CN113730660A - 3D printing porous slow-release vancomycin tricalcium phosphate interstitial biological composite scaffold and preparation method and application thereof - Google Patents

3D printing porous slow-release vancomycin tricalcium phosphate interstitial biological composite scaffold and preparation method and application thereof Download PDF

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CN113730660A
CN113730660A CN202111032477.4A CN202111032477A CN113730660A CN 113730660 A CN113730660 A CN 113730660A CN 202111032477 A CN202111032477 A CN 202111032477A CN 113730660 A CN113730660 A CN 113730660A
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vancomycin
tricalcium phosphate
composite scaffold
release
biological composite
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许刚
何纯青
张飞
买买提克里木·吐松江
尹东峰
吾木尔别克·提劳汉
刘剑
贾勇
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General Hospital Of Xinjiang Military Region Pla
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General Hospital Of Xinjiang Military Region Pla
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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
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/56—Porous materials, e.g. foams or sponges
    • 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
    • A61L27/40—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material
    • A61L27/44—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix
    • A61L27/46—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix with phosphorus-containing inorganic fillers
    • 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
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/54—Biologically active materials, e.g. therapeutic substances
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B33—ADDITIVE MANUFACTURING TECHNOLOGY
    • B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3-D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3-D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
    • B33Y40/00—Auxiliary operations or equipment, e.g. for material handling
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B33—ADDITIVE MANUFACTURING TECHNOLOGY
    • B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3-D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3-D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
    • B33Y70/00—Materials specially adapted for additive manufacturing
    • B33Y70/10—Composites of different types of material, e.g. mixtures of ceramics and polymers or mixtures of metals and biomaterials
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B33—ADDITIVE MANUFACTURING TECHNOLOGY
    • B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3-D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3-D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
    • B33Y80/00—Products made by additive manufacturing
    • 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
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
    • A61L2300/252—Polypeptides, proteins, e.g. glycoproteins, lipoproteins, cytokines
    • 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
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
    • A61L2300/41—Anti-inflammatory agents, e.g. NSAIDs
    • 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
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/60—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a special physical form
    • A61L2300/602—Type of release, e.g. controlled, sustained, slow

Abstract

The invention discloses a 3D printing porous slow-release vancomycin tricalcium phosphate interstitial biological composite stent and a preparation method and application thereof, wherein the composite stent is mainly prepared from polylactic acid-glycolic acid, tricalcium phosphate and vancomycin; the composite scaffold has a three-dimensional porous structure, the porosity is 40% -85%, and the pore connectivity is 95% -99%. The porosity of the obtained biological composite scaffold is 80% by adopting a low-temperature rapid forming 3D printing forming technology, and the composite scaffold material has good vancomycin slow release effect, bacteriostatic effect and compatibility in the application of chronic osteomyelitis treatment.

Description

3D printing porous slow-release vancomycin tricalcium phosphate interstitial biological composite scaffold and preparation method and application thereof
Technical Field
The invention relates to the technical field of biomedical materials, in particular to a 3D printing porous slow-release vancomycin tricalcium phosphate interstitial biological composite scaffold and a preparation method and application thereof.
Background
In clinical practice in orthopedics department, the repair of large bone defects caused by severe trauma, infection of bones and joints, tuberculosis or tumor erosion, extensive surgical resection and the like is one of the most difficult problems in the orthopedics department. Especially the segmental bone defect caused by chronic osteomyelitis has a well-known refractory property. Although autologous bone grafting is the gold standard for treating the bone defect, the autologous bone grafting has a plurality of limitations because the autologous bone grafting has limited sources and is easy to cause complications in a bone supply area, and is difficult to be used for treating large-section bone defects, and allogeneic bone grafting is easy to cause immune rejection. In recent years, the search for alternative materials for bone grafting has become one of the research hotspots in the field of orthopedics. A large class of bioactive ceramics from nature, such as beta-tricalcium phosphate, has good bioactivity, good biodegradability and proper biocompatibility, and is an ideal choice for developing high-efficiency and safe bone repair materials proved by previous researches. Scaffolds are the core and key to the construction of tissue engineered bones.
A3D printing technology belongs to the category of additive manufacturing, is a new technology appearing internationally in the later period of the 20 th century 80 years, integrates the fields of computer aided design and computer aided manufacturing (CAD/CAM), numerical control technology, laser technology, high polymer materials, three-dimensional CT technology and the like into a whole, and mainly utilizes the geometric information determined by a CAD model of a part under the control of a computer according to the principle of discrete/stacking forming to carry out two-dimensional scanning and processing on a layer material or a powder layer, wherein the layer material or the powder layer is stacked layer by layer from point to line from line to surface from surface to body.
Polylactic-co-glycolic acid (PLGA), a polymer approved for implantation in the body by the food and drug administration (CFDA), is a good binder in 3D printing devices. Tricalcium Phosphate (TCP) is also a biological ceramic material approved by CFDA (carbon fiber digital data acquisition), TCP powder can be used as a raw material of a 3D printer, has good bone inductivity, and the mechanical strength of a printed PLGA/TCP stent is higher than that of a pure PLGA stent. Many researches show that the PLGA and TCP composite porous scaffold material has good biocompatibility and high porosity, can be degraded and absorbed in vivo, is applied to bone defect experimental animal models, obtains good repairing and reconstructing effects, and is one of ideal candidate materials for autologous bone transplantation. PLGA and beta-TCP are biological materials approved by the national food and drug administration (CFDA), PLGA is suitable for a low-temperature rapid deposition process, beta-TCP powder can improve the physicochemical properties of the materials and has good bone inductivity when being applied in vivo, and the mechanical strength of the scaffold material printed by combining the two materials is higher than that of a pure PLGA scaffold. However, vancomycin has poor penetration ability in bone tissue, and it is difficult to control infection by intravenous administration for patients with chronic osteomyelitis.
Infection treatment is an essential and important measure in open fractures due to trauma, osteomyelitis and post-surgical treatment. Although conventional anti-infection treatment is mainly carried out by oral administration or intravenous administration, although a certain curative effect can be achieved, for patients with severe bone tissue infection, blood flow around the infected bone tissue is reduced, which easily causes the phenomenon that the concentration of the medicine in blood is high and the concentration of the medicine in local bone tissue is low, and the treatment effect is reduced. Infectious bone defects are mainly found in open fracture of limbs caused by high-energy injuries such as war firearm injuries, traffic injuries and the like, are one of the most common high-disability injuries in the modern times, have the characteristics of high infection rate, serious complications, long treatment period, high disability rate and the like, and how to quickly and effectively treat the infectious bone defects in orthopedics department is a major problem in the department field of bone injury. The research shows that the infectious bacterial spectrum relates to various bacteria including gram negative bacteria, staphylococcus epidermidis, anaerobe and multi-drug resistant bacteria. Researchers have long emphasized the use of broad-spectrum antibiotics (e.g., ampicillin, sulfadimidine, gentamicin, etc.) in the open wound early vein to reduce infection rates. However, intravenous application of antibiotics faces a number of difficulties such as selection, configuration, infusion of antibiotics as soon as possible, and in order to achieve an effective concentration of antibiotics locally, it is necessary to maintain a high concentration of antibiotics in the blood, which increases the toxic side effects of antibiotics. Topical application of antibiotics has become a routine means of treating bone infections. When the artificial material is clinically applied to repair infectious bone defects, cells and tissue fluid are difficult to permeate into the material, and the growth speed of blood vessels is quite low, so that the blood flow around an infected focus is reduced, the local antibiotics cannot reach effective concentration, and the local infection is difficult to be effectively controlled. How to improve the osteogenesis, promote the growth of new blood vessels and simultaneously play a treatment function, such as antibacterial activity and the like, by adjusting the components of the material, such as different calcium-phosphorus content ratios and structural design, such as pore diameter and porosity, or a method of compounding bioactive molecules, is a key problem to be solved by the development of the segmental bone defect prosthesis at present. Therefore, the research on the biodegradable material capable of compounding antibiotics controls infection by means of the local drug slow-release effect after the material is implanted in one period of operation, and meanwhile, the repair of bone defects is not influenced, so that the material becomes one of the research hotspots of interstitial materials.
The preparation methods of the common scaffold materials at the present stage can be divided into the following categories: thermally induced phase separation, solution cast particle leaching, gas foaming, microsphere sintering, and the like. The application of the additive manufacturing emerging technology in the field of bone tissue engineering research greatly promotes the research and development of bone repair biomaterials, and various scaffolds have been developed for various sub-specialty applications in orthopedics. The most important additive manufacturing techniques include three-dimensional inkjet printing, stereolithography, fused deposition modeling, and selective laser sintering. The operating temperature of conventional additive manufacturing printing methods is high. The method is not suitable for directly adding antibiotics into the printing ink solution, and most researches select a secondary adsorption adding mode to add the antibiotic slow-release component, so that the material prepared by the method has unstable slow-release efficiency and is difficult to meet the clinical requirements.
In summary, how to develop a biological composite scaffold capable of slowly releasing antibiotics is a technical problem to be solved urgently.
Disclosure of Invention
Therefore, the invention provides a 3D printing porous slow-release vancomycin tricalcium phosphate interstitial biological composite scaffold and a preparation method and application thereof.
In order to achieve the above purpose, the invention provides the following technical scheme:
the embodiment of the invention provides a 3D printing porous slow-release vancomycin tricalcium phosphate interstitial biological composite scaffold, which is mainly prepared from polylactic acid-glycolic acid, tricalcium phosphate and vancomycin;
the composite scaffold has a three-dimensional porous structure, the porosity is 40% -85%, and the pore connectivity is 95% -99%.
In one embodiment of the invention, the pores comprise pores and micropores.
In one embodiment of the present invention, the diameter of the hole is 390-410 μm;
the diameter of the micropores is 20-40 mu m.
In one embodiment of the present invention, the depth of the micro-holes is 10 to 20 μm.
The invention also provides a preparation method of the 3D printing porous slow-release vancomycin tricalcium phosphate interstitial biological composite scaffold, which comprises the following steps:
s1, dissolving collagen in 1, 4-dioxane, and uniformly mixing to obtain a first solution;
s2, adding vancomycin powder, triple calcium phosphate powder and polylactic acid-glycolic acid powder into 1,4 dioxane, and uniformly mixing to obtain a second solution;
s3, uniformly mixing the first solution and the second solution to obtain printing slurry;
s4, printing and forming the printing slurry in a layered curing mode by a 3D printer according to a preset three-dimensional model to obtain a primary biological composite bracket;
and S5, carrying out low-temperature treatment and freeze drying on the primary biological composite scaffold to obtain the porous slow-release vancomycin tricalcium phosphate interstitial biological composite scaffold.
In one embodiment of the present invention, the mass concentration of the collagen in the first solution is 10%.
In one embodiment of the present invention, in the second solution,
the mass concentration of the vancomycin is 3%;
the mass concentration of the tricalcium phosphate is 3%;
the mass concentration of the polylactic acid-glycolic acid is 12%.
In one embodiment of the invention, the first solution and the second solution are mixed according to a volume ratio of 1:1 to obtain the printing paste.
In one embodiment of the invention, the particle sizes of the vancomycin powder, the tricalcium phosphate powder and the polylactic acid-glycolic acid powder are all 300 meshes.
The application of the 3D printed porous slow-release vancomycin tricalcium phosphate interstitial biological composite scaffold in treatment of infectious bone defect infection and chronic osteomyelitis also belongs to the protection scope of the invention.
The invention has the following advantages:
the porosity of the obtained biological composite scaffold is 80% by adopting a low-temperature rapid forming 3D printing forming technology, and the composite scaffold material has good vancomycin slow release effect, bacteriostatic effect and compatibility in the application of chronic osteomyelitis treatment.
According to the invention, by using Computer Aided Design (CAD) and Computer Aided Manufacturing (CAM) technologies, composite high polymer materials are subjected to layer stacking under the condition of 30-40 ℃ below zero by using a numerical control technology according to a CAD model to obtain a clinically actually required personalized three-dimensional entity, so that brand-new personalized Design of an implanted stent can be realized, the stent with composite requirements can be accurately printed, the stents with different three-dimensional shapes can be printed, on the other hand, the pore diameter structures on the surface and in the implanted stent can be controlled according to requirements, and the pore diameter range can be from 100 micrometers to 1000 micrometers. Thirdly, the biological activity of the artificial bone material can be obviously improved by a method of loading active molecules (such as bone morphogenetic protein, angiogenesis promoting growth factors, natural plant derived small molecules and the like) during the preparation of the scaffold.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It should be apparent that the drawings in the following description are merely exemplary, and that other embodiments can be derived from the drawings provided by those of ordinary skill in the art without inventive effort.
The structures, ratios, sizes, and the like shown in the present specification are only used for matching with the contents disclosed in the specification, so as to be understood and read by those skilled in the art, and are not used to limit the conditions that the present invention can be implemented, so that the present invention has no technical significance, and any structural modifications, changes in the ratio relationship, or adjustments of the sizes, without affecting the effects and the achievable by the present invention, should still fall within the range that the technical contents disclosed in the present invention can cover.
Fig. 1 is a general view and a microscopic electron microscope image of a 3D printed porous slow-release vancomycin tricalcium phosphate interstitial biological composite scaffold provided by an embodiment of the present invention, wherein a1 and a2 are general views of the composite scaffold material of the present invention, and b1 and b2 are characteristic views of pores of the composite scaffold material under an electron microscope;
FIG. 2 is a graph showing the test results of the compatibility test of the 3D printed porous slow-release vancomycin tricalcium phosphate interstitial biological composite scaffold according to the embodiment of the invention;
fig. 3 is a schematic diagram illustrating the result of vancomycin sustained release measurement of a 3D printed porous sustained-release vancomycin tricalcium phosphate interstitial biological composite scaffold provided by an embodiment of the present invention;
fig. 4 is a result graph of a bacteriostasis experiment of the 3D printed porous sustained-release vancomycin tricalcium phosphate matrix biological composite scaffold provided by the embodiment of the present invention.
Detailed Description
The present invention is described in terms of particular embodiments, other advantages and features of the invention will become apparent to those skilled in the art from the following disclosure, and it is to be understood that the described embodiments are merely exemplary of the invention and that it is not intended to limit the invention to the particular embodiments disclosed. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1 preparation of 3D-printed porous sustained-release vancomycin tricalcium phosphate interstitial biological composite scaffold
The embodiment provides a method for 3D printing of a porous slow-release vancomycin tricalcium phosphate interstitial biological composite scaffold, which comprises the following steps:
1. dissolving collagen in 1,4 dioxane, and mixing to obtain a first solution, wherein the concentration of the collagen in the first solution is 10%.
2. Adding vancomycin powder, triple calcium phosphate powder and polylactic acid-glycolic acid powder into 1,4 dioxane, and uniformly mixing to obtain a second solution; wherein the particle sizes of the vancomycin powder, the triple calcium phosphate powder and the polylactic acid-glycolic acid powder are 300 meshes;
in the second solution, the mass concentration of vancomycin is 3%;
the mass concentration of tricalcium phosphate is 3%;
the mass concentration of the polylactic acid-glycolic acid is 12 percent.
3. Mixing the first solution and the second solution according to the volume ratio of 1:1, and uniformly mixing to obtain printing slurry;
4. printing and molding the printing slurry in a layered curing manner by adopting a 3D printer according to a preset three-dimensional model to obtain a primary biological composite bracket;
specifically, a target dimension is counted by using a MEDCAD module in the modular structure software Mimics, a CAD model is created according to analysis and measurement results, for example, the CAD model can be designed into a cuboid porous bone biological composite support with the length multiplied by the width multiplied by the height multiplied by 3 multiplied by 4cm, data containing the CAD model is exported into an STL format file, and then a low-temperature rapid prototyping machine carries out layering processing by using self-provided layering software so as to determine the thickness of each layer, namely the layering thickness. Wherein, the layering thickness is 0.12mm, the nozzle angle is 0 degree/90 degree conversion, one layer is printed according to 0 degree or 90 degree, then the next layer is printed according to 90 degree or 0 degree, the conversion is carried out, and the layered data is exported to be a preset three-dimensional model of the CLI format file.
Pouring the printing slurry into a slurry tank of a low-temperature rapid prototyping printer (the machine model of the low-temperature rapid prototyping printer CLRF-2000-II), inputting a designed three-dimensional model, setting the spinning distance to be 1.2mm, the nozzle speed to be 21mm/s and the liquid outflow speed to be l ml/min according to the CLI format file, and performing three-dimensional printing at the temperature of minus 25 ℃ to form a porous primary biological composite scaffold, wherein the holes of the porous primary biological composite scaffold are circular, the diameter of the holes is 400 mu m, the initial porosity is 68 percent and the hole communication rate is 98 percent.
5. And carrying out low-temperature treatment and freeze drying on the primary biological composite scaffold to obtain the porous slow-release vancomycin tricalcium phosphate interstitial biological composite scaffold.
Specifically, after the primary biological composite scaffold is placed at-40 ℃ for 24min, the primary biological composite scaffold after low-temperature treatment is placed in a freeze-drying machine, the vacuum degree is 10Pa, the heating rate is 1 ℃/h, the temperature is raised to 20 ℃, the primary biological composite scaffold is freeze-dried for 48 hours, after the drying process, the organic reagent 1,4 dioxane is completely removed, and the porous slow-release vancomycin tricalcium phosphate interstitial biological composite scaffold with the pore diameter of 400 microns, the porosity of 80 percent and a large number of micropores with the pore diameter of 20-40 microns distributed on the pore wall is obtained, as shown in figure 1, the depth of the micropores of the biological composite scaffold is about 14 microns.
The porous biological composite scaffold material for compounding the vancomycin/PLGA/TCP organically integrates three types of biological materials with different properties, namely vancomycin, PLGA and beta-TCP to form a biological composite scaffold material, can flexibly prepare porous scaffolds with different microstructures by controlling various physical and chemical properties of the porous scaffolds, and can select a proper scaffold shape according to specific conditions clinically.
As a variable implementation mode, the invention can correspondingly prepare the 3D printing porous slow-release vancomycin tricalcium phosphate interstitial biological composite scaffold with the porosity of 40%, the porosity of 50%, the porosity of 60%, the porosity of 70% and the porosity of 80% by changing different conditions by using the method according to needs.
Test example 1 compatibility test of 3D-printed porous sustained-release vancomycin tricalcium phosphate interstitial biological composite scaffold
The experimental example of the invention is used for verifying that the porous sustained-release vancomycin tricalcium phosphate interstitial biological composite scaffold has no cytotoxicity to human bone marrow mesenchymal stem cells through in vitro biocompatibility test. In order to further explore whether the porous slow-release vancomycin tricalcium phosphate interstitial biological composite scaffold has good biocompatibility, human bone marrow mesenchymal stem cells are used as co-culture objects in the test example, the human bone marrow mesenchymal stem cells and the porous slow-release vancomycin tricalcium phosphate interstitial biological composite scaffold are placed together for co-culture, and the cell proliferation degree is measured for 7 days continuously so as to evaluate the biocompatibility of the material.
The method for testing the compatibility of the biological composite scaffold comprises the following steps:
firstly, human mesenchymal stem cells are separated and cultured, and the human bone marrow extraction solution comes from healthy donors and accords with the guidelines of the ethical committee of general hospitals in the military region of Xinjiang. 10mL of bone marrow fluid was mixed with PBS of equal volume, mononuclear cells in bone marrow were separated by centrifugation at 900 Xg for 30min using Percoll separation medium (density of working fluid 1.073g/mL, Pharmacia, USA), cultured in OriCell human mesenchymal stem cell medium, and cultured at 6X 103/cm2Inoculating into tissue culture flask containing 5% CO at 37 deg.C2The incubator of (2) for primary culture. The liquid was changed 1 to 2 times per week, the cells were digested using 0.25% trypsin/EDTA (HyColne, USA), adherent cells were passaged one third and the third generation of cells was used for the experiment.
Preparing cell suspension from leaching solution of 3 kinds of porosity materials and normal culture medium respectively, with concentration of 1 × 108/L-1Adding 4 suspensions into a 96-well plate, wherein each well is 100 mu L, culturing in a 37-degree incubator for 1, 3, 5 and 7 days, replacing culture solution every 2 days, adding 10 mu L CCK-8 reagent into each well at each time point, and detecting the absorbance value of each well under the wavelength of 450nm by using an enzyme-labeling instrument.
As shown in fig. 2, compared with the normal culture medium group, there is no significant difference in mesenchymal stem cell proliferation (P >0.05) after the leaching solution of the porous slow-release vancomycin tricalcium phosphate mesenchymal composite scaffold with three porosities is cultured for 1, 3, 5 and 7 days, i.e. the porous slow-release vancomycin tricalcium phosphate mesenchymal composite scaffold has no significant cytotoxicity to the bone marrow mesenchymal stem cells.
Test example 2 vancomycin sustained release determination of 3D printed porous sustained release vancomycin tricalcium phosphate interstitial biological composite scaffold
The vancomycin slow release test method for the 3D printing of the porous slow release vancomycin tricalcium phosphate interstitial biological composite scaffold comprises the following steps:
the 3 porosity materials prepared in the invention example are respectively put into 6 test tubes, 10ml of PBS (pH value 7.4) is added into each tube, the solution is replaced for 1 time every 24 hours, 24 hours of eluent in 1 st, 5 th, 10 th, 15 th, 20 th, 25 th and 30 th days is collected, and the concentration of vancomycin in the eluent is measured by a high performance liquid chromatograph.
As shown in FIG. 3, the results show that the drug release degree of the porous slow-release vancomycin tricalcium phosphate interstitial biological composite scaffold with three porosities is equivalent in the first 15 days, and can be kept above the standard of 50g/L, but after the 20 th day, the drug release efficiency of the 40% and 60% porosity groups is significantly lower than that of the 80% porosity group (P <0.05), and cannot be maintained above the standard of 50 g/L. The porous slow-release vancomycin tricalcium phosphate interstitial biological composite scaffold with the porosity of 80% prepared in the embodiment 1 of the invention is proved to have good slow-release effect.
Experimental example 3 bacteriostatic experiment of 3D-printed porous slow-release vancomycin tricalcium phosphate interstitial biological composite scaffold
3 kinds of materials with different porosities are respectively put into 6 pieces of materials uniformly coated with 1.5 multiplied by 108The size of the zone of inhibition was measured after 24h of culture at 37 ℃ in MH agar culture dishes of Staphylococcus aureus. The culture dish is replaced every 3 days, and the size of the inhibition zone on the 10 th, 15 th, 20 th, 25 th and 30 th days is measured.
As shown in fig. 4, the results show that the three porosity porous sustained-release vancomycin tricalcium phosphate interstitial biological composite scaffolds have equivalent bacteriostatic degrees in the first 15 days, and have no obvious difference, but at the 20 th day, the bacteriostatic zones of the 40% and 60% porosity groups begin to shrink, and have obvious difference compared with the 80% porosity group (P < 0.05). The porous slow-release vancomycin tricalcium phosphate interstitial biological composite scaffold with the porosity of 80% is proved to have long-term bacteriostatic effect.
The slow release capability test of vancomycin finds that the three stent materials with different porosities have good drug slow release capability, while the slow release capability of the material with 80% porosity is better and can be maintained at more than 50mg/L within 20 days. In-vitro antibacterial experiments prove that the scaffold material with the porosity of 80 percent has better long-term antibacterial effect than the rest two groups.
Moreover, in animal experiments, the anti-infection effect of the 3D porous scaffold group is equivalent to that of the bone cement bead chain group, and the anti-infection effect of the biological composite scaffold provided by the invention is fully proved to reach the clinical application standard, so that the local infection focus of chronic osteomyelitis can be effectively controlled. In a goat femur infectious bone defect model, the 3D printed porous slow-release vancomycin tricalcium phosphate interstitial biological composite scaffold is implanted, and the 3D printed porous slow-release vancomycin tricalcium phosphate interstitial biological composite scaffold is found to be significantly degraded in 12 weeks after operation compared with 4 weeks, so that the 3D printed porous slow-release vancomycin tricalcium phosphate interstitial biological composite scaffold has an anti-infection effect and also has good biodegradability.
Therefore, when the 3D printing porous slow-release vancomycin tricalcium phosphate interstitial biological composite scaffold is applied to clinical treatment of patients with chronic osteomyelitis with bone defects, the bone carrying scaffold can be installed immediately after the material is implanted in a first stage of debridement, the bone carrying treatment can be carried out synchronously along with complete control of infection and gradual absorption of the material, the step that the bone carrying can be carried out only after the implant is taken out in a second operation is avoided, the trauma caused by multiple operations is avoided, the treatment period and the cost of the patients are obviously shortened, and good social effects are generated.
Although the invention has been described in detail above with reference to a general description and specific examples, it will be apparent to one skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims (10)

1. A3D printing porous slow-release vancomycin tricalcium phosphate interstitial biological composite scaffold is characterized in that,
the composite stent is mainly prepared from polylactic acid-glycolic acid, tricalcium phosphate and vancomycin;
the composite scaffold has a three-dimensional porous structure, the porosity is 40% -85%, and the pore connectivity is 95% -99%.
2. The 3D printed porous slow-release vancomycin tricalcium phosphate matrix biological composite scaffold of claim 1,
the pores include pores and micropores.
3. The 3D printed porous slow-release vancomycin tricalcium phosphate matrix biological composite scaffold of claim 2,
the diameter of the hole is 390-410 mu m;
the diameter of the micropores is 20-40 mu m.
4. The 3D printed porous slow-release vancomycin tricalcium phosphate matrix biological composite scaffold of claim 2,
the depth of the micropores is 10-20 μm.
5. The preparation method of the 3D printed porous slow-release vancomycin tricalcium phosphate matrix biological composite scaffold according to any one of claims 1 to 4, wherein the method comprises the following steps:
s1, dissolving collagen in 1, 4-dioxane, and uniformly mixing to obtain a first solution;
s2, adding vancomycin powder, triple calcium phosphate powder and polylactic acid-glycolic acid powder into 1,4 dioxane, and uniformly mixing to obtain a second solution;
s3, uniformly mixing the first solution and the second solution to obtain printing slurry;
s4, printing and forming the printing slurry in a layered curing mode by a 3D printer according to a preset three-dimensional model to obtain a primary biological composite bracket;
and S5, carrying out low-temperature treatment and freeze drying on the primary biological composite scaffold to obtain the porous slow-release vancomycin tricalcium phosphate interstitial biological composite scaffold.
6. The method of claim 5,
the mass concentration of the collagen in the first solution is 10%.
7. The method of claim 5,
in the second solution, the mass concentration of the vancomycin is 3%;
the mass concentration of the tricalcium phosphate is 3%;
the mass concentration of the polylactic acid-glycolic acid is 12%.
8. The method of claim 5,
and mixing the first solution and the second solution according to the volume ratio of 1:1 to obtain the printing paste.
9. The method of claim 5,
the particle sizes of the vancomycin powder, the calcium phosphate tribasic powder and the polylactic acid-glycolic acid powder are all 300 meshes.
10. The use of the 3D printed porous slow-release vancomycin tricalcium phosphate composite scaffold according to any one of claims 1 to 4 in the treatment of infectious bone defect infection and chronic osteomyelitis.
CN202111032477.4A 2021-09-03 2021-09-03 3D printing porous slow-release vancomycin tricalcium phosphate interstitial biological composite scaffold and preparation method and application thereof Pending CN113730660A (en)

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