CN117942427B - Injectable intelligent controlled release hydrogel and application thereof in treating bone injury - Google Patents
Injectable intelligent controlled release hydrogel and application thereof in treating bone injury Download PDFInfo
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Abstract
The invention belongs to the field of medicine, and relates to wound dressing, in particular to injectable intelligent controlled-release hydrogel and application thereof in treating bone injury, the hydrogel takes an aqueous solution of oxidized hyaluronic acid and an aqueous solution of carboxymethyl chitosan as matrixes, a hydrogel substrate is prepared through chemical reaction, and the hydrogel substrate is internally loaded with Pht@ZIF-8@PDA, and the preparation method of the Pht@ZIF-8@PDA comprises the following steps: firstly, encapsulating the Pht in ZIF-8 nano particles to obtain Pht@ZIF-8 nano particles; then wrapping PDA on the surface of the Pht@ZIF-8 nano particles; wherein, pht is phloretin, ZIF-8 is zinc-based zeolite imidazole skeleton, and PDA is polydopamine. After the hydrogel is subjected to near infrared photothermal treatment, intelligent on-demand release and efficient transportation of Pht and Zn 2+ are realized, so that the medicine can more effectively permeate into bone tissues, more accurate targeted drug delivery is realized, the bone repair effect can be further improved, and the repair of critical-size bone defects is realized.
Description
Technical Field
The invention belongs to the field of medicines, relates to wound dressing, and in particular relates to injectable intelligent controlled-release hydrogel and application thereof in treating bone injury.
Background
Bone is one of the largest organs of the human body, and plays an important role in maintaining the structure of the human body, protecting internal organs, and regulating the metabolic balance of various ions in the body. When bone damage exceeds its ability to repair itself, it will not recover itself, a so-called "critical dimension defect" (CSD). Medical intervention is necessary when the bone exceeds the critical dimension defect, but the existing treatment methods still have difficulty achieving the intended therapeutic effect.
In clinical practice of bone defect repair, autologous bone grafting has been challenged by practical use as an ideal means, mainly due to limited donor sources, high surgical costs, and the possible need for additional surgical incisions. In the face of millions of global patient groups in need of surgical bone grafting, the search for novel bone substitute materials is particularly urgent. This need is not only an important topic of medical research, but is also a significant social issue.
Bone Tissue Engineering (BTE) is a promising new approach to repair bone defects. Injectable hydrogels are considered ideal biomimetic material choices for BTEs. In addition, the injectable hydrogel can form gel in situ at the defect site and fully fill the wound area, thereby effectively overcoming the limitations of the conventional hydrogels.
Phloretin (Pht) is a widely distributed flavonoid. Pht has a variety of pharmacological effects including anti-inflammatory, antibacterial, antioxidant and other pharmacological effects. However, pht has low absolute bioavailability, poor stability, limiting its use. Therefore, there is a need for an injectable hydrogel that can intelligently release bone repair active ingredients as needed, has excellent bone repair effects, and can repair critical-size bone defects.
Disclosure of Invention
In view of the above technical problems and defects, the invention aims to provide an injectable intelligent controlled release hydrogel, which takes Pht as an active ingredient, takes ZIF-8 nano particles as a carrier and takes PDA as a nano particle coating, after near infrared light is irradiated, intelligent release and efficient transportation of Pht and Zn 2+ according to needs are realized, so that the medicament can more effectively permeate into bone tissues, more accurate targeted administration is realized, the bone repair effect can be further improved, and the repair of critical-size bone defects is realized.
In order to achieve the above purpose, the invention adopts the following technical scheme:
The injectable intelligent controlled release hydrogel takes an aqueous solution of oxidized hyaluronic acid and an aqueous solution of carboxymethyl chitosan as matrixes, a hydrogel substrate is prepared through chemical reaction, and the hydrogel substrate is internally loaded with Pht@ZIF-8@PDA, and the preparation method of the Pht@ZIF-8@PDA comprises the following steps: firstly, encapsulating the Pht in ZIF-8 nano particles to obtain Pht@ZIF-8 nano particles; then wrapping PDA on the surface of the Pht@ZIF-8 nano particles; wherein, pht is phloretin, ZIF-8 is zinc-based zeolite imidazole skeleton, and PDA is polydopamine.
As the preferable mode of the invention, the preparation method of the injectable intelligent controlled release hydrogel comprises the following steps:
(1) Mixing hyaluronic acid and sodium periodate in water, and performing oxidation reaction to obtain oxidized hyaluronic acid;
(2) Mixing Pht with 2-methylimidazole methanol solution at room temperature, dissolving zinc nitrate hexahydrate in deionized water, adding into the mixed solution containing Pht, stirring, standing, centrifuging to recover suspension, washing, and drying to obtain Pht@ZIF-8 nanoparticles;
(3) Suspending the Pht@ZIF-8 nano particles in a Tris-HCl buffer solution, performing ultrasonic treatment, dropwise adding dopamine hydrochloride, standing for a period of time, continuing ultrasonic treatment, dropwise adding dopamine hydrochloride, stirring, centrifugally collecting, and drying to obtain the Pht@ZIF-8@PDA nano particles;
(4) Dissolving oxidized hyaluronic acid in deionized water, adding Pht@ZIF-8@PDA nanoparticles, and then mixing with carboxymethyl chitosan aqueous solution to prepare the OHA/CMCS/Pht@ZIF-8@PDA, thereby obtaining the injectable intelligent controlled-release hydrogel.
As a preferred aspect of the present invention, the specific steps of step (2) are: 35 mg of Pht and 5ml of 0.97M 2-methylimidazole methanol solution were mixed at room temperature and stirring continued until dissolved; then 0.1g of zinc nitrate hexahydrate is dissolved in 5mL deionized water and added into the mixed solution containing Pht, after stirring for 80 minutes and standing for 1 hour, the suspension is recovered by centrifugation, washed with methanol solution and dried at room temperature, and pht@zif-8 nano particles are obtained.
As a preferred aspect of the present invention, the specific steps of step (3) are: 8 mg of synthesized Pht@ZIF-8 nanoparticles were suspended in 40 ml of Tris-HCl buffer solution and sonicated for 5 minutes, then 5 mg of dopamine hydrochloride was added dropwise and left at room temperature for 2 hours; after 5 minutes of ultrasound, 2 mg of dopamine hydrochloride is added dropwise, stirred for 2 hours at room temperature, and the final product Pht@ZIF-8@PDA nanoparticles are collected by centrifugation, washed with methanol and dried at room temperature.
As a preferred aspect of the present invention, the specific steps of step (4) are: dissolving hyaluronic acid in deionized water to prepare 5% aqueous solution of oxidized hyaluronic acid; the concentration of the carboxymethyl chitosan aqueous solution is 5%, and the volume ratio of the oxidized hyaluronic acid aqueous solution to the carboxymethyl chitosan aqueous solution is 1:1.
The injectable intelligent controlled release hydrogel provided by the invention is beneficial to osteoblast proliferation, can promote osteoblast osteogenic differentiation and calcium deposition capacity, up-regulates osteogenic markers such as osteogenic gene alkaline phosphatase (ALP), bone Silicon Protein (BSP), osteopontin (OPN), osterix (OSX) and the like, and has positive effects on repairing bone defects and forming new bones; in addition, the in vivo bone reconstruction experiment shows that the rat subjected to PZPG +NIR irradiation has excellent curative effect in accelerating in vivo bone formation and repair, and the critical bone defect is basically completely healed after 8 weeks of treatment, so that the rat can be applied to the preparation of the biological scaffold for treating bone injury.
The injectable intelligent controlled release hydrogel provided by the invention can be heated to a proper temperature after being irradiated by near infrared light (NIR), and can not damage surrounding healthy cells, and has excellent photo-thermal conversion efficiency and good photo-thermal stability, so that the injectable intelligent controlled release hydrogel can be applied to preparation of photo-thermal conversion materials.
The injectable intelligent controlled release hydrogel provided by the invention can obviously improve the expression of BMP2, SMAD1/5/8, p-SMAD1/5/8 and Runx2, and activates BMP/SMAD channels by up-regulating the expression of BMP2, SMAD1/5/8, p-SMAD1/5/8 and Runx 2; therefore, the compound can be applied to the preparation of BMP/Smad signal channel activators, and the bone trabecula and cortical bone are promoted to be combined and regenerated by activating the BMP/Smad signal channel, so that bone reconstruction is realized, and bone defect repair is accelerated.
The invention has the advantages and beneficial effects that:
(1) The hydrogel provided by the invention consists of oxidized hyaluronic acid, carboxymethyl chitosan and Pht@ZIF-8@PDA nanoparticles encapsulated in PDA, wherein the Pht is encapsulated in ZIF-8, so that the release speed of the Pht can be delayed, the PDA is wrapped on the surface of the Pht@ZIF-8, the stability of the Pht can be further improved, the PDA and the ZIF-8 cooperate, the sustained release duration of the Pht can be prolonged to 30 days or longer, the Pht is released at a proper speed and concentration in the whole bone reconstruction process, and the active ingredients can play roles for a long time and continuously, thereby accelerating bone defect repair, and avoiding surgical wounds caused by secondary drug administration.
(2) According to the hydrogel provided by the invention, through coating (packaging) Polydopamine (PDA) on the surface of the nano particles, the Pht@ZIF-8 nano particles can be uniformly dispersed in the hydrogel, so that the problem of poor ZIF-8 dispersibility is solved, meanwhile, the PDA can effectively absorb near infrared light to realize photo-thermal conversion, and effectively generate heat to be transferred to different positions of a bone defect part, so that Bone Mesenchymal Stem Cells (BMSCs) are accelerated to be stimulated to be converted to an osteogenic differentiation direction, thereby promoting the repair and regeneration of bone tissues and improving the bone repair curative effect.
(3) The uniformly dispersed ZIF-8 in the hydrogel can release Zn 2+, the early-stage abrupt release of Zn 2+ is more beneficial to destroying the integrity of bacterial exosomes, plays antibacterial activity and contributes to early-stage anti-inflammatory, the hydrogel realizes antibacterial effect through the synergistic effect of ZIF-8, pht and NIR-induced photo-thermal radiation, the inhibition capability of the hydrogel on staphylococcus aureus is up to 99.39%, the inhibition rate of the hydrogel on escherichia coli is up to 97.53 +/-0.57%, and the hydrogel has excellent antibacterial performance and can effectively prevent infection during bone defect repair; in addition, the synergistic effect of Zn 2+ and Pht released in the early stage and the photo-thermal radiation induced by NIR can fully play the osteogenesis role, so that the bone reconstruction efficiency is improved.
(4) The hydrogel provided by the invention has a uniform porous structure inside, a stable network structure and a small pore diameter, and the addition of the Pht@ZIF-8@PDA nanoparticles enhances the mechanical property of the hydrogel, so that the hydrogel has good elasticity and stronger deformation resistance, and is more beneficial to protecting the tissues below the skull defect part and preventing secondary damage.
(5) The hydrogel provided by the invention has strong near infrared light absorption, high photo-thermal conversion efficiency and good photo-thermal stability, can realize intelligent regulation and control of active ingredient release through remotely controlled photo-thermal property, can be used for photo-thermal skull defect treatment, namely near infrared photo-thermal treatment after being implanted into the hydrogel, can enhance the antibacterial effect and bone repair effect under the synergistic effect of photo-thermal therapy and the hydrogel, and can also furthest reduce unnecessary side effects.
(6) The hydrogel provided by the invention has good injectability, can be customized according to the specific form of the skull defect part, can well connect nutrient substances and waste exchange between the bone defect part and surrounding natural bone tissues, and is beneficial to osteoblast proliferation and differentiation.
(7) The hydrogel provided by the invention can effectively promote differentiation and regeneration processes of in-vivo and in-vitro bone tissues, and realize intelligent regulation and control of release of zinc ions and Pht under the periodic near infrared illumination condition, so that multiple effects of anti-inflammation, antibacterial and bone repair are synchronously exerted, and a stable microenvironment is constructed for the defect part.
(8) The hydrogel provided by the invention has osteogenic differentiation capability, can promote osteogenic differentiation and calcium deposition capability of osteoblasts, up-regulates osteogenic genes alkaline phosphatase (ALP), bone Silicon Protein (BSP), osteopontin (OPN) and Osterix (OSX), and has the advantages that a rat subjected to PZPG + NIR irradiation shows excellent curative effect in accelerating in-vivo osteogenesis and repair, and is substantially completely healed after 8-week critical bone defect treatment, and the hydrogel is obviously superior to the existing hydrogel material.
(9) The hydrogel provided by the invention has excellent antioxidant activity, can remove ROS generated at the bone defect position, and can promote bone healing.
(10) The hydrogel provided by the invention has excellent biodegradability, biocompatibility and remarkable swelling capacity, and can ensure safety and no toxicity in the use process.
Drawings
FIG. 1 is a representation of nanoparticles; where a is a TEM image of ZIF-8, PZ, PZP (scale bar=100 μm, 50 μm); b. XRD spectra of Pht, ZIF-8, pht@ZIF-8 and Pht@ZIF-8@PDA; c-e are the particle size distribution of ZIF-8, pht@ZIF-8 and Pht@ZIF-8@PDA respectively; f is the Zeta potential of ZIF-8, pht@ZIF-8 and Pht@ZIF-8@PDA; g is the infrared spectrum of Pht, ZIF-8, pht@ZIF-8 and Pht@ZIF-8@PDA.
FIG. 2 is a representation of hyaluronic acid and nanocomposite hydrogels; wherein a is the infrared spectrum of HA and OHA; b is 1 H NMR spectrum of HA and OHA; c is a scanning electron microscope image of the nanocomposite hydrogel (scale bar = 200 μm, 100 μm, 50 μm); d. e is the rheological analysis of the nanocomposite hydrogels.
FIG. 3 is a mild photothermal effect analysis of PZPG; wherein a is an in vitro infrared heat map; b is a photo-thermal temperature rise curve of the PZP particle hydrogels with different concentrations; c is PZPG heating and cooling curves; d is ln (q) linear time data obtained from the cooling cycle; e is cooling cycle time data;
FIG. 4 is a graph of release and degradation of nanocomposite hydrogels; wherein a is the accumulated drug release of PG, PZG, PZPG + NIR; b is the accumulated release amount of zinc ions; c is the degradation rate of the hydrogel; d is the swelling rate of the hydrogel.
FIG. 5 is an in vitro activity study of nanocomposite hydrogels; wherein a is an inhibition image of escherichia coli and staphylococcus aureus; b is the inhibition ratio of escherichia coli and staphylococcus aureus; c is antioxidant activity; d is a hemolysis test; e is the cell viability of the composite hydrogel; f is EDU image of the composite hydrogel; p <0.05, < p < 0.01).
FIG. 6 is in vitro osteogenic differentiation of nanocomposite hydrogels; wherein a is the ALP and ARS images of the composite hydrogel; b is a protein band of a Western blot analysis chart; c-f is the WB analysis of ALP, BSP, OPN and OSX, respectively, and the protein expression level; * p <0.05, p <0.01.
FIG. 7 is a in vivo surgical and Micor-CT analysis of nanocomposite hydrogels; wherein a is a skull defect model operation schematic diagram; b is PZPG near infrared thermal imaging of the defect part; c is a CT image of the defect part; d is the bone trabecular spacing (Tb. Sp); e is bone trabecular thickness (Tb. Th); f is the number of bone trabeculae (Tb. N); g is bone tissue volume/total tissue volume (BV/TV).
FIG. 8 is an image of H & E and Masson staining of bone tissue.
FIG. 9 Western blot analysis; wherein a is a protein band; b-i is ALP, BSP, OPN, OSX, BMP2, SMAD1/5/8, p-SMAD1/5/8 and RUNX2, respectively; * p <0.05, p <0.01.
Detailed Description
The invention will be further described with reference to the accompanying drawings and specific examples, to which embodiments of the invention are not limited. For process parameters not specifically noted, reference may be made to conventional techniques. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
1. Materials and methods
1.1 Material
Pht, purity not less than 98.0%, carboxymethyl chitosan (CMCS, mw=2240 Kda), sodium periodate (NaIO 4, purity not less than 99.5%), 2-methylimidazole (2-MeIm, purity not less than 98.0%), dopamine hydrochloride (DA, purity not less than 98.0%), methanol (MeOH, purity not less than 99.5%), triton X-100 were supplied by the company of the chemical company of wheat Lin Shengwu. Hyaluronic acid (HA, mw=20000-40000 Kda, purity ≡99.0%) was purchased from ALADDIN CHEMICALS co. Tribasic was purchased from Beijing Jin Taihong to biotechnology Co. Zinc nitrate hexahydrate (Zn (NO 3) 2-6H 2O) with purity greater than or equal to 99.0% was purchased from the chemical industry Co., ltd. Hydrochloric acid was purchased from beijing chemical plant limited. Phosphate Buffered Saline (PBS) was purchased from sora biotechnology limited. Anti-alkaline phosphatase (ALP), anti-bone ialoprotein (BSP), anti-Osteopontin (OPN), anti-Osterix (OSX), anti-collagen-I (COL I), anti-bone morphogenic protein 2 (BMP 2), anti-SMAD 1/5/8 (Smad 1/5/8), anti-Phospho-SMAD 1/5/8 (p-Smad 1/5/8), anti-Runt-related transcription factor 2 (Runx 2) and secondary antibodies were purchased from proteintech Biotechnology Inc.
1.2 Preparation and characterization of OHA, pht@ZIF-8 and Pht@ZIF-8@PDA
(1) HA (3 g) was dissolved in deionized water (100 g) and then sodium periodate (1 g) was added. The mixture was stirred at 25℃for 6 hours in the dark, then 2 mLCH 2 OH was added. Then, dialyzing with deionized water for 3 days to obtain OHA, and freeze-drying the product.
(2) Pht (35 mg) and 5 ml of 0.97M 2-MeIm methanol solution were mixed at room temperature and stirring continued until dissolved. Then, 0.1g of Zn (NO 3)2-6H2 O was dissolved in 5 mL deionized water and rapidly added to the above mixed solution, and after stirring for 80 minutes and standing for 1 hour, the suspension was recovered by centrifugation, washed with methanol solution and dried at room temperature to give Pht@ZIF-8 (PZ).
(3) The synthesized PZ (8 mg) was suspended in 40 ml Tris-HCl buffer (10 mmol, ph=8.5) and sonicated for 5 minutes, then 5mg dopamine hydrochloride was added dropwise and left at room temperature for 2 hours. After 5 minutes of sonication, 2 mg of dopamine hydrochloride was slowly added dropwise, stirred at room temperature for 2 hours, and the final product pht@zif-8@pda (PZP) was collected by centrifugation, washed 3 times with methanol and dried at room temperature.
The morphology and shape of the nanoparticles were determined by TEM (JEM-2100) imaging. The composition of the nanoparticles was determined by fourier transform infrared spectroscopy (FT-IR). The nanoparticles were subjected to X-ray diffraction (XRD) analysis using an X-ray diffractometer fitted with a copper sealed tube. The particle size distribution and zeta potential of the nanoparticles were analyzed with Malvern Zetasizer Nano ZS dynamic light scattering analyzer (DLS).
1.3 Construction and characterization of hydrogels
Dissolving OHA in deionized water to prepare 5% OHA solution, and then adding Pht, PZ, PZP parts of OHA solution into the solution respectively and stirring uniformly. Then, an equal amount of 5% CMCS aqueous solution was added to the above mixture to prepare an OHA/CMCS/Pht hydrogel (PG), an OHA/CMCS/Pht@ZIF-8 hydrogel (PZG), an OHA/CMCS/Pht@ZIF-8@PDA hydrogel (PZPG), and the Pht concentrations of the three hydrogels were 290. Mu.g/ml. In addition, OHA/CMCS hydrogel (BG) was also prepared without Pht, PZ, PZP added.
And (3) observing the surface morphology of the section of the freeze-dried metal spraying treated composite hydrogel sample by adopting an ultra-high resolution Scanning Electron Microscope (SEM). The composition of the nanocomposite hydrogel was determined by Fourier transform infrared spectroscopy (FT-IR), scanning wavenumbers of 400-4000 cm -1, and resolution of 2 cm -1. Finally, the present invention obtains the rheological properties of the hydrogel samples by measuring the storage modulus (G') and loss modulus (G ") using an Anton Paar instrument (PHYSICA MCR, germany). When the strain is 1%, the angular frequency functions of G 'and G' are measured under the conditions of 25 ℃ and frequency sweeping of 1-100 rad/s. The time effect was tested under conditions of strain 1%, frequency 1 Hz, temperature 25 ℃, time from 0s to 600 s.
1.4 In vitro drug and Zn 2+ release
1 Ml of the nanocomposite hydrogel was placed in 5 ml of PBS (ph=7.4) and shaken in a constant temperature shaker at 37 ℃ and 100 revolutions per minute, once every predetermined time. The in vitro drug release behavior of hydrogels was studied. Release of Pht was determined using UV-VIS method. The Pht release rate calculates the cumulative release percentage (1) according to the following formula:
（1）
V d is the displacement volume of PBS: 5 ml;
v is the volume of release solution PBS: 5. milliliters;
c i is the concentration of Pht in the released solution (micrograms/milliliter) at the ith exchange of sample;
m s is the mass (micrograms) of Pht in the drug delivery material for release;
n is the number of PBS changes;
the free zinc ions released by the nanoparticles were measured under neutral conditions (PBS, pH 7.4) using the method and formula described above. Finally, a portion of the liquid was taken at various time points for thermal concentration, and then the zinc ions were quantified by ICP-MS.
1.5 In vitro Release and expansion
The in vitro swelling and degradation characteristics of the nanocomposite hydrogels were tested using a weighing method. A weight of hydrogel was first weighed, fully immersed in PBS solution, and then the hydrogel was removed and weighed at selected time points. The swelling and degradation conditions were calculated from the mass ratio of the front and rear hydrogels.
1.6 Photothermal properties of hydrogels
To investigate the photothermal effect of the nanocomposite hydrogels, PZPG min at different concentrations (0, 0.5, 1, 2 mg/mL) were irradiated with a near infrared laser (1.0W/cm 2, 808 nm). The temperature of each sample was then monitored with a thermal near infrared imager and an infrared thermographic image was recorded. The pure H 2 O control group was also measured under the same conditions. To verify the photostability of the formulation, PZPG suspensions were exposed to an on/off cycle of a near infrared laser (1.0W/cm 2, 808 nm) for 10 minutes and then cooled to room temperature. The irradiation cycle was repeated three times. The temperature of the entire heating and cooling process was recorded using a thermal near infrared imager. The photothermal conversion efficiency (. Eta.) is calculated as (2, 3) below:
(2)
I is the power density of the laser: 1W/cm 2;
A 808 is absorbance at 808 nm wavelength: 0.443;
Q diss is the heat obtained by the quartz cell under irradiation, measured independently as 20.6 mW;
；
t max is the maximum temperature under irradiation, T surr is the ambient temperature;
To obtain hs:
(3)
m is the mass of the hydrogel: 1. g;
C p is the specific heat capacity of the measured material: 4.2 J g -1 ℃-1;
；
t is the time of rising and falling before and after irradiation, ts is the slope of the linear regression equation of-lnθ and T;
i is the number of samplings.
1.7 In vitro Activity Studies
1.7.1 Hemolysis test
To assess the compatibility of the hydrogel with blood, the hydrogel was added to whole blood, incubated at 37 ℃ for 60 minutes, and then absorbance was measured at 545nm wavelength. The negative and positive controls consisted of PBS and Triton X-100, respectively, and the rate of hemolysis was calculated as follows (4):
(4)
A s is the absorbance of the test sample;
A n is the absorbance of the negative control;
A p is the absorbance of the positive control;
1.7.2 Antioxidant Activity
To investigate the antioxidant activity of the hydrogels and to evaluate the ability of ABTS to scavenge free radicals, the hydrogel extract was added to 3 milliliters ABTS working solution. The culture was performed in the dark. ABTS solution without hydrogel extract was added as a control. The absorbance of the solution was measured at 734 nm wavelength.
1.7.3 Antibacterial activity
In vitro antibacterial efficacy values of BG, PG, PZG and PZPG on staphylococcus aureus (s. Aureus) (from shanghai yaji biotechnology ltd) and escherichia coli (e. Coli) (from shanghai yaji biotechnology ltd) were tested according to plate counting. Briefly, bacterial suspensions (800. Mu.l, 1X 107 CFU/mL) were co-cultured with each sample in 24 well plates, and then PZPG groups were irradiated with 808 nm near infrared laser (1W/cm 2 min). After a period of co-incubation in shake flasks at 37 ℃, 30 μl of the suspension was incubated on solid agar plates for a period of time.
1.7.4 Cell culture
Mouse osteogenic MC3T3-E1 cells (available from Shanghai Pont Sci Co.) were cultured in an environment of 5% CO 2 at 37℃using alpha-MEM complete medium containing 10% Fetal Bovine Serum (FBS) and 1% penicillin-streptomycin. Cell culture medium was changed every 2-3 days. Cells were subjected to five different treatments, divided into five groups, including untreated (Control) and BG, PG, PZG, PZPG +NIR.
1.7.5 Cell viability
To assess the cell viability and biocompatibility of the hydrogels, colorimetric 3- (4, 5 dimethyl-2-thiazolyl) -2,5 diphenyl tetrazolium bromide (MTT) assays were performed using the EDU cell proliferation assay kit after corresponding treatments.
1.8 Osteogenic differentiation of MC3T3-E1
Mouse osteoblast-like MC3T3-E1 cells were cultured in an osteogenic medium containing 10% FBS, 0.1. Mu.M dexamethasone, 10 mM. Mu.M beta. -glycerophosphate and 50. Mu.g/ml ascorbic acid, the number of cells being 5X 10 4 cells/cm2. After cell fixation, alkaline phosphatase (ALP) staining and detection was performed on day 7 and alizarin red staining was performed on day 21. After 7 days of culture using osteogenic medium, the cells were harvested for western blotting.
1.9 In vivo experiments
1.9.1 Surgical procedure
Animal experiments were performed strictly under the authority approval of the animal laboratory at Jilin university and it was ensured that all experimental procedures strictly followed the guidelines and procedures set by the local animal welfare agency. Throughout the feeding process, a Strict Pathogen Free (SPF) environment is maintained. Female Sprague Dawley (SD) rats (body weight: 200-220 g) were purchased from vinca laboratory animal technologies Co.
Mice were randomly divided into five groups (n=10), including a bone defect group without any treatment (control group), BG group, PG group, PZG group, PZPG +nir group. Before the operation, the mice were anesthetized with 10% chloral hydrate (1 mL kg-1) by intraperitoneal injection, and the operation was performed under aseptic conditions. Two circular trans-periosteal defects (phi=5 mm) were made in the periosteum of the mice with a puncture needle mounted on a dental drill, and then hydrogels were injected into each defect. The incision was carefully sutured layer by layer with 3-0 nylon thread and rats were injected daily with 40 ten thousand IU of penicillin sodium for five consecutive days. Within 8 weeks after implantation, rats were irradiated with near infrared radiation every 7 days for 10 minutes and allowed to eat and drink freely in the cages. All experimental procedures were approved by the institutional animal care and utilization board of Jilin university (accession number 2023-07-19-011).
1.9.2 Micro CT
After 8 weeks of implantation, each group of rats (n=10/group) was sacrificed and intact skull was removed and samples of the skull were subjected to a micro CT (PerkinElmer, quantum GX2, usa) scan. After scanning, the scanned image is reconstructed three-dimensionally using CTVol software (SkyScan). Quantitative analysis of and calculation of new bone formation, including bone volume/tissue volume (BV/TV) and bone mini Liang Shu (Tb.), using DataViewer software (SkyScan) and CT An program (SkyScan)
1.9.3 Histological study
New bone tissue was harvested from the bone defect area at week 8, immersed in 4% paraformaldehyde solution, embedded in paraffin, sectioned, stained with H & E and Masson (zeiss, axio ver A1, germany), and observed for tissue recovery using fluorescence microscopy.
1.9.4 Immunohistochemical staining analysis
After 8 weeks, harvested rat skull was fixed with 4% paraformaldehyde by volume fraction, paraffin embedded, sectioned, and immunohistochemical staining (IHC staining) was performed. To study bone regeneration and remodeling, runt-related transcription factor 2 (Runx 2), osteogenin (OPN), osteocalcin (OCN), alkaline phosphatase (ALP), bone morphogenic protein (BMP 2), and collagen-I (COL I) were immunohistochemically stained at 8 weeks.
1.9.5 Immunoblotting
Total protein is lysed to extract new bone in the bone-deficient tissue. Antibodies to the osteogenic related genes ALP, bone-silicon protein (BSP), OPN, osterix (OSX) and BMP/Smad signaling pathway related proteins (BMP 2, anti-SMAD 1/5/8 (Smad 1/5/8), p-Smad1/5/8, RUNX 2) were incubated with PVDF membrane for 12 hours at 4 ℃. Finally, the signals on the membrane were developed using an enhanced chemiluminescent kit and a Western blot detection system.
1.9.6 Statistical analysis
To ensure the reliability of the experimental results, each experiment was repeated at least three times, and the results were finally displayed with the average value matched with the Standard Deviation (SD). In addition, the invention firstly performs normal distribution test on the collected data, and further applies a unitary analysis of variance (ANOVA) method and subsequent multiple comparison test to strictly judge the significance difference among the groups of data. If p <0.05, the difference is considered significant (< p <0.05, < p <0.01or, < p < 0.01).
2. Results
2.1 Synthesis and characterization of ZIF-8, pht@ZIF-8 and Pht@ZIF-8@PDA
In experiments, pht was encapsulated in ZIF-8 using in situ synthesis to make pht@zif-8 NPs (PZ). Immersing the prepared PZ into alkaline DA, and self-polymerizing the DA to form surface PDA functionalized Pht@ZIF-8@PDA (PZP). The effect of in situ drug loading and PDA coating on ZIF-8 NPs was evaluated. Research shows that the surface structure of the nano medicine carrying material has great influence on the slow release performance, and the slow release characteristic plays a crucial role in the long healing process of bone repair.
The invention is characterized by (transmission electron microscope) TEM. As shown in FIG. 1a, the TEM image shows that loading the Pht in situ does not destroy the morphology of ZIF-8, and a thin gray translucent PDA is wrapped on the PZ surface.
Powder X-ray diffraction (XRD) analysis shows that the spectrum of PZ is similar to that of ZIF-8, the spectrum is not obviously different, the peak positions are the same, and the crystal structure of ZIF-8 nano particles is not influenced by loading Pht. The characteristic peak intensity of ZIF-8 was reduced because PDA was encapsulated at the PZ surface, thereby limiting the detection depth of XRD (fig. 1 b).
DLS results showed that the particle sizes of ZIF-8 and PZ were not greatly different before and after drug delivery, while the particle size of PZP was more uniform, possibly due to its enhanced hydrophilicity (FIGS. 1 c-e). Subsequently, the present invention uses zeta potential assays to study the effect of PDA on Nanoparticle (NPs) surface charge. As shown in fig. 1f, PDA modification resulted in a significant drop in potential from 17.81 mV (PZ) to-2.86 mV (PZP), probably due to the presence of amine groups and phenolic hydroxyl groups in the PDA structure. The negative potential value is favorable for the dispersion of PZP, so that PZP can be uniformly dispersed in hydrogel, near infrared light is effectively absorbed, and heat generated effectively is transferred to different positions of a bone defect part, thereby improving bone reconstruction efficiency.
After coating the PDA, the infrared spectrum (FIG. 1 g) showed a new peak at 1262 cm -1, representing the stretching vibration of the phenolic hydroxyl group (C-O) in the PDA. At the same time, the peak at 3437 cm -1 was enhanced and broadened, a change indicating that the O-H in the PDA had undergone telescopic vibration absorption. These results clearly demonstrate that surface PDA was successfully modified at the surface of PZ NPs, thereby forming PZP NPs. The coverage of the PDA coating does not change the original particle size of PZ to a great extent, and the nanoscale biological materials can simulate the structural characteristics of bone tissues due to the unique microscopic morphology and dimensional characteristics, so that the nanoscale biological materials are very suitable for being applied to the related fields of drug delivery and bone tissue regeneration.
2.2 Preparation and characterization of nanocomposite hydrogels
First, the present invention gives oxidized hyaluronic acid and is verified by fourier transform infrared spectroscopy and 1 H NMR (fig. 2a, b). In the 1 H NMR spectrum, the absorbance peak at 1720, cm -1 was c=o stretching vibration in the OHA aldehyde group, indicating that HA HAs been successfully oxidized, based on the OHA ring-opened D-glucuronic acid proton peaks (4.88, 4.99, 5.09, ppm) and fourier transform infrared spectra. Next, OHA/CMCS hydrogels, pht loaded hydrogels, and nanoparticle hydrogels were prepared using schiff base reaction. By observing the SEM image (fig. 2 c), it was concluded that each set of hydrogels had a porous microstructure. The surface topography of BG group hydrogels showed some Xu Xizhou due to partial collapse of the polymer network during drying. The PG and PZPG groups showed smoother pore walls, with the PZPG group having more uniform network pore sizes. The pore structure within the hydrogel matrix creates channels for efficient diffusion of the drug, which makes the hydrogel a very potential candidate material in drug delivery systems and can give it better and controllable drug release properties. In the angular frequency range of 1-100 rad/s, the storage modulus G' was higher than the loss modulus G″ for each group of samples, indicating that the hydrogel was more elastic (FIG. 2 d). After measurement for 10 minutes at a frequency of 1Hz, the G 'and G' of each group are hardly changed, which indicates that the formed hydrogel network has stable structure and stronger deformation resistance (figure 2 e), thereby protecting the tissues below the skull defect and preventing secondary damage. Compared with PZG groups, the G' of PZPG groups is obviously increased, the mechanical property of the hydrogel is enhanced by adding PZP particles, probably because more coordination bonds are formed between Zn 2+ and amino groups of PDA, and the fact that intermolecular interaction, hydrogen bond formation and chain entanglement among molecular chains can enable the hydrogel to present better elasticity and excellent mechanical property is also indicated.
2.3 Photothermal properties of nanocomposite hydrogels
To explore the photothermal conversion capability of the material, hydrogels (PZPG) of different particle concentrations were placed under 808 nm laser (1W/cm 2) irradiation to record the temperature change of 10 min. It can be seen that the temperature of the hydrogels containing 0, 0.5, 1, 2mg/ml PZP increased to 25.5, 41.1, 52.9, 60.3deg.C (FIGS. 3a, b), respectively. In contrast, the blank hydrogel group (BG) only increased 3.3 ℃, while the hydrogel temperature containing PZP particles at different concentrations increased regularly and moderately, which phenomenon also appears in thermal imaging pictures. Further, from the negative natural logarithm of the cooling time data ln (q), the value of ts (ts= 414.9121) can be obtained, so that the value of the light-heat conversion efficiency (η) is calculated to be about 44.6% (fig. 3c, d), η being a key feature of the photothermal treatment, which directly determines the irradiation light intensity during the treatment. The excellent light-heat conversion efficiency and the low intensity required by the excellent light-heat conversion efficiency are almost free from damage to surrounding healthy tissue cells. The photostability was also measured subsequently, and the pulsed irradiation for 3 cycles (30 minutes total irradiation) did not significantly change the gel state of the hydrogel, indicating good temperature stability (fig. 3 e). In addition, the thermal stability of the hydrogel also ensures that a minimally invasive surgery (implantation of the hydrogel) can provide adequate photothermal therapy in vivo experiments. Therefore, the nano particle hydrogel wrapped by the PDA has strong near infrared light absorption, high photo-thermal conversion efficiency and good photo-thermal stability, can be used for efficient photo-thermal conversion and storage, and is a potential photo-thermal material.
2.4 Physicochemical characterization of hydrogels
The calculated Drug Loading (DLC) of Pht@ZIF-8 was 29.90%. The Pht percentages for three sets of PG, PZG, and PZPG +nir cumulative release are shown in figure 4 a. The Pht release of PZG and PZPG + NIR is controllable relative to the rapid release of PG. Due to the instability of Pht under physiological conditions, the release rate of Pht in PG group reached equilibrium very fast, whereas the release rate of Pht in PZG group in the packet was much slower, which might be related to the stability of ZIF-8 structure in physiological medium. Most importantly, the sustained release duration of the Pht in the PZPG +NIR group is prolonged to 30 days or longer, so that surgical wounds caused by secondary administration are avoided, the stability of the Pht is further improved due to the packaging of the PDA, and the intelligent on-demand release of the Pht is realized due to the synergistic effect of the PDA and the ZIF-8. These results demonstrate that encapsulation of Pht in ZIF-8 by in situ synthesis delays the release rate of Pht and that the use of PDA to encapsulate pht@zif-8 further ensures that Pht is released at the proper rate and concentration throughout the bone remodeling process.
The present invention also investigated the release of PG, PZG and PZPG +nir group Zn 2+ and the results are shown in figure 4 b. Over time, the amount of Zn 2+ released in group 3 increased. The PZPG + NIR group was burst earlier and equilibrated on day four. The amount of Zn 2+ released by PG and PZG groups reached equilibrium on day 7. Zn 2+ also has the effect of promoting bone growth, and is helpful for generating high-quality new bone tissue. In addition, ZIF-8 also exhibits anti-inflammatory activity by slowly releasing the Pht component. When the Pht is loaded into the ZIF-8@PDA, zinc ions and the Pht are released under the triggering of NIR illumination, so that the multiple effects of anti-inflammation, antibacterial and bone repair are synchronously exerted, and a stable microenvironment is constructed for the defect.
The degradation behavior of the hydrogel in the bone regeneration process is also very important, so that not only the medicine should be released in time to treat the defect, but also the hydrogel bracket should be gradually degraded in the bone regeneration period, so that the new bone is not interfered by the hydrogel in the process of generating. As shown in fig. 4c, the degradation performance of the hydrogel was hardly affected after drug loading, and each group could be reduced to about 40% after 30 days. In addition, the test results show that each group has good swelling properties, can absorb tissue exudates and fully fill the defect, and the swelling rate of each group is not significantly changed (fig. 4 d). In conclusion, the hydrogel shows excellent bone scaffold characteristics, can realize stable slow release of Pht, has controllable degradation performance and remarkable swelling capacity, and ensures safety and no toxicity in the use process.
2.5 In vitro Activity
To evaluate the ability of the material to inhibit bacteria under near infrared irradiation at 808nm, two common bacteria were selected as agar plate subjects, namely staphylococcus aureus and escherichia coli (fig. 5 a). The inhibition rates of the BG group, PG group, PZG group and PZPG + NIR group on escherichia coli were 55.79 ±3.24%, 68.99 ±2.37%, 88.04 ±0.40% and 97.53 ±0.57%, respectively, compared to the control group. And the inhibition rate of each group on staphylococcus aureus is consistent with that of escherichia coli. Correspondingly, the quantitative data show that the antibacterial rate of BG, PG, PZG, PZPG +NIR groups is 57.11 + -1.52%, 72.15 + -1.89%, 88.44 + -0.86% and 99.39+ -0.32%, respectively. PG has higher antibacterial rate than BG, and it can be seen that Pht can kill part of thallus, and has certain antibacterial effect (figures 5 a-b). And the bacteriostatic rate of PZG groups is obviously higher than that of BG and PG groups. The ZIF-8 material is composed of a cation Zn 2+ and an organic ligand 2-methylimidazole through bonding. This means that Zn 2+ released in ZIF-8 is likely to interact with enzymes inside the bacteria, thereby effectively blocking the respiratory metabolic pathways of the bacteria. Meanwhile, the organic component (2-methylimidazole) can be combined with bacterial cell walls to further trigger cell membrane rupture, so that bacterial cells cannot be kept alive to die, and electrostatic attraction generated between positive charges carried by ZIF-8 Nano Particles (NPs) and negative charges on the surfaces of bacteria can also enhance antibacterial activity. The PZPG +NIR group has more remarkable antibacterial effect, has remarkable antibacterial efficiency (> 96%) on two bacteria, and improves the antibacterial effect under NIR illumination. Further disclosed is the synergistic bactericidal effect of phloretin, zn 2+ with NIR-induced photothermal radiation. Notably, PZPG +NIR can inhibit staphylococcus aureus by up to 99.39%. Overall, PZPG composite hydrogel systems exhibit excellent antimicrobial properties under moderate photothermal irradiation, and are effective in preventing infection during bone defect repair.
In the bone regeneration process, not only the infection is to be avoided, but also the oxidization problem of the defect part is to be concerned, and free radicals are cleared in time to avoid oxidative stress. Excessive ROS production not only further promotes the process of osteoclast production and enhances its activity, but also inhibits osteogenic differentiation, which in turn ultimately leads to the onset of osteoporosis, manifested by high levels of bone loss and lower bone strength. In the interface region where bone tissue meets the implant, the large amount of ROS generated at the site of the bone defect significantly weakens the osteogenic bioefficacy of the implant, with the end result that bone union is not successfully achieved. In summary, scavenging excess ROS plays a key role in the bone healing process. Thus, the present invention evaluates the antioxidant activity of the material by the ABST test (fig. 5 c). BG. The IC50 values of PG, PZG, and PZPG + NIR for ABTS were cleared at 15.09+ -0.42 μg/ml, 7.69+ -0.23 μg/ml, 8.02+ -0.08 μg/ml, and 7.50+ -0.48 μg/ml, respectively. The study results indicate that these four groups have excellent efficacy against ABTS radicals.
2.6 Cell viability
Good blood compatibility is critical for biological materials. As shown in FIG. 5d, the hemolysis rate of each group of hydrogels was very low, ranging from 0.76% to 0.91%, and the results were in accordance with the clinical approval requirements (not more than 5%) of biomedical materials. It can be seen that the hydrogel has good blood compatibility and is suitable for being used as a biological material.
To examine the effect of hydrogels on MC3T3-E1 cell proliferation, the proliferation potency of each group of cells was examined using MTT and EDU, as can be seen in FIG. 5E, the PZG and PZPG + NIR groups grew faster on day 7. EDU (5-ethynyl-2' -deoxyuridine) positive cells were increased in the PZPG + NIR group compared to the other four groups, with greater proliferation capacity (FIGS. 5 e-f).
2.7 Osteogenic differentiation of MC3T3-E1
Osteoblasts are the primary functional cells in the bone regeneration process. Thus, after demonstrating the cytocompatibility of the hydrogels in vitro, the present invention further tested their osteogenic effect. In order to explore the in vitro osteogenesis of hydrogels, the present invention investigated the osteogenic differentiation capacity of MC3T3-E1 co-cultured with hydrogel extracts by measuring ALP activity, ARS mineralization and protein expression of osteogenesis related genes. First, the activity of early osteogenic marker ALP was evaluated. After induction of osteogenic differentiation and incubation of 14 d, the ALP staining intensity was sequentially increased for the remaining four groups compared to the control group, with the ALP staining intensity being strongest for the PZPG +nir group (fig. 6 a). Bone mineralization is an important process for bone formation. The mineralization ability was reflected by alizing the cells by inducing MC3T3-E1 cells 21 d with osteoblast differentiation, alizarin red staining. The results show that the PZPG +nir group had the most calcium nodules, the strongest mineralization, followed by PZG group. These two staining results essentially confirm that the material has osteogenic differentiation capacity. The present invention then evaluates the ability of the material to induce osteogenesis from the protein level. After stimulation of osteoblasts 7d with the material extract, bottom cells were collected for Westen bolt analysis. As can be seen from FIGS. 6b-f, the remaining four groups all up-regulated osteogenic genes ALP, BSP, OPN and OSX to a different extent than the control group. Alkaline phosphatase (ALP) is an early osteogenic marker of cell maturation and calcification. Consistent with previous staining data, ALP expression in PZPG + NIR group was about 1.5 times that in control group. Bone Silica Protein (BSP) is an extracellular matrix involved in bone metabolism, appears after ALP expression, is located in mineralized matrix, and can promote in vitro osteogenesis by hydroxyapatite mineralization, and increase calcium binding and calcium nodule formation. Four groups showed increased BSP expression compared to the control group, while PZG and PZPG + NIR groups showed significantly increased BSP expression. Osteopontin (OPN) is one of the non-collagenous proteins contained in the bone matrix produced by osteoblasts and osteoclasts, and can effectively stimulate the osteoclastic generation and absorption activities of mature osteoblasts. Osterix (OSX) is an osteoblast-specific transcription factor, critical for osteoblast proliferation, differentiation and bone formation. Regarding the osteogenic genes OPN and OSX, the protein expression levels of BG group, PG group, PZG group and PZPG +nir group were increased as compared to the control group, wherein the protein expression levels of PZG group and PZPG +nir group were increased by about 2-fold. Through researches, the four treatment groups can effectively improve the osteogenic differentiation and calcium deposition capacity of MC3T3-E1 cells and up-regulate osteogenic genes ALP, BSP, OPN, OSX. Of these groups, the PZPG +NIR group exhibited more remarkable improvement.
2.7 In vivo bone remodeling
Inspired by the experimental results in vitro, the invention further researches the healing promotion effect of PZPG hydrogel on critical bone defects in a rat model. Thermal imaging of PZPG + NIR group rats before and after 10 minutes of irradiation (fig. 7 b) shows that near infrared responsive hydrogels can be locally warmed to the appropriate temperature after irradiation without damaging surrounding healthy cells, and thus can exert their therapeutic effect as well. With the continued advancement of imaging technology, bone healing is being evaluated more deeply and accurately. At the same time, micro-CT has become the "gold standard" for assessing bone morphology and bone microstructure. The images show that free Pht in PG is able to properly promote skull healing after 8 weeks of treatment (fig. 7 c). In contrast, the therapeutic effect was more pronounced in PZG groups of rats, as the PZ particles released Pht slowly, thus achieving the therapeutic effect. Of particular interest, rats that received PZPG + NIR radiation exhibited superior efficacy in accelerating in vivo osteogenesis and repair compared to any other treatment, essentially complete healing of 8 week critical bone defects, as a result of intelligent release of Pht, zn 2+ and NIR-based photothermal co-therapy.
In order to confirm the tissue regeneration results under different treatments, the present invention also performed further quantitative analysis of the bone formation microstructure at the defect site. Trabecular bone assessment has been widely used to analyze bone function and strength, bone implant healing, bone diseases (such as osteoporosis), and the like. Trabeculae are porous network structures within cortical bone, with extensions into cancellous bone to provide support to the bone marrow cavity. It is directly related to bone healing and bone strength, and therefore the importance of this structure should be emphasized. An increase in bone volume fraction (BV/TV) indicates that bone anabolism is greater than bone catabolism. In this study, the present analysis quantified bone trabecular morphology and structural index (bone trabecular spacing (tb.sp), bone trabecular thickness (tb.th) and bone trabecular number (tb.n)), as well as bone volume fraction (BV/TV). As shown in fig. 7d-g, the trabecular spacing was lower for BG, PG, PZG, and PZPG + NIR groups, the bone mass was higher, bone size was smaller Liang Zengcu, and new bone formation was more (tb.sp was lower, tb.th was higher, tb.n was more, BV/TV was more) than for the control group. Of note, of the above data, the PZPG +nir group showed the best osteogenic effect, again verifying that intelligent release of Pht, zn 2+ and NIR-based photothermal co-therapy was effective in promoting bone regeneration in a rat bone defect model.
To further investigate the effect of PZPG hydrogels on bone remodeling, the present invention performed H & E and Masson staining analyses on the collected skull samples. As can be seen in fig. 8, the control and BG groups were essentially fibrous tissue, while the BG group showed a small amount of new bone at one end of the defect. In the PG and PZG groups, more new bone was formed at both ends of the defect, and more fibrous tissue was formed in the middle. The PZPG +nir group showed intramembrane bone formation. Both ends of the skull defect have been bridged by newly formed bone tissue, and the defect area has been restored to substantially the original state. The results were again verified by mackerel staining. BG and PG groups are mainly composed of fibrous tissue with small amounts of new bone mass. In contrast, PZG contains a relatively rich new bone tissue, PZPG + NIR group is almost entirely composed of new bone tissue, and these bone tissues almost achieve a comprehensive connection. By contrast, the staining results are consistent with CT detection results, and further prove that PZPG hydrogel has a remarkable promotion effect on bone regeneration.
In order to further investigate the mechanism of action of PZPG hydrogels on bone regeneration in severely bone-deficient rats, the present invention explored the bone repair activity of Pht through BMP/Smad signaling pathways. For ALP, BSP, OPN and OSX proteins, the expression trend of each group was approximately the same as the results of the cell WB analysis. Runx2 is a key upstream transcription factor in the process of osteoblast differentiation. In addition, the key transcription factors such as Runx2 and OSX play an important role in regulating and controlling the osteogenic differentiation process, but if OSX is not present, ossification does not occur. Even without expression of OSX, expression of Runx2 is present in bone forming cells. Thus, OSX acts downstream of Runx2, indicating that Runx2 promotes differentiation of immature osteoblasts in the osteoblast lineage, whereas OSX is necessary for differentiation of mature osteoblasts. It was found that mice lacking the Runx2 gene had impaired osteogenic capacity. This indirectly confirms that early osteoblast differentiation is mainly regulated by Runx2 gene expression. Genetic studies have found that Bmp2 promotes osteoblast differentiation by targeting Runx2 downstream. BMP/Smad signaling pathway is a key signaling pathway for classical regulation of osteogenic differentiation, and its absence can lead to bone-related diseases such as osteoporosis. Binding of extracellular BMP to BMP receptors (BMPR) on the cell membrane results in receptor kinase activation, which phosphorylates Smad1/5/8 in the cell, forming a complex with Smad4, and then enters the nucleus to bind to DNA sequences, regulating transcription of BMP target genes. BMP2, SMAD1/5/8, p-SMAD1/5/8, and Runx2 protein expression were up-regulated in the BG, PG, PZG, PZG, and PZPG + NIR groups compared to the control group, wherein the PZPG + NIR groups expressed 3, 2, 3, and 4 fold higher BMP2, SMAD1/5/8 (BMP 2 downstream BMP/SMAD pathway signature), p-SMAD1/5/8, and Runx2 proteins, respectively, than the control group. From this, it was determined that the hydrogels provided by the present invention can activate BMP/SMAD pathway by up-regulating BMP2, SMAD1/5/8, p-SMAD1/5/8, and Runx2, thereby promoting bone trabecular and cortical bone to combine and regenerate, and achieve bone remodeling.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements within the spirit and scope of the present invention.
Claims (8)
1. The injectable intelligent controlled release hydrogel is characterized in that the hydrogel takes an aqueous solution of oxidized hyaluronic acid and an aqueous solution of carboxymethyl chitosan as matrixes, a hydrogel substrate is prepared through chemical reaction, the hydrogel substrate is internally loaded with Pht@ZIF-8@PDA, and the preparation method of the Pht@ZIF-8@PDA comprises the following steps: firstly, encapsulating the Pht in ZIF-8 nano particles to obtain Pht@ZIF-8 nano particles; then wrapping PDA on the surface of the Pht@ZIF-8 nano particles; wherein, pht is phloretin, ZIF-8 is zinc-based zeolite imidazole skeleton, and PDA is polydopamine.
2. The injectable intelligent controlled release hydrogel according to claim 1, wherein the preparation method of the hydrogel comprises the following steps:
(1) Mixing hyaluronic acid and sodium periodate in water, and performing oxidation reaction to obtain oxidized hyaluronic acid;
(2) Mixing Pht with 2-methylimidazole methanol solution at room temperature, dissolving zinc nitrate hexahydrate in deionized water, adding into the mixed solution containing Pht, stirring, standing, centrifuging to recover suspension, washing, and drying to obtain Pht@ZIF-8 nanoparticles;
(3) Suspending the Pht@ZIF-8 nano particles in a Tris-HCl buffer solution, performing ultrasonic treatment, dropwise adding dopamine hydrochloride, standing for a period of time, continuing ultrasonic treatment, dropwise adding dopamine hydrochloride, stirring, centrifugally collecting, and drying to obtain the Pht@ZIF-8@PDA nano particles;
(4) Dissolving oxidized hyaluronic acid in deionized water, adding Pht@ZIF-8@PDA nanoparticles, and then mixing with carboxymethyl chitosan aqueous solution to prepare the OHA/CMCS/Pht@ZIF-8@PDA, thereby obtaining the injectable intelligent controlled-release hydrogel.
3. The injectable intelligent controlled release hydrogel according to claim 2, wherein the specific steps of step (2) are: 35mg of Pht and 5ml of 0.97M 2-methylimidazole in methanol were mixed at room temperature and stirring continued until dissolved; then 0.1g of zinc nitrate hexahydrate is dissolved in 5mL deionized water and added into the mixed solution containing Pht, after stirring for 80 minutes and standing for 1 hour, the suspension is recovered by centrifugation, washed with methanol solution and dried at room temperature, and pht@zif-8 nano particles are obtained.
4. The injectable intelligent controlled release hydrogel according to claim 2, wherein the specific steps of step (3) are: 8 mg of synthesized Pht@ZIF-8 nanoparticles were suspended in 40 ml of Tris-HCl buffer solution and sonicated for 5 minutes, then 5 mg of dopamine hydrochloride was added dropwise and left at room temperature for 2 hours; after 5 minutes of ultrasound, 2 mg of dopamine hydrochloride is added dropwise, stirred for 2 hours at room temperature, and the final product Pht@ZIF-8@PDA nanoparticles are collected by centrifugation, washed with methanol and dried at room temperature.
5. The injectable intelligent controlled release hydrogel according to claim 2, wherein the specific steps of step (4) are: dissolving hyaluronic acid in deionized water to prepare 5% aqueous solution of oxidized hyaluronic acid; the concentration of the carboxymethyl chitosan aqueous solution is 5%, and the volume ratio of the oxidized hyaluronic acid aqueous solution to the carboxymethyl chitosan aqueous solution is 1:1.
6. Use of an injectable smart controlled release hydrogel according to any one of claims 1 to 5 for the preparation of a bioscaffold for the treatment of bone injuries.
7. Use of the injectable intelligent controlled release hydrogel according to any one of claims 1 to 5 in the preparation of photothermal conversion materials.
8. Use of the injectable intelligent controlled release hydrogel according to any one of claims 1 to 5 in the preparation of BMP/Smad signaling pathway activators to promote bone trabecular and cortical bone regeneration, achieve bone remodeling, and accelerate bone defect repair by activating BMP/Smad signaling pathway.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114848506A (en) * | 2017-06-27 | 2022-08-05 | 强生消费者公司 | Multifunctional personalized patch with therapeutic properties |
| CN114939163A (en) * | 2022-04-06 | 2022-08-26 | 同济大学 | Preparation method and application of composite gel system for multi-mode synergistic tumor treatment |
| CN116807964A (en) * | 2023-03-28 | 2023-09-29 | 安徽省立医院(中国科学技术大学附属第一医院) | Hydrogel spray liquid for preventing traumatic ectopic ossification, preparation method and spraying device |
| CN117122727A (en) * | 2023-08-28 | 2023-11-28 | 浙江大学 | Nanocomposite hydrogel dressing with reversible adhesion-photo-thermal sterilization function and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114939163A (en) * | 2022-04-06 | 2022-08-26 | 同济大学 | Preparation method and application of composite gel system for multi-mode synergistic tumor treatment |
| CN116807964A (en) * | 2023-03-28 | 2023-09-29 | 安徽省立医院(中国科学技术大学附属第一医院) | Hydrogel spray liquid for preventing traumatic ectopic ossification, preparation method and spraying device |
| CN117122727A (en) * | 2023-08-28 | 2023-11-28 | 浙江大学 | Nanocomposite hydrogel dressing with reversible adhesion-photo-thermal sterilization function and preparation method thereof |
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| CN117942427A (en) | 2024-04-30 |
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