material;
step 3, filling the antibacterial porous nano hydroxyapatite composite material into the hollow part of the mesh-shaped hollow support body 1;
the step 3 specifically comprises the following steps:
heating the antibacterial porous nano-hydroxyapatite composite material prepared in the step 2.4 to 95 ℃ in an aseptic environment, putting the mesh-shaped hollow support body 1 with the connecting blocks 3 and the fixing holes 4 printed in the step 1 in a manufactured mould, heating to 95 ℃, pressing the antibacterial porous nano-hydroxyapatite composite material at 95 ℃ into the mould from one side of the mould under the pressure of 3.3Mpa, stopping filling the material after the antibacterial porous nano-hydroxyapatite composite material on the other side of the mould is extruded from a flash port of the mould, maintaining the pressure at 1Mpa for 1.5 hours at the temperature of 95 ℃, correcting to remove the redundant antibacterial porous nano-hydroxyapatite composite material after cooling and solidification, cleaning, freeze-drying and sterilizing;
step 4, uniformly inoculating the cultured stem cells of the patient on the antibacterial porous nano-hydroxyapatite composite material, and then carrying out aseptic packaging to obtain the bone repair scaffold with self-repairing and antibacterial effects;
the step 4 specifically comprises the following steps:
extracting appropriate amount of bone marrow from patient, separating stem cells from patient, culturing, growing to fuse, preparing stem cells into cell suspension at 2 × 10 8 L -1 The cell density is 2mL, the cell suspension is evenly inoculated on the antibacterial porous nano-hydroxyapatite composite material, and the cell suspension is subjected to multiplication culture for 22 hours in a carbon dioxide incubator with the volume fraction of 5% of carbon dioxide and the humidity of 95% and the constant temperature of 37.5 ℃, and then is subjected to aseptic packaging and storage at the temperature of 4 ℃ to obtain the bone repair scaffold with self-repairing and antibacterial effects.
Example 6
A manufacturing method of a bone repair support with self-repairing and antibacterial effects comprises the following steps:
step 1, manufacturing a reticular hollow support body 1 with a connecting block 3 and a fixing hole 4, and culturing stem cells of a patient;
the step 1 of culturing the stem cells of the patient comprises the following steps: taking the bone marrow of a patient, separating out the stem cells of the patient, and carrying out sterile culture on the stem cells at 37 ℃;
step 1 preparation has netted fretwork support body 1 of connecting block 3 and fixed orifices 4 specifically to be:
obtaining a skeleton model of a diseased thigh part of a patient according to medical image data, manufacturing a mesh-shaped hollow support body 1 model which accords with the shape and size of a diseased part of the patient, adding a connecting block 3 on the mesh-shaped hollow support body 1 model and a fixing hole 4 on the connecting block 3, then printing the mesh-shaped hollow support body 1 with the connecting block 3 and the fixing hole 4 by a metal 3D printer, manufacturing a mold which can be arranged in the mesh-shaped hollow support body 1 and is attached to the mesh-shaped hollow support body 1, and selecting a medical grade titanium alloy material for printing by the metal 3D printer;
step 2, preparing an antibacterial porous nano hydroxyapatite composite material;
the step 2 specifically comprises the following steps:
step 2.1, dissolving collagen in deionized water, and stirring for 5 hours at normal temperature to prepare a collagen solution of 0.5 mg/ml;
step 2.2, adding a calcium chloride solution with the concentration of 0.1mol/L into the prepared collagen solution according to the volume ratio of 7.5:50, mixing and stirring for 30min at normal temperature, then slowly adding a sodium dihydrogen phosphate solution with the concentration of 0.1mol/L, adjusting the pH value with 0.1mol/L sodium hydroxide to 8.0, beginning to precipitate a compound A, keeping the pH value of the solution containing the compound A at normal temperature to 8.0, and keeping the solution for 24 h;
step 2.3, centrifuging the solution containing the compound A by using a centrifuge, wherein the rotating speed of the centrifuge is 6000rpm, removing supernatant after the centrifuge rotates for 5min, repeatedly washing the solution by using deionized water for 1.5h to remove salt ions to obtain a sample of the compound A, freezing the sample in a refrigerator at the temperature of-60 ℃ for 24h, taking out the sample, and drying the sample for 24h to obtain a white powdery nano hydroxyapatite-collagen composite material;
step 2.4, mixing the obtained nano hydroxyapatite-collagen composite material powder with deionized water according to the mass-to-volume ratio of 1.5:25, stirring for 15min by using a magnetic stirrer to prepare white turbid liquid B, mixing silver nitrate with the deionized water according to the mass-to-volume ratio of 1.5:250 to obtain silver nitrate solution C, then dropping the silver nitrate solution C into the liquid B under stirring at a constant speed to obtain a mixed solution D, continuously stirring the mixed solution D7h at the temperature of 38 ℃, centrifuging the mixed solution D by using a centrifuge, wherein the rotating speed of the centrifuge is 6000rpm, removing supernatant after the centrifuge rotates for 5min, repeatedly washing the mixed solution with deionized water for 1.5h to remove salt ions to obtain a sample of the mixed solution D, freezing the sample in a refrigerator at the temperature of-60 ℃ for 24h, taking out the frozen sample, and drying the frozen sample for 24h to obtain a yellow powdery silver phosphate-nano hydroxyapatite-collagen composite material;
mixing the obtained silver phosphate-nano hydroxyapatite-collagen composite material with medical polycaprolactone powder according to a mass ratio of 7:1, then placing the mixture into a torque rheometer, stirring the mixture for 5min at a normal temperature at a rotating speed of 85r/min to fully mix the mixed solution, and then carrying out melt banburying on the mixture for 10min in the torque rheometer at a rotating speed of 100r/min at a temperature of 105 ℃ until the torque reaches balance to obtain the silver phosphate-nano hydroxyapatite-collagen-polycaprolactone composite material, namely the antibacterial porous nano hydroxyapatite composite material;
step 3, filling the antibacterial porous nano hydroxyapatite composite material into the hollow part of the mesh hollow bracket body 1;
the step 3 specifically comprises the following steps:
heating the antibacterial porous nano-hydroxyapatite composite material prepared in the step 2.4 to 100 ℃ in an aseptic environment, putting the mesh-shaped hollow support body 1 with the connecting blocks 3 and the fixing holes 4 printed in the step 1 in a manufactured mould, heating to 100 ℃, pressing the antibacterial porous nano-hydroxyapatite composite material at 100 ℃ into the mould from one side of the mould under the pressure of 3.5Mpa, stopping filling the material after the antibacterial porous nano-hydroxyapatite composite material on the other side of the mould is extruded from a flash port of the mould, maintaining the pressure at 1.1 for 2 hours at the temperature of 100 ℃, correcting to remove the redundant antibacterial porous nano-hydroxyapatite composite material under the pressure of 100 Mpa after cooling and solidification, then cleaning, freeze-drying and sterilizing;
step 4, uniformly inoculating the cultured stem cells of the patient on the antibacterial porous nano-hydroxyapatite composite material, and then carrying out aseptic packaging to obtain the bone repair scaffold with self-repairing and antibacterial effects;
the step 4 specifically comprises the following steps:
extracting appropriate amount of bone marrow from patient, separating stem cells from patient, culturing, growing to fuse, preparing stem cells into cell suspension at 2 × 10 8 L -1 The cell density is 2mL, the cell suspension is evenly inoculated on the antibacterial porous nano-hydroxyapatite composite material, the cell suspension is subjected to proliferation culture for 24 hours in a carbon dioxide incubator with the volume fraction of 5% of carbon dioxide and the humidity of 95% and the constant temperature of 38 ℃, and then the cell suspension is subjected to aseptic packaging and storage at the temperature of 6 ℃ to obtain the bone repair scaffold with self-repairing and antibacterial effects.

Claims (2)

1. The manufacturing method of the bone repair support with the self-repairing and antibacterial effects is characterized in that the bone repair support with the self-repairing and antibacterial effects comprises a netted hollow support body (1), the netted hollow support body (1) is made of a titanium alloy material, connecting blocks (3) are arranged at two ends of the netted hollow support body (1), two fixing holes (4) are formed in each connecting block (3), a filler (2) is filled in the netted hollow support body (1), the filler (2) is located at a hollow part of the netted hollow support body (1), the filler (2) is a porous nano-hydroxyapatite composite material, and patient stem cells are inoculated on the porous nano-hydroxyapatite composite material;
the manufacturing method comprises the following steps:
step 1, manufacturing a reticular hollow support body (1) with connecting blocks (3) and fixing holes (4), and culturing stem cells of a patient;
the step 1 of culturing the stem cells of the patient comprises the following steps: taking bone marrow of a patient, separating stem cells of the patient, and performing sterile culture on the stem cells at 36-37 ℃;
step 1 preparation has netted fretwork support body (1) of connecting block (3) and fixed orifices (4) specifically does:
obtaining a skeleton model of a lesion part of a patient according to medical image data, manufacturing a mesh-shaped hollow support body (1) model which accords with the shape and size of the lesion part of the patient, adding a connecting block (3) on the mesh-shaped hollow support body (1) model and a fixing hole (4) on the connecting block (3), then printing the mesh-shaped hollow support body (1) with the connecting block (3) and the fixing hole (4) by a metal 3D printer, manufacturing a mold which can be arranged in the mesh-shaped hollow support body (1) and is attached to the mesh-shaped hollow support body (1), and selecting a medical-grade titanium alloy material for printing by the metal 3D printer;
step 2, preparing an antibacterial porous nano hydroxyapatite composite material;
the step 2 specifically comprises the following steps:
step 2.1, mixing collagen and deionized water solution containing 3% silver phosphate by mass percent in a mass volume ratio of 5-6:10, stirring for 4-5 hours at normal temperature, and preparing into 0.6mg/ml collagen solution;
step 2.2, taking a calcium chloride solution with the concentration of 0.1mol/L, and mixing the calcium chloride solution with the weight ratio of 7-7.5: adding 50 volume ratio into prepared collagen solution, mixing and stirring for 15-30min at normal temperature, slowly adding 0.1mol/L sodium dihydrogen phosphate solution, adjusting pH value with 0.1mol/L sodium hydroxide to 7.0-8.0 to start precipitation of compound A, and keeping pH value of the solution containing compound A at normal temperature to 7.0-8.0 for 23-24 h;
step 2.3, centrifuging the solution containing the compound A by using a centrifuge, wherein the rotating speed of the centrifuge is 6000rpm, removing supernatant after the centrifuge rotates for 4-5min, repeatedly washing the solution by using deionized water for 1-1.5h to remove salt ions to obtain a sample of the compound A, freezing the sample in a refrigerator at-70 to-60 ℃ for 23-24h, taking out the sample, and drying the sample for 23-24h to obtain a yellow powdery silver phosphate-nano hydroxyapatite-collagen composite material;
step 2.4, mixing the obtained silver phosphate-nano hydroxyapatite-collagen composite material powder and medical polycaprolactone powder according to a mass ratio of 8-7:2-1, then placing the mixture into a torque rheometer, stirring the mixture for 4-5min at a normal temperature at a rotating speed of 80-85r/min to fully mix the mixture, and then melting and banburying the mixture for 8-10min in the torque rheometer at a rotating speed of 110-100r/min and a temperature of 95-105 ℃ until the torque is balanced to obtain the silver phosphate-nano hydroxyapatite-collagen-polycaprolactone composite material, namely the antibacterial porous nano hydroxyapatite composite material;
step 3, filling the antibacterial porous nano hydroxyapatite composite material into the hollow part of the mesh hollow support body (1);
the step 3 specifically comprises the following steps:
heating the antibacterial porous nano-hydroxyapatite composite material prepared in the step 2.4 to 90-100 ℃ in an aseptic environment, putting the net-shaped hollowed-out support body (1) with the connecting blocks (3) and the fixing holes (4) printed in the step 1 into a manufactured mould, heating to 90-100 ℃, pressing the antibacterial porous nano-hydroxyapatite composite material at 90-100 ℃ into the mould from one side of the mould under the pressure of 3-3.5Mpa, stopping filling the material after the antibacterial porous nano-hydroxyapatite composite material on the other side of the mould is pressed out from a material overflowing port of the mould, keeping the pressure of 0.9-1.1Mpa for 1-2h at the temperature of 90-100 ℃, cooling and solidifying, correcting to remove the redundant antibacterial porous nano-hydroxyapatite composite material, cleaning, freeze-drying, and packaging, Sterilizing;
and 4, uniformly inoculating the cultured stem cells of the patient on the antibacterial porous nano-hydroxyapatite composite material, and then carrying out aseptic packaging to obtain the bone repair scaffold with self-repairing and antibacterial effects.
2. The method for manufacturing the bone repair scaffold with self-repairing and antibacterial effects as claimed in claim 1, wherein the step 4 specifically comprises:
extracting appropriate amount of bone marrow from patient, separating stem cells from patient, culturing, growing to fuse, preparing stem cells into cell suspension at 2 × 10 8 L -1 The cell density of the scaffold is determined, 2mL of cell suspension is evenly inoculated on the antibacterial porous nano-hydroxyapatite composite material, the cell suspension is subjected to enrichment culture for 23-24h in a carbon dioxide incubator with the volume fraction of 5% of carbon dioxide and the humidity of 95% and the constant temperature of 37-38 ℃, and then the cell suspension is subjected to aseptic packaging and storage at the temperature of 2-6 ℃ to obtain the bone repair scaffold with self-repairing and antibacterial effects.
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