CN114432503A - Drug-loaded bone repair material and preparation method and application thereof - Google Patents
Drug-loaded bone repair material and preparation method and application thereof Download PDFInfo
- Publication number
- CN114432503A CN114432503A CN202210376608.9A CN202210376608A CN114432503A CN 114432503 A CN114432503 A CN 114432503A CN 202210376608 A CN202210376608 A CN 202210376608A CN 114432503 A CN114432503 A CN 114432503A
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- Prior art keywords
- bone
- solution
- calcium carbonate
- containing calcium
- magnesium
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- A61L2300/216—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials with other specific functional groups, e.g. aldehydes, ketones, phenols, quaternary phosphonium groups
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Abstract
The invention discloses a drug-loaded bone repair material and a preparation method and application thereof, wherein the material comprises bone-promoting factor 1, hydroxybutyl chitosan and polydopamine-coated magnesium-containing calcium carbonate microspheres loaded with bone-promoting factor 2; polydopamine-coated magnesium-containing calcium carbonate microspheres loaded with bone factor 2 and hydroxybutyl chitosan are dispersed in a bone factor 1 solution to obtain the drug-loaded bone repair material; the drug-loaded bone repair material can realize anti-inflammatory drugs and promote accurate sequence delivery of bone protein after being implanted into a bone defect part, thereby relieving early inflammatory reaction after bone injury, promoting formation of new bone tissues and improving bone regeneration efficiency.
Description
Technical Field
The application relates to the technical field of biomedicine, in particular to a drug-loaded bone repair material and a preparation method and application thereof.
Background
The oral craniomaxillofacial bone defect has high morbidity, various causes, complex forms and high bone regeneration difficulty, and seriously influences the life quality and physical and psychological health of patients. The bone injury repair process is a highly complex and dynamically variable process, and currently, bone tissue engineering usually uses different types of bone repair materials to load and deliver various bioactive factors to act on different stages of bone injury repair so as to regulate and control the bone regeneration process. For example, the bone repair material sequence can effectively regulate and control the inflammatory reaction in the early stage of bone injury by delivering the anti-inflammatory factor and the osteogenesis induction factor, and is favorable for converting inflammatory tissues into repair tissues, forming new vessels and regenerating bone tissues. However, existing bone repair materials are still deficient in the strategy of delivering bioactive factors: 1) the different degradation characteristics of different biological materials are mostly utilized to deliver various bioactive factors, so that the method has uncontrollable property and is still difficult to accurately regulate and control the release efficiency of different bioactive factors; 2) the existing research shows that the late inflammatory reaction period in the bone injury repair process is the optimal treatment window for delivering the osteogenesis inducing factor, and the most common sustained-release delivery strategy of the existing bone repair material is difficult to accurately regulate and control the release time and the action concentration of the osteogenesis inducing factor; 3) the efficacy of treatment with biologically active factors, such as bone morphogenic protein-2 (BMP-2), is highly dependent on the method of delivery to the target site, and rapid hydration or lysis of the carrier after contact with the biological tissue may result in elution of cytokines, which are often administered at high concentrations to compensate for this loss, further increasing the risk of bone tissue overgrowth, ectopic ossification, and other complications.
Disclosure of Invention
In order to solve the problems, the invention designs a novel dual-stimulation responsive drug-loaded bone repair material which can efficiently deliver anti-inflammatory drugs (such as aspirin) at the early stage of bone defect implantation to regulate and control local tissue inflammatory reaction, and meanwhile, the bone repair material can controllably deliver bone growth factors (such as BMP-2) under near infrared light stimulation so as to regulate and control the inflammatory reaction and bone regeneration at the defect position in a time sequence.
The invention provides a drug-loaded bone repair material, which comprises bone-promoting factor 1, hydroxybutyl chitosan and polydopamine-coated magnesium-containing calcium carbonate microspheres loaded with bone-promoting factor 2; polydopamine-coated magnesium-containing calcium carbonate microspheres loaded with bone factor 2 and hydroxybutyl chitosan are dispersed in a bone factor 1 solution to obtain the drug-loaded bone repair material; after the drug-loaded bone repair material is implanted into a body, the hydroxybutyl chitosan is gelatinized and releases osteogenesis promoting factors 1; the release of the bone factor 2 from the polydopamine-coated magnesium-containing calcium carbonate microspheres loaded with the bone factor 2 can be controlled through near infrared light irradiation.
Specifically, (1) the bone factor 1 is a medicine or nutrient capable of promoting the bone repair process in the early bone repair stage, and the bone factor 2 is a medicine or nutrient capable of promoting the bone repair process in the later bone repair stage;
(2) dispersing magnesium-containing calcium carbonate microspheres in a dopamine salt solution to obtain polydopamine-coated magnesium-containing calcium carbonate microspheres;
(3) dispersing polydopamine-coated magnesium-containing calcium carbonate microspheres in a solution for promoting bone factor 2 to obtain the polydopamine-loaded magnesium-containing calcium carbonate microspheres for promoting bone factor 2;
(4) the near infrared light stimulation intensity is 0.25W/cm2；
(5) Will contain
To a solution containing casein
And
obtaining magnesium-containing calcium carbonate microspheres in the solution;
(6) adding bone factor 1 promoting solution into polydopamine-coated magnesium-containing calcium carbonate microspheres loaded with bone factor 2, repeatedly blowing and beating to uniformly mix, and adding hydroxybutyl chitosan to fully dissolve to obtain the drug-loaded bone repair material.
Further, (1) the mass ratio of the dopamine salt solution to the magnesium-containing calcium carbonate microspheres is 2: 5;
(2) the dopamine salt solution is a dopamine-Tris hydrochloride solution;
(3) dispersing the magnesium-containing calcium carbonate microspheres in the dopamine salt solution by adopting ultrasonic oscillation and/or magnetic stirring, and then centrifuging to remove unreacted dopamine molecules to obtain polydopamine-coated magnesium-containing calcium carbonate microspheres;
(4) the osteogenesis promoting factor 1 is an anti-inflammatory drug, and the osteogenesis promoting factor 2 is osteogenesis promoting protein;
(5) the polydopamine-coated magnesium-containing calcium carbonate microspheres are soaked in alcohol for disinfection and washed by deionized water, and then dispersed in a solution for promoting bone factor 2.
Specifically, the bone promoting factor 1 is aspirin, and the bone promoting factor 2 is BMP-2.
More specifically, (1) the preparation method of the magnesium-containing calcium carbonate microspheres comprises the following steps:
will be provided with
Adding into 2mg/mL casein solution to obtain solution containing 50 mM
Solution A of (1); and was prepared to contain 47.5 mM
And 2.5 mM
Solution B of (1); quickly pouring the solution A into the solution B, reacting at room temperature for 20 min, then obtaining calcium carbonate microsphere sediment with 5% of magnesium content by high-speed centrifugation, washing the sediment by deionized water to obtain magnesium-containing calcium carbonate microspheres；
(2) Adopting 4 mM HCl solution to prepare BMP-2 solution with the concentration of 10 mug/mL; and (3) fully mixing the BMP-2 solution with polydopamine-coated magnesium-containing calcium carbonate microspheres which are sterilized by alcohol and washed by deionized water according to the mass ratio of 10000:1-1000:1, and centrifuging after oscillation reaction to obtain polydopamine-coated magnesium-containing calcium carbonate microsphere particles loaded with bone growth promoting factors 2 (BMP-2).
The invention also provides a preparation method of the drug-loaded bone repair material, which comprises the following steps:
(1) dispersing the magnesium-containing calcium carbonate microspheres in a dopamine salt solution to obtain polydopamine-coated magnesium-containing calcium carbonate microspheres;
(2) dispersing polydopamine-coated magnesium-containing calcium carbonate microspheres in a bone factor 2 promoting solution to obtain polydopamine-loaded bone factor 2 promoting magnesium-containing calcium carbonate microspheres;
(3) adding bone factor 1 promoting solution into polydopamine-coated magnesium-containing calcium carbonate microspheres loaded with bone factor 2, repeatedly blowing and beating to uniformly mix, and adding hydroxybutyl chitosan to fully dissolve to obtain the drug-loaded bone repair material.
Specifically, (1) the bone factor 1 is a medicine or nutrient capable of promoting the bone repair process in the early bone repair stage, and the bone factor 2 is a medicine or nutrient capable of promoting the bone repair process in the later bone repair stage;
(2) will contain
To a solution containing casein
And
obtaining magnesium-containing calcium carbonate microspheres in the solution;
(3) the mass ratio of the dopamine salt solution to the magnesium-containing calcium carbonate microspheres is 2: 5;
(4) the dopamine salt solution is a dopamine-Tris hydrochloride solution;
(5) dispersing the magnesium-containing calcium carbonate microspheres in the dopamine salt solution by adopting ultrasonic oscillation and/or magnetic stirring, and then centrifuging to remove unreacted dopamine molecules to obtain polydopamine-coated magnesium-containing calcium carbonate microspheres;
(6) the polydopamine-coated magnesium-containing calcium carbonate microspheres are soaked in alcohol for disinfection and washed by deionized water, and then dispersed in a solution for promoting bone factor 2.
More specifically, (1) the osteogenesis promoting factor 1 is aspirin and the osteogenesis promoting factor 2 is BMP-2;
(2) when the bone promoting factor 2 is BMP-2, the mass ratio of the BMP-2 to the polydopamine coated magnesium-containing calcium carbonate microspheres is 10000:1-1000: 1;
(3) the preparation method of the magnesium-containing calcium carbonate microspheres comprises the following steps:
will be provided with
Adding into 2mg/mL casein solution to obtain solution containing 50 mM
Solution A of (1); and was prepared to contain 47.5 mM
And 2.5 mM
Solution B of (1); quickly pouring the solution A into the solution B, reacting at room temperature for 20 min, then obtaining calcium carbonate microsphere sediment with the magnesium content of 5% through high-speed centrifugation, and washing the sediment with deionized water to obtain magnesium-containing calcium carbonate microspheres;
(4) when the bone promoting factor 2 is BMP-2, preparing a BMP-2 solution with the concentration of 10 mu g/mL by adopting a 4 mM HCl solution; and (3) fully mixing the BMP-2 solution with polydopamine-coated magnesium-containing calcium carbonate microspheres which are sterilized by alcohol and washed by deionized water according to the mass ratio of 10000:1-1000:1, and centrifuging after oscillation reaction to obtain polydopamine-coated magnesium-containing calcium carbonate microsphere particles loaded with bone growth promoting factors 2 (BMP-2).
The invention also provides application of the medicine-carrying bone repair material or the preparation method in the fields of bone defect repair and bone regeneration.
Specifically, the application is to inject the drug-loaded bone repair material or the bone repair material prepared by the preparation method into the vicinity of a part to be repaired.
The beneficial effects of the invention include:
(1) according to the invention, hydroxybutyl chitosan (HBC) temperature-sensitive hydrogel is used as a carrier of the bone repair material, and the hydroxybutyl chitosan can realize sol-gel conversion only through temperature conversion, does not depend on conversion modes such as ultraviolet crosslinking and chemical crosslinking, and has good biocompatibility and proper degradation characteristics; meanwhile, compared with other temperature-sensitive hydrogels, the sol-gel conversion temperature of the hydrogel is 37 ℃, and the temperature is closest to the body temperature. The hydroxybutyl chitosan is in a sol state below the critical solution temperature, is suitable for being used as an injection formulation, is suitable for repairing bone defects of irregular and non-bearing parts, is stored and transported in a liquid state under the condition of being lower than the body temperature, is converted into a gel state after being implanted into a body, quickly fills the bone defects in a sol state immediately after being injected, and is molded and kept at the local bone defects after the gel conversion. In the invention, with the gelation and in vivo degradation of the hydroxybutyl chitosan, the loaded hydroxybutyl chitosan contributes to the release of bone factor 1.
(2) The invention adopts near infrared light (NIR) as an excitation mode for controlling release, and the near infrared light stimulated release is milder than other stimulated release modes (such as PH change, temperature change, ultrasonic oscillation and the like), so that the invention has no adverse effect on surrounding healthy tissues, and the near infrared light has a certain bone-promoting effect.
(3) The polydopamine-coated magnesium-containing calcium carbonate microspheres prepared by the method are used as near-infrared response materials, can release loaded drugs under the stimulation of near-infrared light, have a proper in-vivo degradation speed, effectively release calcium and magnesium ions in the degradation process, and promote bone tissue regeneration. In addition, polydopamine as a bionic biological macromolecule has good biocompatibility and negligible toxicity, and has no obvious antigen reaction after being injected or implanted into a living organism.
(4) The drug-loaded bone repair material can realize anti-inflammatory drugs and promote accurate sequence delivery of bone protein after being implanted into a bone defect part, thereby relieving early inflammatory reaction after bone injury, promoting formation of new bone tissues and improving bone regeneration efficiency.
Drawings
FIG. 1 shows HBC
Nuclear magnetic resonance spectrogram and infrared spectrogram, wherein A in FIG. 1 is the content of hydroxybutyl chitosan in heavy water
B is an infrared spectrogram of chitosan and hydroxybutyl chitosan;
FIG. 2 is an infrared spectrogram and scanning electron microscope observation result of magnesium-containing calcium carbonate microspheres (5 MCM) with a magnesium content of 5% and polydopamine-coated magnesium-containing calcium carbonate microspheres (5 MP), wherein A in FIG. 2 is an infrared spectrogram of casein (CCO), 5MCM and 5 MP; b is the result of observing 5MCM and 5MP by a scanning electron microscope;
FIG. 3 is a scanning electron microscope used for observing the internal appearance of various bone repair materials;
FIG. 4 shows the degradation rate detection and SEM observation results of different bone repair materials in PBS, lysozyme and lipase solutions, wherein A-C in FIG. 4 are degradation rate detection results, and D is SEM observation results;
FIG. 5 is a graph of storage modulus for different bone repair materials
And loss modulus
The change with temperature and the transformation diagram of 'sol-gel', wherein A in figure 5 is the transformation diagram of 'sol-gel', and B is the storage modulus
And a loss modeMeasurement of
Graph of change with temperature;
FIG. 6 is a graph of temperature change with time of an HBC composite 5MP hydrogel (HBC + Asp +5 MP-BMP) loaded with aspirin and BMP-2 at different NIR stimulation intensities, wherein A is a thermal imaging result and B is a temperature change curve at different NIR stimulation intensities in FIG. 6;
FIG. 7 is a graph of the release of Asp and BMP-2 by different bone repair materials, wherein A is the graph of the release of Asp and B is the graph of the release of BMP-2 in FIG. 7;
FIG. 8 shows the effect of different bone repair materials on the proliferation potency and adhesion of human mesenchymal stem cells (hBMMSCs), and in FIG. 8, A is CCK-8 to detect the effect of different bone repair materials on the proliferation potency of hBMMSCs; b is an adhesion result of hBMMSCs on the surfaces of different bone repair materials observed by a scanning electron microscope;
FIG. 9 shows the results of alkaline phosphatase (ALP) and Alizarin Red (ARS) staining on day 14 of culturing hBMMSCs on different types of bone repair materials;
FIG. 10 shows Micro-CT scanning images of different kinds of bone repairing materials after 8 weeks of SD rat skull defect repairing operation and analysis of bone volume/tissue volume, trabecular bone thickness and trabecular bone number, wherein in FIG. 10, A is the Micro-CT scanning image, and B is the analysis result of bone volume/tissue volume, trabecular bone thickness and trabecular bone number
；
Fig. 11 is a schematic view of the preparation and release process of the bone repair material of example 1.
Detailed Description
The present invention is further illustrated and described below in conjunction with the following examples, but the examples described are only some, and not all, of the present invention. All other inventions and embodiments based on the present invention and obtained by a person of ordinary skill in the art without any creative effort belong to the protection scope of the present invention.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1A bone repair Material
The preparation method comprises the following steps:
1. preparation of hydroxybutyl chitosan
The preparation of the hydroxybutyl chitosan (HBC) comprises three steps of alkalization, modification and post-treatment of the chitosan:
(1) 1 g of chitosan (Sigma, USA) powder is weighed and dispersed in 20 mL of NaOH solution (50%, w/w) for alkalization, stirred at room temperature for 48 h, and filtered to remove excess lye.
(2) And adding the alkalized chitosan into an isopropanol/water (1: 1, v/v) solution for dispersing for 24 h at room temperature until the alkalized chitosan is completely dispersed in the isopropanol/water solution. 20 mL of 1, 2-butylene oxide (Aladdin, China) was slowly added dropwise to the dispersion obtained in the previous step, and the mixture was placed in a heated magnetic stirrer to react at 55 ℃ for 72 hours. After the reaction is finished, cooling to room temperature, and dropwise adding 0.1M HCl solution into the reaction solution to adjust the pH of the system to be neutral.
(3) The solution is filled into a dialysis bag (8000- + 14000D), and the distilled water is fully dialyzed for 3 to 4 days and replaced every 8 hours. And (3) taking out the dialyzed liquid after the dialysis is finished, filtering to remove insoluble impurities, placing in a refrigerator with the temperature of 20 ℃ below zero for freezing for more than 24 hours, and freeze-drying for more than 60 hours to obtain the HBC sample.
The elemental composition of HBC was analyzed by a Vario EL-III elemental analyzer, and the substitution degree of hydroxybutyl (DS) was calculated by examining the percentage (C, N, H) of each element in the molecule, and the result showed that the substitution degree of hydroxybutyl in the side chain of HBC molecule was 1.64. By passing
Nuclear magnetic resonance spectroscopy (measurement of nuclear magnetic resonance spectrum: (
NMR) chemical Shift reference D of the atoms in the Hydrogen Spectrum2O signal by Fourier transformInfrared spectroscopy (FTIR) detects chemical bonds and groups contained in HBC molecules, thereby presuming whether successful substitution of the molecular structure occurs. Analysis of the NMR spectrum of HBC (A in FIG. 1) revealed that HBC showed strong signal peaks at 0.4 ppm and 1.0 ppm, and the ratio of the integrated areas of the two peaks was 3:2, i.e., -CH-of successfully substituted hydroxybutyl3and-CH2-protons on, demonstrating the successful substitution of hydroxybutyl groups on the side chains of the chitosan molecule. FTIR patterns (B in FIG. 1) of HBC and chitosan are shown at 2876-2920
And 1462
Obvious C-H stretching vibration absorption peak appears, which indicates that-CH is added in the molecular structure of the modified product3and-CH2Successful substitution of the hydroxybutyl group. At the same time, original C6C-O on-OH at 1023
The absorption peak is shifted to 1026-1054
New absorption doublet is formed, which shows that the substitution reaction mainly occurs in C6-OH.
Preparation of polydopamine-coated magnesium-containing calcium carbonate microspheres (MCM @ PDA)
(1) 200 mg of casein was weighed and dissolved in 100 mL of deionized water to prepare a casein solution with a concentration of 2mg/mL, and 529.95 mg of anhydrous sodium caseinate was added
Sufficiently dissolved to obtain a solution containing 50 mM
Solution A of (1).
(2) Separately weighing 527.20 mg
And 23.80 mg
The powder was dissolved in 100 mL of deionized water to obtain a solution containing 47.5 mM
And 2.5 mM
Solution B of (1).
(3) And quickly pouring the solution A into the solution B, reacting at room temperature for 20 min (600 rpm), then obtaining magnesium-containing calcium carbonate microspheres (5 MCM) precipitate with the magnesium content of 5% by high-speed centrifugation (6500 rpm, 10 min), washing the obtained precipitate for 3 times by using deionized water, removing supernatant by high-speed centrifugation (6500 rpm, 10 min) each time, removing the unreacted casein and inorganic salt components, and then freeze-drying the precipitate to obtain the magnesium-containing calcium carbonate microspheres (5 MCM).
(4) 31.52 mg of Tris-HCl powder were weighed and dissolved in 20 mL of deionized water to obtain a 10 mM Tris-HCl salt solution, and the pH of the Tris-HCl salt solution was adjusted to 8.5 using 1M NaOH solution. 40 mg of dopamine hydrochloride powder is weighed and dissolved in 20 mL of Tris-HCl salt solution to obtain 2mg/mL of dopamine-Tris-HCl solution.
(5) Weighing 100 mg of 5MCM microsphere powder, dispersing the powder in 20 mL of dopamine-Tris-HCl solution, carrying out ultrasonic oscillation for 10 min to uniformly disperse the 5MCM microspheres in the dopamine-Tris-HCl solution, and then continuously reacting for 6 h at room temperature by using magnetic stirring (600 rpm).
(6) After the reaction, unreacted dopamine molecules were removed by high-speed centrifugation (6500 rpm, 20 min), the obtained precipitate was washed 3 times with deionized water, and the supernatant was removed by high-speed centrifugation (6500 rpm, 10 min) each time, to obtain Polydopamine (PDA) -coated magnesium-containing calcium carbonate microsphere particles (5 MCM @ PDA, abbreviated as 5 MP).
Detecting chemical bonds and groups contained in 5MCM and 5MP by Fourier transform infrared spectroscopy (FTIR)And observing the surface morphology of the microsphere particles by a Scanning Electron Microscope (SEM). FTIR results are shown in FIG. 2, A, at 870
And 1400-1500 cm-1Is obviously shown
Absorption Peak, 706
The absorption peak at (b) represents the characteristic peak of the calcite crystal form. The scanning electron microscope observation result is shown in B in FIG. 2, and the SEM observation shows that the 5MCM microsphere particles are in a short rod shape and are about 1 μm long. Dopamine molecules with catechol and amino functional groups can spontaneously polymerize on the surface of the 5MCM microsphere to form a polydopamine coating under the weak base condition, the particle size of the 5MP particles is increased compared with that of the 5MCM particles, and the 5MP particles tend to agglomerate due to the fact that the surfaces of the dopamine molecules are rich in functional groups such as catechol and the like.
Polydopamine-coated magnesium-containing calcium carbonate microsphere loaded BMP-2
50 mg of 5MP was weighed out, soaked in 75% alcohol for 3 hours for sterilization, and then washed 3 times with deionized water. BMP-2 (Proteintech) solution was prepared at a concentration of 10. mu.g/mL using 4 mM HCl solution. 500 μ L of BMP-2 solution was mixed well with the washed 5 MP. And then placing the mixture in a rotary shaking table (60 rpm), reacting for 6 h at room temperature, centrifuging at 6500rpm for 5min, discarding the supernatant, and freeze-drying to obtain the poly-dopamine coated magnesium-containing calcium carbonate microsphere particles (5 MP-BMP) loaded with BMP-2.
Preparation of temperature and near-infrared dual-responsiveness bone repair material loaded with aspirin and BMP-2
Aspirin (Asp) solution with a concentration of 200. mu.g/mL was prepared using 1 XPBS solution as solvent. And then adding 1 mL of Asp solution into 50 mg of 5MP-BMP particles, repeatedly blowing and beating the particles to fully and uniformly mix the particles, then adding 50 mg of freeze-dried HBC, standing the mixture at 4 ℃ overnight to fully dissolve the HBC, and thus preparing the HBC composite 5MP hydrogel (HBC + Asp +5 MP-BMP) loaded with aspirin and BMP-2.
Meanwhile, HBC + Asp and HBC +5MP-BMP are prepared by the same method to be used as a control group. Several bone repair material compositions are listed in the following table:
example 2 detection of physicochemical Properties of bone repair Material
(1) Topography Observation of bone repair materials
And (3) freeze-drying the bone repair material of HBC, HBC + Asp, HBC +5MP-BMP and HBC + Asp +5MP-BMP by using a vacuum freeze-drying machine, cutting the exposed section of the bone repair material by using a blade, spraying gold in vacuum, and observing the section appearance and the internal microstructure of the bone repair material by using a Scanning Electron Microscope (SEM), wherein the voltage is 5.0 kV.
The result is shown in figure 3, the HBC + Asp +5MP-BMP bone repair material is loose and porous inside, has good appearance, and has pore sizes of 70-100 microns, which is favorable for cell creeping-in, nutrient absorption and cell metabolic waste elimination. The addition of the Asp and 5MP-BMP microspheres has no obvious influence on the internal structure of the bone repair material, the internal pore walls of the bone repair material containing the 5MP-BMP microspheres are thickened, and the uniform distribution of the microsphere particles in the pore walls can be observed under SEM.
(2) Detection of in vitro degradation capability of bone repair material
And (3) observing the in-vitro degradation capability of the bone repair material by adopting a weight loss method. Weighing the self-made nylon bag and recording the weight
Injecting HBC, HBC +5MP-BMP and HBC + Asp +5MP-BMP into nylon bags, injecting about 100 microliter of bone repair material into each nylon bag, weighing after the bone repair material is fully gelatinized, and recording as
. Subsequently, lysozyme and lipase solutions each having a concentration of 500. mu.g/mL were prepared using 1 XPBS solution as a solvent, respectively. The nylon bags were soaked in 1 XPBS solution and the above lysozyme and lipase solutions, respectively, and placed in a constant temperature shaker (60 rpm) at 37 ℃. In 1 st, 4 th, 7 th, 14 th, 21 th and 2 ndThe nylon bag is taken out after 8 days, and the weight is recorded
. The degradation rate of the bone repair material is calculated by the following formula: rate of degradation
. The results are shown in fig. 4, a-C, where all three bone repair materials were able to degrade efficiently and their mass was decreasing with time in PBS, lysozyme and lipase solutions. Wherein, in lysozyme and lipase solution, the bone repair material is degraded more quickly than in PBS solution, the reduction of the quality is more obvious, and when the bone repair material is degraded to 28 days, the weight loss of the bone repair material is about 80%.
In addition, in order to observe the change of the internal structure in the degradation process of the bone repair material, the bone repair material was obtained on the 7 th day of degradation of the bone repair material, freeze-dried, the cross section of the exposed bone repair material was cut with a blade, and the internal structure morphology was observed by SEM. As a result, as shown in D in FIG. 4, the change in the internal structure of the bone repair material was observed, and when the degradation reached day 7, the pores in the lysozyme and lipase-treated bone repair material were reduced as compared with the PBS-treated group, and the internal structure was more dense.
Example 3 measurement of stimulus response characteristics and factor Release characteristics of bone repair Material
(1) Detection of temperature-sensitive characteristics of bone repair materials
The rheological behavior changes of the four groups of bone repair materials of HBC, HBC + Asp, HBC +5MP-BMP and HBC + Asp +5MP-BMP along with the temperature change are observed by adopting an HAAKE MARS 40 rheometer. The measuring method comprises the following steps: placing the sol bone repair material on a detection flat plate with the diameter of 25 mm, setting the testing distance between parallel plates to be 0.1 mm, measuring the temperature range to be 4-40 ℃, the heating rate to be 1 ℃/min, the fixed strain to be 1 percent and the frequency to be 1 Hz, and recording the storage modulus of a bone repair material sample along with the temperature change
And loss modulus
A change in (c). In the experimental process, a silicon oil layer is coated around the bone repair material for sealing, so that the evaporation of water in the temperature rising process is prevented.
The results are shown in FIG. 5, panel B, the sol-gel transition temperature of the HBC hydrogel is the storage modulus
And loss modulus
I.e. 21.46 deg.c. Of hydrogels at temperatures below 21.46 deg.C
Is less than
The sample is in a liquid state with stronger fluidity, and when the temperature is higher than 21.46 ℃,
rises rapidly and is greater than
Indicating that the hydrogel is in a sol-gel conversion state and is converted into a solid gel state. The phase transition temperature at 21.46 ℃ is lower than the normal physiological temperature, which indicates that the HBC hydrogel can show a stable gel state under physiological conditions. Similarly, the bone repair materials of HBC + Asp, HBC +5MP-BMP and HBC + Asp +5MP-BMP show similar rheological characteristics, and the phase transition temperatures are respectively 20.16 ℃, 19.87 ℃ and 19.33 ℃, which are lower than the physiological temperature. In addition, compared with simple HBC hydrogel, the bone repair material containing Asp and 5MP-BMP microspheres
And
smaller distance therebetween, indicatesThe loading of Asp and 5MP-BMP microspheres increases the hydrogel viscosity.
Meanwhile, under the condition of 4 ℃, the bone repair materials are all sol with good fluidity, and the liquid level of the bone repair materials is kept parallel to the horizontal plane when the glass bottle is inclined (see A in figure 5). The glass bottle is placed in a constant-temperature water bath kettle at 37 ℃, the hydrogel system is gradually turbid along with time increase, the glass bottle is inclined by 45 degrees, the liquid level of the hydrogel does not move any more, the glass bottle is inverted, the hydrogel is displayed as non-flowable solid gel, the conversion from a flowing sol state to a forming gel state is successfully realized, and the sol-gel conversion characteristic of the HBC is not influenced by Asp and 5MP-BMP microsphere particles.
(2) Near infrared light response characteristic of bone repair material
In order to detect the relationship between the near infrared light (NIR) stimulation responsiveness and the illumination intensity of the bone repair material, a near infrared laser exciter with the wavelength of 808 nm (New Changchun industry, China) is used for carrying out near infrared illumination with different intensities on the HBC + Asp +5MP-BMP bone repair material in an experiment, and meanwhile, a thermal imager (Testo, Germany) is used for recording the temperature change of the bone repair material.
As shown in fig. 6 a-B, the bone repair material temperature increased significantly with increasing NIR stimulation time, showing desirable photothermal properties. Wherein, the concentration is 0.25W/cm2Under the infrared stimulation intensity, the temperature of the bone repair material is not increased by more than 10 ℃, the bone repair material is considered to be applied to the body, the local tissue is heated too high to be beneficial to bone tissue regeneration, and therefore the near infrared light stimulation intensity selected in subsequent experiments is 0.25W/cm2。
(3) Detection of Asp and BMP-2 releasing characteristics of bone repairing material sequence
Respectively injecting 200 mu of LHBC + Asp and HBC + Asp +5MP-BMP bone repair materials into a 12-hole plate, and incubating for 30 min in a constant-temperature incubator at 37 ℃ to ensure that the bone repair materials are fully crosslinked. Subsequently 1 mL of 1 × PBS solution (pH = 7.4) was added per well and the 12-well plate was placed in a 37 ℃ constant temperature shaker (60 rpm) to detect the release of Asp at different time points. At each time point to be tested (2 h, 6 h, 1 d, 2d, 3 d, 5d, 7 d, 10 d and 14 d) 200. mu.L of the well plate was aspirated and 200. mu.L of fresh 1 XPBS solution was added again. Using ultraviolet-visible lightThe concentration of Asp in the solution supernatant at different time points was determined by a spectrophotometer and the cumulative Asp release rate was calculated from the following equation: accumulated Asp Release Rate (%) =
. Wherein M istFor the total mass of Asp released at time t, M0Is the total mass of Asp contained within the bone repair material.
Meanwhile, in order to detect the influence of near NIR stimulation on BMP-2 release of the bone repair material, 200 mu of LHBC + Asp +5MP-BMP bone repair material is injected into a 12-hole plate adaptive upper chamber of a Transwell chamber, incubated in a constant-temperature incubator at 37 ℃ for 30 min, added into 1 mL of 1 XPBS solution in a lower chamber of the Transwell chamber after being fully crosslinked, and the 12-hole plate is placed in a constant-temperature shaking table at 37 ℃ (60 rpm) to detect the release of BMP-2 at different time points. BMP-2 release curves were calculated and plotted by measuring the concentration of BMP-2 in the solution at different time points using an ELISA kit (RayBiotech) with NIR stimulation applied on days 3, 7 and 11, respectively, and with the group without NIR stimulation as a control group.
Bone repair material release Asp profile As shown in A of FIG. 7, the bone repair material of HBC + Asp and HBC + Asp +5MP-BMP released Asp rapidly and continuously with Asp release of more than 60% in 48 h and over 75% Asp release on day 14. In addition, the HBC + Asp +5MP-BMP bone repair material can start the rapid release of BMP-2 under the NIR stimulation according to the required time (B in figure 7), and shows the on-demand NIR stimulation response release characteristic. While in the absence of NIR stimulation, the bone repair material releases BMP-2 slowly. Therefore, the bone repair material HBC + Asp +5MP-BMP can quickly release low-concentration Asp in the early stage, is beneficial to reducing local tissue inflammatory reaction in the early stage of bone defect occurrence, and then responds to NIR stimulation to release BMP-2 as required so as to accurately regulate the differentiation of mesenchymal stem cells to osteogenesis and promote bone tissue regeneration.
Example 4 cellular compatibility and bone-enabling efficacy testing of bone repair materials.
(1) Effect of bone repair materials on proliferation potency of human mesenchymal Stem cells (hBMMSCs)
Adding different kinds of bone repairing materials into 48-hole plate with each hole at 37 deg.C and 50 μ LIncubating in a constant temperature incubator for 30 min to ensure that the cells are fully crosslinked, and taking the blank control group without adding bone repair materials in the pore plates. Then obtaining hBMMSCs cell suspension, adjusting cell concentration to 2
Inoculating hBMMSCs into bone repair material or blank well plate, adding 200 μ L cell suspension per well, standing at 37 deg.C and 5% CO2And carrying out static culture in a constant-temperature incubator with saturated humidity, replacing a fresh culture medium every other day, and detecting the proliferation condition of hBMMSCs by adopting a CCK-8 method when culturing 1 st, 3 rd, 5 th and 7 th, wherein the specific operation is as follows: mixing CCK-8 reagent (North Kernel chemical technology Co., Ltd.) and basal medium at a ratio of 1:10 to prepare CCK-8 working solution, completely sucking the original culture medium in a 48-well plate, adding 1 XPBS solution to clean cells for 1 time, completely sucking 1 XPBS solution, then adding 200 microliter of the working solution into each well, culturing for 1 hour in a constant-temperature incubator at 37 ℃, sucking 100 microliter of upper culture solution into a new 96-well plate for each well, measuring the absorbance value of the cells at 450nm by using a full-wavelength microplate reader, and drawing a cell proliferation curve of hBMMSCs according to the absorbance value.
The results are shown in a in fig. 8, when hBMMSCs are cultured on the surface of the bone repair material, the cell number is low on day 1 of culture, and then the cell number of the hBMMSCs increases with time, meanwhile, no significant effect of different types of bone repair materials on the proliferation capacity of the hBMMSCs can be observed, no inhibition of the bone repair materials on the proliferation capacity of the hBMMSCs is observed, which proves that several groups of bone repair materials have good cell compatibility characteristics, and the proliferation capacity of the cells is not affected by the addition of Asp and 5MP-BMP microspheres.
(2) SEM observation of adhesion of hBMMSCs on surface of bone repair material
Respectively dripping 100 mu L of HBC, HBC + Asp, HBC +5MP-BMP and HBC + Asp +5MP-BMP bone repair materials on the surface of a 24-pore plate adaptive cell slide, and incubating for 30 min in a constant-temperature incubator at 37 ℃ to ensure that the materials are fully crosslinked. Then obtaining hBMMSCs cell suspension, adjusting cell concentration to 2
Dropping 100 mu L of cell suspension on the surface of the bone repair material after full cross-linking, placing the bone repair material in a constant-temperature incubator at 37 ℃ for incubation for 30 min, and supplementing 500 mu L of proliferation culture medium into each hole after the cells are attached to the wall. At days 3 and 7 of cell culture, the culture medium in the pore plate is completely sucked, the cells are washed 1 time by 1 XPBS solution, 500 mu L of 4% paraformaldehyde solution is added into each pore to fix the cells for 30 min, the paraformaldehyde solution is discarded, the cells are washed 1 time by the 1 XPBS solution, and then, the sample is fully frozen and dried and the adhesion condition of the cells on the upper surface of the bone repair material is observed by using SEM.
In fig. 8, B shows the adhesion condition of the hBMMSCs cells on the bone repair material as photographed by SEM, as shown by arrows in the figure, the cells adhere well to the surfaces of different bone repair materials and show more pseudopodia, no obvious difference is observed in the cell adhesion morphology among groups, which indicates that different kinds of drug-loaded bone repair materials can effectively support the adhesion, migration and proliferation of cells.
(3) Detection of osteogenic differentiation capability of hBMMSCs (human bone marrow stromal cells) promoted by bone repair material
Extracting cells according to 5
The cells were seeded at a density of one/mL in 6-well plates, and the addition of cell proliferation medium (PM, main component α -MEM, containing 10% fetal bovine serum, 1% streptomycin) was started when the cells were about 80% confluent, while a 6-well plate adapted Transwell chamber (Corning, 0.4 μm) was added to the well plate and 500 μ L of different kinds of bone repair materials was added to the upper chamber of the chamber. The well plate is placed in a constant-temperature incubator at 37 ℃ for incubation for 30 min, and 500 mu LPM culture medium is added on the surface of the bone repair material.
A negative control group (PM group) and a positive control group (OM group) were set simultaneously, cells in the PM group were cultured using only PM, and cells in the OM group were cultured using an osteogenic induction medium (mainly composed of α -MEM containing 10% fetal bovine serum, 1% streptomycin, 100 nM dexamethasone, 0.2 mM ascorbic acid, and 10 mM β -GP).
NIR laser stimulation (0.25 cm/W) is applied to a part of the HBC +5MP-BMP, HBC + Asp +5MP-BMP bone repair material group on days 3, 7 and 11 of cell culture 210 min). On day 14 of cell culture, cells were analyzed for alkaline phosphatase (ALP) staining and Alizarin Red (ARS) staining.
The staining results are shown in fig. 9, and different types of bone repair materials can promote osteogenic differentiation of hBMMSCs to different degrees compared to the PM group without the addition of the bone repair material component. The bone repair material loaded with the 5MP-BMP microspheres (comprising HBC +5MP-BMP, HBC + Asp +5MP-BMP, HBC +5MP-BMP + NIR and HBC + Asp +5MP-BMP + NIR) has more obvious capacity of promoting the expression of the hBMMSCs alkaline phosphatase. Meanwhile, compared with HBC +5MP-BMP and HBC +5MP-BMP + NIR groups, the release of BMP-2 can be effectively promoted by adding NIR laser stimulation in the cell culture process, so that the expression of hBMMSCs alkaline phosphatase is further promoted. In addition, the Asp-loaded bone repair material can further induce the expression increase of alkaline phosphatase, shows the capability of synergistically promoting osteogenic differentiation of hBMMSCs, and has the strongest capability of promoting the expression of hBMMSCs alkaline phosphatase in an HBC + Asp +5MP-BMP + NIR group.
Example 5 in vivo injectable application example of bone repair Material and in vivo osteogenesis promoting Effect test
A bilateral skull defect model was prepared using 6-8 week-old male SD rats, with a defect diameter of 5 mm, and bone repair material injected into the defect site, but not implanted in the Blank control group (Blank). Near infrared light stimulation is carried out on the defect part on the 3 rd, 7 th and 11 th days after injection. After 8 weeks, the materials are taken and subjected to Micro-CT analysis to compare the capacity of different types of bone repair materials for promoting the regeneration of new bones of SD rats. Micro-CT scanning three-dimensional reconstruction shows (A in figure 10), only a small amount of new bone tissue formation at the edge of a bone defect area is observed in a Blank group without bone repair material filling, and the establishment of the SD rat critical skull defect model is successful. When different types of bone repair materials are used for repairing SD rat skull defects, new bones are formed from the edge to the center of the skull defect part to different degrees, wherein the bone repair materials loaded with 5MP-BMP have more formation amount than blank groups and bone repair materials not loaded with 5MP-BMP, the new bone tissues of the groups stimulated by NIR laser are obviously more, the new bone formation amount of HBC + Asp +5MP-BMP + NIR groups is the most, and the new bone tissues can basically cover the bone defect area.
Meanwhile, analysis is performed on bone volume/tissue volume (BV/TV), trabecular bone thickness (tb.th) and trabecular bone number (tb.n) of each group, and the results show (B in fig. 10), that the enhancement of the capacity of the HBC hydrogel alone and the Asp-loaded HBC hydrogel for promoting new bone formation is not significant as compared with the blank group, the capacity of the bone repair material containing 5MP-BMP for promoting new bone formation is significantly enhanced, in addition, the release of BMP-2 is properly regulated and controlled by NIR laser stimulation, osteogenic differentiation of MSCs can be effectively regulated and bone regeneration is enhanced, the bone repair material simultaneously delivers Asp and BMP-2 to further exhibit the effect of synergistically promoting bone tissue regeneration, and the BV/TV and tb.n of the HBC + Asp +5MP-BMP + NIR group are significantly higher than the blank group, and have faster and better bone integration capacity to promote the formation of new bone.
Claims (10)
1. The drug-loaded bone repair material is characterized by comprising bone-promoting factor 1, hydroxybutyl chitosan and polydopamine-coated magnesium-containing calcium carbonate microspheres loaded with bone-promoting factor 2; polydopamine-coated magnesium-containing calcium carbonate microspheres loaded with bone factor 2 and hydroxybutyl chitosan are dispersed in a bone factor 1 solution to obtain the drug-loaded bone repair material; after the drug-loaded bone repair material is implanted into a body, the hydroxybutyl chitosan is gelatinized and releases osteogenesis promoting factors 1; the release of the bone factor 2 from the polydopamine-coated magnesium-containing calcium carbonate microspheres loaded with the bone factor 2 can be controlled through near infrared light irradiation.
2. The drug-loaded bone repair material according to claim 1, comprising one or more of the following (1) to (6):
(1) the osteogenesis promoting factor 1 is a medicine or nutrient capable of promoting the bone repair process in the early bone repair stage, and the osteogenesis promoting factor 2 is a medicine or nutrient capable of promoting the bone repair process in the later bone repair stage;
(2) dispersing magnesium-containing calcium carbonate microspheres in a dopamine salt solution to obtain polydopamine-coated magnesium-containing calcium carbonate microspheres;
(3) dispersing polydopamine-coated magnesium-containing calcium carbonate microspheres in a solution for promoting bone factor 2 to obtain the polydopamine-loaded magnesium-containing calcium carbonate microspheres for promoting bone factor 2;
(4) the near infrared light stimulation intensity is 0.25W/cm2；
(5) Will contain
Adding the casein solution to the solution containing
And
obtaining magnesium-containing calcium carbonate microspheres in the solution;
(6) adding bone factor 1 promoting solution into polydopamine-coated magnesium-containing calcium carbonate microspheres loaded with bone factor 2, repeatedly blowing and beating to uniformly mix, and adding hydroxybutyl chitosan to fully dissolve to obtain the drug-loaded bone repair material.
3. The drug-loaded bone repair material according to claim 2, comprising one or more of the following (1) to (5):
(1) the mass ratio of the dopamine salt solution to the magnesium-containing calcium carbonate microspheres is 2: 5;
(2) the dopamine salt solution is a dopamine-Tris hydrochloride solution;
(3) dispersing the magnesium-containing calcium carbonate microspheres in the dopamine salt solution by adopting ultrasonic oscillation and/or magnetic stirring, and then centrifuging to remove unreacted dopamine molecules to obtain polydopamine-coated magnesium-containing calcium carbonate microspheres;
(4) the osteogenesis promoting factor 1 is an anti-inflammatory drug, and the osteogenesis promoting factor 2 is osteogenesis promoting protein;
(5) the polydopamine-coated magnesium-containing calcium carbonate microspheres are soaked in alcohol for disinfection and washed by deionized water, and then dispersed in a solution for promoting bone factor 2.
4. The drug-loaded bone repair material according to any one of claims 1 to 3, wherein the osteogenesis-promoting factor 1 is aspirin and the osteogenesis-promoting factor 2 is BMP-2.
5. The drug-loaded bone repair material according to claim 4, comprising one or more of the following (1) - (2):
(1) the preparation method of the magnesium-containing calcium carbonate microspheres comprises the following steps:
will be provided with
Adding into 2mg/mL casein solution to obtain solution containing 50 mM
Solution A of (1); and was prepared to contain 47.5 mM
And 2.5 mM
Solution B of (1); quickly pouring the solution A into the solution B, reacting at room temperature for 20 min, then obtaining calcium carbonate microsphere sediment with the magnesium content of 5% through high-speed centrifugation, and washing the sediment with deionized water to obtain magnesium-containing calcium carbonate microspheres;
(2) preparing a BMP-2 solution with the concentration of 10 mu g/mL by adopting a 4 mM HCl solution; and (3) taking the BMP-2 solution and polydopamine coated magnesium-containing calcium carbonate microspheres which are sterilized by alcohol and washed by deionized water, fully mixing the polydopamine coated magnesium-containing calcium carbonate microspheres according to the mass ratio of 10000:1-1000:1, and centrifuging after oscillation reaction to obtain the polydopamine coated magnesium-containing calcium carbonate microsphere particles loaded with BMP-2.
6. The preparation method of the drug-loaded bone repair material is characterized by comprising the following steps:
(1) dispersing the magnesium-containing calcium carbonate microspheres in a dopamine salt solution to obtain polydopamine-coated magnesium-containing calcium carbonate microspheres;
(2) dispersing polydopamine-coated magnesium-containing calcium carbonate microspheres in a bone factor 2 promoting solution to obtain polydopamine-loaded bone factor 2 promoting magnesium-containing calcium carbonate microspheres;
(3) adding bone factor 1 promoting solution into polydopamine-coated magnesium-containing calcium carbonate microspheres loaded with bone factor 2, repeatedly blowing and beating to uniformly mix, and adding hydroxybutyl chitosan to fully dissolve to obtain the drug-loaded bone repair material.
7. The production method according to claim 6, characterized by comprising one or more of the following (1) to (6):
(1) the osteogenesis promoting factor 1 is a medicine or nutrient capable of promoting the bone repair process in the early bone repair stage, and the osteogenesis promoting factor 2 is a medicine or nutrient capable of promoting the bone repair process in the later bone repair stage;
(2) will contain
To a solution containing casein
And
obtaining magnesium-containing calcium carbonate microspheres in the solution;
(3) the mass ratio of the dopamine salt solution to the magnesium-containing calcium carbonate microspheres is 2: 5;
(4) the dopamine salt solution is a dopamine-Tris hydrochloride solution;
(5) dispersing the magnesium-containing calcium carbonate microspheres in the dopamine salt solution by adopting ultrasonic oscillation and/or magnetic stirring, and then centrifuging to remove unreacted dopamine molecules to obtain polydopamine-coated magnesium-containing calcium carbonate microspheres;
(6) the polydopamine-coated magnesium-containing calcium carbonate microspheres are soaked in alcohol for disinfection and washed by deionized water, and then dispersed in a solution for promoting bone factor 2.
8. The method according to claim 7, characterized by comprising one or more of the following (1) to (4):
(1) the osteogenesis promoting factor 1 is aspirin, and the osteogenesis promoting factor 2 is BMP-2;
(2) when the bone promoting factor 2 is BMP-2, the mass ratio of the BMP-2 to the polydopamine coated magnesium-containing calcium carbonate microspheres is 10000:1-1000: 1;
(3) the preparation method of the magnesium-containing calcium carbonate microspheres comprises the following steps:
will be provided with
Adding into 2mg/mL casein solution to obtain solution containing 50 mM
Solution A of (1); and was prepared to contain 47.5 mM
And 2.5 mM
Solution B of (1); quickly pouring the solution A into the solution B, reacting at room temperature for 20 min, then obtaining calcium carbonate microsphere sediment with the magnesium content of 5% through high-speed centrifugation, and washing the sediment with deionized water to obtain magnesium-containing calcium carbonate microspheres;
(4) when the bone promoting factor 2 is BMP-2, preparing a BMP-2 solution with the concentration of 10 mu g/mL by adopting a 4 mM HCl solution; and (3) taking the BMP-2 solution and polydopamine coated magnesium-containing calcium carbonate microspheres which are sterilized by alcohol and washed by deionized water, fully mixing the polydopamine coated magnesium-containing calcium carbonate microspheres according to the mass ratio of 10000:1-1000:1, and centrifuging after oscillation reaction to obtain the polydopamine coated magnesium-containing calcium carbonate microsphere particles loaded with BMP-2.
9. The drug-loaded bone repair material according to any one of claims 1 to 5 or the preparation method according to any one of claims 6 to 8 for use in the fields of bone defect repair and bone regeneration.
10. The use according to claim 9, wherein the medicament-loaded bone repair material according to any one of claims 1 to 5 or the bone repair material prepared by the preparation method according to any one of claims 6 to 8 is injected near a site to be repaired.
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