CN110665069A

CN110665069A - Preparation method of melt electrospinning composite fiber scaffold for bone repair and infection resistance

Info

Publication number: CN110665069A
Application number: CN201910973026.7A
Authority: CN
Inventors: 白见福; 王晗; 汤亚东; 梁峰; 汪子煦; 蓝兴梓; 周颖; 苗小敏
Original assignee: Guangdong University of Technology
Current assignee: Guangdong University of Technology
Priority date: 2019-10-14
Filing date: 2019-10-14
Publication date: 2020-01-10
Anticipated expiration: 2039-10-14
Also published as: CN110665069B

Abstract

The invention provides a preparation method of a melt electrospinning composite fiber scaffold for bone repair and infection resistance, which comprises the following steps: a) uniformly mixing polycaprolactone, polyethylene glycol and macrolide antibiotics in an organic solvent, evaporating the organic solvent, and then drying in vacuum to obtain a solid mixed material; the mass ratio of the polycaprolactone to the polyethylene glycol to the macrolide antibiotics is 90: (0.1-20): (0.1 to 20); b) and (b) carrying out melt electrospinning on the solid mixed material obtained in the step a) to obtain the melt electrospinning composite fiber scaffold for bone repair and infection resistance. Compared with the prior art, the preparation method provided by the invention can obtain the novel anti-infection melt electrospinning composite fiber scaffold for bone repair, which has the advantages of ordered structure, excellent biocompatibility, complete degradation, antibiosis, promotion of bone cell proliferation and capability of meeting the physical property requirement of bone repair, and solves the problems that the bone repair implant needs to be taken out for the second time in operation and the bone implant is infected.

Description

Preparation method of melt electrospinning composite fiber scaffold for bone repair and infection resistance

Technical Field

The invention relates to the technical field of biological materials, in particular to a preparation method of a melt electrospinning composite fiber scaffold for bone repair and infection resistance.

Background

The infection rate of plastic and bone trauma surgery accounts for 15% of all hospital infections. In the united states, bone infections associated with implantation (suppurative arthritis and osteomyelitis) occur in 5% of 200 ten thousand cases of fracture fixation each year, resulting in $ 15,000 to $ 50,000 per event. Several steps are commonly experienced in the treatment of bone infections: treatment usually goes through several stages: local drainage, debridement of necrotic tissue, management of bone dead space, soft tissue coverage, and restoration of blood supply. The management of the bone dead zone is the most important step, and the effective management of the bone dead zone can effectively prevent the reinfection of a bone defect part and the physical integrity of bone tissues, prevent the growth of soft tissues in the bone defect part, and generally fill the bone defect part with an implant.

Currently, Polymethylmethacrylate (PMMA) is commonly used as an implant for treating bone defects, and infection resistance at the site of a bone defect can be achieved by mixing a broad spectrum antibiotic such as gentamicin in PMMA. However, since PMMA is a non-degradable material, the release of the antibacterial drug is limited, so that the antibacterial effect is not desirable, and it may be difficult to achieve infection resistance at the bone defect site. Moreover, due to the non-degradability of PMMA, PMMA needs to be taken out after the bone defect is treated, which greatly increases the treatment cost and is easy to cause secondary infection at the bone defect part. Therefore, how to manufacture a bone implant capable of preventing infection of a bone defect site and effectively treating the bone defect site is a great technical problem to be solved in the field of bone implants.

Disclosure of Invention

In view of the above, the invention aims to provide a preparation method of a melt electrospinning composite fiber scaffold for bone repair and infection resistance, which can solve the technical problems that the existing bone repair implant needs to be taken out by a secondary operation and is susceptible to infection, and can promote the proliferation of bone cells and accelerate the bone repair.

The invention provides a preparation method of a melt electrospinning composite fiber scaffold for bone repair and infection resistance, which comprises the following steps:

a) uniformly mixing polycaprolactone, polyethylene glycol and macrolide antibiotics in an organic solvent, evaporating the organic solvent, and then drying in vacuum to obtain a solid mixed material; the mass ratio of the polycaprolactone to the polyethylene glycol to the macrolide antibiotics is 90: (0.1-20): (0.1 to 20);

b) and (b) carrying out melt electrospinning on the solid mixed material obtained in the step a) to obtain the melt electrospinning composite fiber scaffold for bone repair and infection resistance.

Preferably, the macrolide antibiotic in step a) is selected from one or more of roxithromycin, clarithromycin, clindamycin and azithromycin.

Preferably, the organic solvent in step a) is tetrahydrofuran.

Preferably, the mixing in step a) is performed by stirring; the stirring temperature is 45-55 ℃, the rotating speed is 350-450 r/min, and the time is 22-26 h.

Preferably, the temperature of the vacuum drying in the step a) is 75-85 ℃, and the time is 2-5 h.

Preferably, the melt electrospinning process in step b) specifically comprises:

b1) cutting the solid mixed material into pieces and putting the pieces into a charging barrel; cleaning and drying the needle head; connecting the positive electrode of a high-voltage direct-current power supply to a glass plate of the collection platform, and connecting the ground to the needle head; then the grounded needle head is arranged on the material barrel, and the material barrel is placed in the heating cavity;

b2) enabling the melt spinning machine, and positioning a collection platform loaded with glass sheets below the needle head; controlling a motor in the z-axis direction, and adjusting the distance from the needle head to the glass sheet;

b3) and (3) setting electrostatic spinning parameters, and performing electrostatic spinning to obtain the melt electrospinning composite fiber scaffold for bone repair and infection resistance.

Preferably, the inner diameter of the needle in the step b2) is 0.4 mm-0.5 mm.

Preferably, the distance between the adjusting needle and the glass sheet in the step b2) is 15 mm-20 mm.

Preferably, the speed of the collection platform of the electrostatic spinning in the step b3) is 6 mm/s-80 mm/s, the spinning air pressure is 1.2 kPa-5 kPa, and the voltage is 2.2 kV-2.7 kV.

Preferably, the temperature of the melt electrospinning in the step b) is 80-110 ℃, and the time is 2-10 h.

The invention provides a preparation method of a melt electrospinning composite fiber scaffold for bone repair and infection resistance, which comprises the following steps: a) uniformly mixing polycaprolactone, polyethylene glycol and macrolide antibiotics in an organic solvent, evaporating the organic solvent, and then drying in vacuum to obtain a solid mixed material; the mass ratio of the polycaprolactone to the polyethylene glycol to the macrolide antibiotics is 90: (0.1-20): (0.1 to 20); b) and (b) carrying out melt electrospinning on the solid mixed material obtained in the step a) to obtain the melt electrospinning composite fiber scaffold for bone repair and infection resistance. Compared with the prior art, the preparation method provided by the invention takes polycaprolactone, polyethylene glycol and macrolide antibiotics as melt electrospinning materials to obtain the novel bone repair anti-infection melt electrospinning composite fiber scaffold which has an ordered structure, excellent biocompatibility, complete degradation, antibacterial property, capability of promoting bone cell proliferation and meeting the physical property requirement of bone repair; the degradable fiber scaffold prepared by melt electrostatic spinning solves the problem that the bone repair implant needs to be taken out for the second time in an operation; the problem of bone implant infection is solved by loading macrolide antibiotics in the fibers, and long-acting antibiosis can be realized because the medicament is dispersed in the fibers; the hydrophobicity of polycaprolactone can be improved by adding hydrophilic substances into the fibers, and the hydrophilicity of the stent can be adjusted by adjusting the content of polyethylene glycol, so that the adhesion and proliferation of cells can be increased, and the bone repair is accelerated; meanwhile, compared with other bone repair implants, the bone repair implant provided by the invention can effectively prevent bone infection, avoid the removal of the implant after operation, promote the proliferation of bone cells and accelerate the bone repair; the macrolide antibiotics are adopted as raw materials, trace amount of the macrolide antibiotics is adopted, the macrolide antibiotics has high bacteriostasis rate on gram-negative bacteria represented by escherichia coli and gram-positive bacteria represented by golden yellow staphylococcus (1.67-5 mu g/ml), and the macrolide antibiotics is safe and harmless to human bodies; the micron-sized material is quickly obtained by using a melt electrostatic spinning technology, and the prepared micron-sized fiber scaffold has the advantages of broad spectrum, high-efficiency antibacterial activity, no toxicity, large specific surface area, high porosity, high safety, high cell proliferation rate, structure controllability and the like.

In addition, the melt electrospinning composite fiber scaffold obtained by the preparation method provided by the invention is used as a new generation of bone repair anti-infection implant, and has a wide application prospect in the field of bone repair for treating suppurative arthritis, osteomyelitis and the like caused by the implant.

Drawings

FIG. 1 is a flow chart of a method of preparation provided by an embodiment of the present invention;

fig. 2 is a scanning electron microscope image of the melt electrospun composite fiber scaffold for bone repair anti-infection obtained by the preparation method provided in embodiment 1 of the invention;

fig. 3 is a morphology diagram of the melt electrospun composite fiber scaffold for bone repair anti-infection obtained by the preparation method provided in embodiment 1 of the invention;

FIG. 4 is a graph showing the inhibition effect of the melt electrospun composite fiber scaffold obtained by the preparation methods provided in examples 1 to 3 and comparative examples 1 to 2 of the present invention on Escherichia coli;

FIG. 5 is a graph showing the effect of the melt electrospun composite fiber scaffold obtained by the preparation methods provided in examples 1 to 3 and comparative examples 1 to 2 of the present invention on the inhibition of Staphylococcus aureus;

FIG. 6 is a measurement result of hydrophilic and hydrophobic contact angles of the melt electrospun composite fiber scaffold obtained by the preparation methods provided by examples 1-3 and comparative examples 1-2 of the invention;

FIG. 7 is a diagram showing cell proliferation effects of the melt electrospun composite fibrous scaffolds obtained by the preparation methods provided in examples 1-3 and comparative examples 1-2 of the present invention.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. 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.

The invention firstly mixes polycaprolactone, polyethylene glycol and macrolide antibiotics in organic solvent evenly, evaporates the organic solvent and dries in vacuum to obtain solid mixed material. The sources of the Polycaprolactone (PCL) and polyethylene glycol (PEG) are not particularly limited in the present invention, and commercially available products or self-prepared products known to those skilled in the art can be used.

In the invention, the Polycaprolactone (PCL) is also called poly epsilon-caprolactone, is a high molecular organic polymer formed by ring opening polymerization of epsilon-caprolactone monomers under the catalysis of a metal anion complex catalyst, and different molecular weights can be obtained by controlling polymerization conditions; the appearance of the material is white solid powder, is nontoxic, is made of hydrophobic materials, is insoluble in water, and is easily soluble in various polar organic solvents; the PCL has good biocompatibility, good organic polymer compatibility and good biodegradability, and is widely applied to controllable drug-releasing carriers, cells and tissue culture base frames. In the present invention, the molecular weight of the polycaprolactone is preferably 5 to 8 ten thousand, more preferably 5 ten thousand.

In the present invention, the polyethylene glycol (PEG), also known as poly (ethylene oxide) (PEO) or Polyoxyethylene (POE), refers to oligomers or polymers of ethylene oxide; polyethylene glycol is soluble in water, methanol, benzene, and dichloromethane, and insoluble in diethyl ether and n-hexane; polyethylene glycol is an artificially synthesized polymer, has good hydrophilicity, dissolubility, nontoxicity, immunogenicity, no rejection reactivity, biocompatibility and anti-thrombosis capability, and is approved by the U.S. food and drug administration to be used in human bodies; hydroxyl groups at two ends of a molecular chain of the functional modifier have good reactivity and can react with a plurality of compounds to achieve the purpose of modification or functionalization; because a large number of ethoxy groups exist in polyethylene glycol molecules, the polyethylene glycol can form hydrogen bonds with water, and has good hydrophilicity, the polyethylene glycol can be combined with a material which is difficult to be hydrophobic by utilizing the good hydrophilicity, and the hydrophilicity of the hydrophobic material can be effectively increased. In the invention, the molecular weight of the polyethylene glycol is preferably 4000-8000, and more preferably 6000. In the invention, the PEG contained in the melt electrospinning composite fiber scaffold for bone repair and infection resistance can regulate the release of drugs and promote the proliferation of bone cells by changing the hydrophilicity of the surface of the scaffold, thereby realizing the infection resistance, promoting the proliferation of bone cells and accelerating the bone repair.

In the invention, the Macrolide Antibiotics (MA) are a generic name of antibacterial drugs with 12-16 carbon lactone rings in molecular structures, inhibit the synthesis of bacterial proteins by blocking the activity of peptidyl transferase in 50s ribosome, and belong to rapid bacteriostat; mainly used for treating infection of aerobic gram positive coccus and negative coccus, certain anaerobe, legionella, mycoplasma, chlamydia and the like. In the present invention, the macrolide antibiotic is preferably selected from one or more of roxithromycin, clarithromycin, clindamycin and azithromycin, more preferably roxithromycin, clarithromycin, clindamycin or azithromycin; wherein:

roxithromycin, which is also called Claramid and Rulid in English, is a new generation macrolide antibiotic and a semi-synthetic 14-membered macrolide drug; the action mechanism is the same as that of erythromycin, is mainly combined with bacterial 50s ribosome subunits, and inhibits the synthesis of bacterial protein by blocking transpeptidation and mRNA shift, thereby playing an antibacterial role; it is characterized in that the medicament can rapidly enter macrophages, lung cells, alveoli and polymorphonuclear leukocytes; the antibacterial spectrum of the roxithromycin is similar to that of erythromycin, the in vitro antibacterial effect of the roxithromycin is similar to that of the erythromycin, and the in vivo antibacterial effect of the roxithromycin is 1-4 times stronger than that of the erythromycin; the roxithromycin has better antibacterial action on most gram-positive bacteria, and has similar antibacterial activity to common pathogenic bacteria infected by respiratory tract and skin, such as staphylococcus aureus, staphylococcus epidermidis, pneumococcus, streptococcus pyogenes, bacillus influenzae, mycoplasma pneumoniae, legionella and the like as the erythromycin; the roxithromycin absorption rate is not influenced by age, generally, the adverse reaction is less, the toxicity is low, and the effective rate of treating infection is high; according to the report at home and abroad, the effective rate of the roxithromycin for treating the upper respiratory tract infection is 96%, the effective rate of the roxithromycin for treating the acute attack of the chronic bronchitis in the lower respiratory tract infection is 94%, the effective rate of the pneumonia is 95%, the effective rates for acute pharyngolaryngitis, tonsillitis, sinusitis and otitis media are 97%, 96% and 96%, respectively, the effective rates for acute attack of the bronchitis, chronic bronchitis and pneumonia are 97%, 94% and 95%, respectively, and the incidence rate of adverse reactions is 4%.

Clarithromycin a 14-membered ring macrolide antibiotic; the antibacterial spectrum is the same as that of erythromycin, roxithromycin and the like, but the antibacterial effect on gram-positive bacteria such as streptococcus, pneumococcus and staphylococcus is slightly excellent, and the antibacterial peptide also has certain antibacterial activity on erythromycin drug-resistant strains generated by induction; the clarithromycin has an inhibiting effect on gram-positive bacteria such as staphylococcus aureus, streptococcus, pneumococcus and the like, and also has an inhibiting effect on part of gram-negative bacteria such as haemophilus influenzae, bordetella pertussis, diplococcus gonorrhoeae, legionella pneumophila and part of anaerobic bacteria such as bacteroides fragilis, streptococcus digestans, propionibacterium acnes and the like; in addition, the compound also has an inhibitory effect on mycoplasma; clarithromycin is characterized by similar antibacterial activity to erythromycin in vitro, but stronger antibacterial activity to some bacteria such as staphylococcus aureus, streptococcus, haemophilus influenzae and the like in vivo than erythromycin.

Clindamycin is a chemical with white crystal powder appearance and has a chemical formula of C₁₈H₃₃ClN₂O₅S, molecular weight is 424.98300, and melting point is 141-143 ℃; clindamycin is an antibiotic drug, is mainly used for abdominal cavity and gynecological infection caused by anaerobic bacteria clinically, and is the first choice for treating staphylococcus aureus osteomyelitis.

Azithromycin (Azithromycin) is a semi-synthetic pentadeca-membered macrolide antibiotic, has an expanded antibacterial spectrum compared with erythromycin, and obviously enhances the activity of gram-negative cocci, bacilli and anaerobic bacteria besides retaining the action on gram-positive bacteria; in addition, the product has good activity on other pathogenic microorganisms such as mycoplasma pneumoniae, chlamydia trachomatis, treponema pallidum and the like. Can be used for treating infection of respiratory tract, urinary tract, skin and soft tissue.

The source of the macrolide antibiotic in the present invention is not particularly limited, and commercially available products of the aforementioned roxithromycin, clarithromycin, clindamycin and azithromycin known to those skilled in the art may be used.

In the invention, the mass ratio of the polycaprolactone to the polyethylene glycol to the macrolide antibiotic is 90: (0.1-20): (0.1 to 20), preferably 90: (1-20): (1-10), more preferably 90: (5-17): (5-6). In a preferred embodiment of the present invention, the mass ratio of the polycaprolactone to the polyethylene glycol to the macrolide antibiotic is 90: 5: 5; the weight percentage of the components are respectively as follows: 90%, 5% and 5%; in another preferred embodiment of the present invention, the mass percentages of the polycaprolactone, the polyethylene glycol and the macrolide antibiotic are as follows: 85%, 10% and 5%, and the mass ratio of the polycaprolactone, the polyethylene glycol and the macrolide antibiotic is calculated to be 90: 10.6: 5.3; in another preferred embodiment of the present invention, the mass percentages of the polycaprolactone, the polyethylene glycol and the macrolide antibiotic are as follows: 80%, 15% and 5%, and the mass ratio of the polycaprolactone, the polyethylene glycol and the macrolide antibiotic is calculated to be 90: 16.875: 5.625.

in the present invention, the organic solvent is preferably tetrahydrofuran; the source of the tetrahydrofuran is not particularly limited in the present invention, and commercially available products known to those skilled in the art may be used. In the invention, the dosage ratio of the organic solvent to the polycaprolactone, the polyethylene glycol and the macrolide antibiotic which are raw materials is preferably (30 mL-50 mL): 5g, more preferably 30 mL: 5g of the total weight. In the present invention, the organic solvent is completely evaporated before the subsequent printing of the stent, which only plays a role in mixing; the present invention is not particularly limited in this regard.

In the present invention, the mixing is preferably performed by stirring; the stirring mode is not particularly limited, and the technical scheme of manual stirring or mechanical stirring which is well known by the technical personnel in the field can be adopted; in a preferred embodiment of the invention, the stirring is performed by mechanical stirring, in particular by a magnetic stirrer. In the present invention, the stirring temperature is preferably 45 ℃ to 55 ℃, and more preferably 50 ℃; the rotating speed of the stirring is preferably 350 r/min-450 r/min, and more preferably 400 r/min; the stirring time is preferably 22 to 26 hours, and more preferably 24 hours.

In the present invention, the manner of evaporating the organic solvent is preferably specifically:

pouring the uniformly mixed materials into a glass vessel, standing in a fume hood for 2-6 h to evaporate the organic solvent;

more preferably:

the uniformly mixed material was poured into a glass dish and placed in a fume hood and left for 4h to evaporate the organic solvent.

In the present invention, the temperature of the vacuum drying is preferably 75 to 85 ℃, more preferably 80 ℃; the time for vacuum drying is preferably 2 to 5 hours, and more preferably 3 hours.

After the solid mixed material is obtained, the melt electrospinning composite fiber scaffold for bone repair and infection resistance is obtained by carrying out melt electrospinning on the obtained solid mixed material. In the invention, the solid mixed material does not contain an organic solvent, so that the subsequent spinning process is different from solution electrostatic spinning; the melt electrostatic spinning process does not involve organic solvent, so that the prepared fiber has higher safety than solution electrostatic spinning.

In the invention, the melt electrostatic spinning is a manufacturing process for preparing micro-nano fibers, and the heated melt is subjected to jet spinning in a strong electric field; according to the invention, the solid mixed material is firstly heated and melted in the syringe, then the material at the needle head changes from a spherical shape to a conical shape (namely a Taylor cone) under the action of an electric field, and when the electric field force is larger than the surface tension force of the material, the material extends from the conical tip to obtain fiber filaments which are then deposited on the collection platform. In the present invention, the melt electrospinning process is preferably embodied as follows:

Firstly, shearing the solid mixed material and putting the solid mixed material into a charging barrel; cleaning and drying the needle head; connecting the positive electrode of a high-voltage direct-current power supply to a glass plate of the collection platform, and connecting the ground to the needle head; the grounded needle is then attached to the cartridge, which is then placed into the heating chamber. In the present invention, the inner diameter of the needle is preferably 0.4mm to 0.5mm, more preferably 0.45 mm.

After the preparation process is finished, the melt spinning machine is enabled, and the collection platform loaded with the glass sheets is positioned below the needle head; and controlling a motor in the z-axis direction to adjust the distance from the needle head to the glass sheet. In the present invention, the process of enabling the melt spinning machine is preferably controlled by opening the melt spinning control software on a computer.

In the present invention, the distance from the adjustment needle to the glass sheet is specifically preferably 15mm to 20mm, and more preferably 18 mm.

And then, setting electrostatic spinning parameters, and carrying out electrostatic spinning to obtain the melt electrospinning composite fiber scaffold for bone repair and infection resistance. In the invention, the electrostatic spinning parameters comprise the speed of a collecting platform, the spinning air pressure and the voltage. In the invention, the speed of the collection platform of the electrostatic spinning is preferably 6-80 mm/s, more preferably 40-45 mm/s; particularly, the adjustment is carried out by setting spinning motion parameters. In the present invention, the spinning air pressure for the electrospinning is preferably 1.2 to 5kPa, more preferably 3 to 4 kPa; specifically, the air pressure valve is opened, and the knob of the pressure regulating valve is rotated to perform regulation. In the present invention, the voltage of the electrostatic spinning is preferably 2.2kV to 2.7kV, and more preferably 2.5 kV; specifically, the high-voltage direct-current power supply is turned on, and the voltage regulating knob is rotated to regulate.

In the present invention, the temperature of the melt electrospinning is preferably 80 to 110 ℃; the time for the melt electrospinning is preferably 2 to 10 hours, more preferably 6 hours.

At the end of spinning, the present invention preferably further comprises:

firstly, adjusting the voltage of a high-voltage direct-current power supply to zero, then closing the high-voltage direct-current power supply, then adjusting a pressure regulating valve to zero, then closing a gas pressure valve, and closing the platform to enable; and taking down the glass sheet with the multilayer micron fiber scaffold to obtain the melt electrospinning composite fiber scaffold for bone repair and infection resistance.

The melt electrospinning is adopted, and the prepared micro-nano fiber has certain excellent performances, such as large specific surface area, high porosity, strong stability and slow release effect of loaded active ingredients. In addition, ordered fibers can be obtained in the melt electrostatic spinning process provided by the invention by controlling the distance between the needle head and the collecting platform, so that the printing of fiber supports with different structures can be realized; therefore, the nanofiber can be applied to many fields, such as wound dressing, drug release, vascular repair, bone repair and the like, but the application of the nanofiber in resisting infection and promoting the proliferation of bone cells in bone repair is relatively new, so that the microfiber prepared by melt electrospinning has great research value in the field.

To further illustrate the present invention, the following examples are provided for illustration. The reagents used in the following examples of the invention are all commercially available or self-made; wherein the molecular weight of the polycaprolactone is 5 ten thousand, and the molecular weight of the polyethylene glycol is 6000.

Example 1

(1) Placing 90% by mass of PCL, 5% by mass of PEG and 5% by mass of roxithromycin into an erlenmeyer flask, wherein the total mass of the raw materials is 5 g; and then adding 30mL of organic solvent tetrahydrofuran, stirring for 24 hours in a magnetic stirrer with the temperature of 50 ℃ and the rotating speed of 400r/min, pouring the uniformly mixed material into a glass dish, standing in a fume hood for 4 hours to evaporate the organic solvent tetrahydrofuran, then putting the glass dish filled with the material into a vacuum drying oven with the temperature of 80 ℃ for drying for 3 hours, taking out the material after drying, standing and cooling to obtain the solid mixed material of PCL, PEG and roxithromycin.

(2) Carrying out melt electrospinning on the solid mixed material obtained in the step (1), wherein the specific process is as follows:

shearing the solid mixed material by using scissors and putting the sheared solid mixed material into a charging barrel; taking a needle head with the inner diameter of 0.45mm, cleaning the needle head with clear water, and drying the needle head; connecting the positive electrode of a high-voltage direct-current power supply to a glass plate of the collection platform, and connecting the ground to the needle head; then the grounded needle head is arranged on the material barrel, and the material barrel is placed in the heating cavity; opening control software of melt spinning on a computer, enabling a melt spinning machine, and positioning a collection platform loaded with glass sheets below a needle head; controlling a motor in the z-axis direction, and moving the needle head to 18mm of the glass sheet foil; setting spinning motion parameters, and adjusting the speed of a spinning collecting platform to 43 mm/s; opening the air pressure valve, rotating a knob of the pressure regulating valve and regulating the air pressure to 3.1 kPa; turning on a high-voltage direct-current power supply, and rotating a voltage regulating knob to regulate the voltage to 2.5 kV; electrostatic spinning for 6 h; when spinning is finished, firstly, adjusting the voltage of a high-voltage direct-current power supply to zero, then, closing the high-voltage direct-current power supply, then, adjusting a pressure regulating valve to zero, then, closing a gas pressure valve, and closing the platform to enable; and taking down the glass sheet with the multilayer micron fiber scaffold to obtain the melt electrospinning composite fiber scaffold for bone repair and infection resistance.

The melt electrospun composite fiber scaffold obtained in example 1 was analyzed by a scanning electron microscope, and a scanning electron microscope image thereof is shown in fig. 2. Fig. 2 is a scanning electron microscope image of the melt electrospun composite fiber scaffold for bone repair anti-infection obtained by the preparation method provided in embodiment 1 of the invention; as can be seen from fig. 2, the obtained nanofibers had uniform size and high porosity. In addition, a morphology of the melt electrospinning composite fiber scaffold for bone repair anti-infection obtained by the preparation method provided in embodiment 1 of the present invention is shown in fig. 3.

Comparative example 1

Pure PCL fiber scaffolds were prepared as controls using the same procedure as in example 1.

Comparative example 2

The same procedure as in example 1 was used to prepare 95% by mass of PCL, 0% by mass of PEG, and 5% by mass of roxithromycin fibrous scaffold.

Example 2

The same procedure as in example 1 was used to prepare a fiber scaffold comprising PCL 85% by mass, PEG 10% by mass, and roxithromycin 5% by mass.

Example 3

The same procedure as in example 1 was used to prepare 80% by mass of PCL, 15% by mass of PEG, and 5% by mass of roxithromycin fibrous scaffold.

The melt electrospun composite fiber scaffolds prepared in examples 1 to 3 and comparative examples 1 to 2 were used as objects, and the bacteriostatic effect and the cell proliferation promoting effect of the scaffolds were measured.

The bacteriostatic experiment steps are as follows: the bacteriostatic activity of the nanofiber membrane on escherichia coli (gram-negative bacteria) and staphylococcus aureus (gram-positive bacteria) was evaluated by using the zone inhibition method. The frozen bacteria were removed from the-80 ℃ freezer and incubated overnight in solid medium to recover the bacterial species. Then, an amount of bacteria was extracted from the medium with a inoculating loop, and the density was diluted to 10^4CFU/mL with physiological saline. 50 mu L of diluent is taken to be coated on a solid culture medium, then a sterilized 6mm circular fiber bracket is placed in the center of a solid culture dish, and the diameter of a bacteriostatic circle is measured after 24 hours of culture so as to evaluate the bacteriostatic effect of the substance.

The bacteriostatic activity of the drug-loaded fibrous scaffolds against escherichia coli (gram-negative bacteria) and staphylococcus aureus (gram-positive bacteria) was evaluated using nephelometry. The frozen bacteria were removed from the-80 ℃ freezer and incubated overnight in solid medium to recover the bacterial species. Then, an amount of bacteria was extracted from the medium with a inoculating loop, and the density was diluted to 10^5CFU/mL with physiological saline. Then 100. mu.L of the diluted bacterial solution was added to the liquid medium to obtain a bacterial solution with a concentration of 10^4 CFU/mL. Then, the experimental group added sterilized fiber scaffolds containing active ingredients with different concentrations to the bacterial solution, while the control group added no fiber scaffold, and both groups were incubated in a shaker at 37 ℃ for 12h with an oscillation frequency of 200 r/min. Then, the above solutions cultured for 12 hours were taken, shaken up, 100. mu.L of each solution was pipetted into a 96-well plate with 3 duplicate wells, and the bacterial concentration was measured at a wavelength of 620nm with a microplate reader, and the subsequent dilution factor was calculated. Then, the bacterial suspension was serially diluted 10-fold in saline, and 50. mu.L of the diluent was applied to the solid medium. After incubation overnight in a 37 ℃ incubator, the bacteria on each solid medium were counted. The experiment was repeated three times and the average of CFU was recorded.

Qualitative measurement of cell viability of melt electrospun composite fibrous scaffolds for bone repair anti-infection using the MTT method, to compare the effects of different hydrophilic scaffolds on cell proliferation. Firstly, recovering and passaging MG63 cells, verifying the cell activity through three passages, and starting an MTT experiment when the activity is good. First, the fiber scaffolds were sterilized under UV for 30min, then the scaffolds were placed in 6-well plates, then the cells that had been passaged three times were seeded on the scaffolds and on blank glass slides at 10^4cells/mL (blank control), after 2h of fixation 3mL of complete medium were added to 24-well plates and placed in an incubator for 24h and 48 h. Six-well plates were removed according to time, the supernatant was discarded, 300. mu.L of MTT at a concentration of 5mg/mL and 2.7mL of complete medium were added to the six-well plates, and placed in an incubator for 4 hours. Then, removing supernatant, adding 1mL of DMSO (dimethyl sulfoxide), developing and dissolving, shaking for 10min, respectively sucking 100 mu L of solutions into a 96-well plate by using a pipette gun, making 3 multiple wells for each solution, measuring OD (optical density) values by using an enzyme-labeling instrument at a wavelength of 570nm, and comprehensively analyzing the influence of the hydrophilicity and hydrophobicity of the scaffold on the cell activity by combining a contact angle of the scaffold shown in FIG. 5.

The detection results are shown in FIGS. 4-7:

fig. 4 is a diagram showing the inhibition effect of the melt electrospinning composite fiber scaffold obtained by the preparation methods provided in examples 1 to 3 and comparative examples 1 to 2 of the present invention on escherichia coli, wherein a, b, c, d, and e in fig. 4 correspond to comparative examples 1 and 2, and examples 1, 2, and 3, respectively, and it can be seen from the experimental results that the antibacterial effect is gradually enhanced along with the increase of PEG content in the scaffold and along with the better antibacterial effect of roxithromycin on escherichia coli.

Fig. 5 is a diagram showing the inhibitory effect of the melt electrospun composite fiber scaffold obtained by the preparation methods provided in examples 1 to 3 and comparative examples 1 to 2 of the present invention on staphylococcus aureus, where a, b, c, d, and e in fig. 5 correspond to comparative examples 1 and 2, and examples 1, 2, and 3, respectively, and it can be seen from the experimental results that the antibacterial effect is gradually enhanced with the increase of PEG content in the scaffold and with the significant antibacterial effect of roxithromycin on staphylococcus aureus.

Fig. 6 is a measurement result of hydrophilic and hydrophobic contact angles of the melt electrospun composite fiber scaffolds obtained by the preparation methods provided in examples 1 to 3 and comparative examples 1 to 2 of the present invention, wherein a, b, c, d, and e in fig. 6 correspond to comparative examples 1 and 2 and examples 1, 2, and 3, respectively, and it can be seen from the experimental result that the hydrophilicity of the scaffold is better and better with the increase of PEG content.

Fig. 7 is a diagram showing the cell proliferation effect of the melt electrospinning composite fiber scaffold obtained by the preparation methods provided in examples 1 to 3 and comparative examples 1 to 2 of the present invention, and a, b, c, d, and e in fig. 7 correspond to comparative examples 1, 2, and 3, respectively, and it can be seen by combining the results of the contact angle experiment that the cell proliferation increases first and then decreases with the increase of hydrophilicity in the scaffold, but the cell proliferation is higher than that of the blank group and the fiber group prepared in comparative example 1, which indicates that the composite scaffold has good biocompatibility and cell compatibility, and is a potential composite scaffold applied to bone repair anti-infection.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A preparation method of a melt electrospinning composite fiber scaffold for bone repair and infection resistance comprises the following steps:

2. The process according to claim 1, wherein the macrolide antibiotic in step a) is one or more selected from the group consisting of roxithromycin, clarithromycin, clindamycin and azithromycin.

3. The method according to claim 1, wherein the organic solvent in step a) is tetrahydrofuran.

4. The method according to claim 1, wherein the mixing in step a) is performed by stirring; the stirring temperature is 45-55 ℃, the rotating speed is 350-450 r/min, and the time is 22-26 h.

5. The preparation method according to claim 1, wherein the temperature of the vacuum drying in the step a) is 75-85 ℃ and the time is 2-5 h.

6. The method according to claim 1, wherein the step b) of melt electrospinning comprises:

7. The method of claim 6, wherein the needle of step b2) has an inner diameter of 0.4mm to 0.5 mm.

8. The method of claim 6, wherein the distance from the adjusting needle to the glass sheet in step b2) is in particular 15mm to 20 mm.

9. The method according to claim 6, wherein the electrostatic spinning in step b3) has a collection platform speed of 6mm/s to 80mm/s, a spinning air pressure of 1.2kPa to 5kPa, and a voltage of 2.2kV to 2.7 kV.

10. The method according to claim 1, wherein the melt electrospinning in step b) is carried out at a temperature of 80 ℃ to 110 ℃ for 2h to 10 h.