CN117547511A - Sugar response liposome, oriented electrospun membrane for bone regeneration in diabetes environment and preparation method thereof - Google Patents
Sugar response liposome, oriented electrospun membrane for bone regeneration in diabetes environment and preparation method thereof Download PDFInfo
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- CN117547511A CN117547511A CN202311338375.4A CN202311338375A CN117547511A CN 117547511 A CN117547511 A CN 117547511A CN 202311338375 A CN202311338375 A CN 202311338375A CN 117547511 A CN117547511 A CN 117547511A
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Abstract
The invention discloses a glucose-responsive liposome, an oriented electrospun membrane for bone regeneration in a diabetes environment and a preparation method thereof, wherein the oriented electrospun membrane comprises an oriented nanofiber scaffold and a self-assembled substance compounded by the oriented nanofiber scaffold, and the self-assembled substance is formed by collagen and a glucose-responsive liposome preparation. The PCLA isotactic electrospun membrane of the embodiment of the application remodels the pathological diabetes microenvironment into a regeneration microenvironment by blocking IRE alpha/NOD-like/NF kappa B signal channels, and simultaneously reprograms the transition of macrophages from an M1 to an M2 phenotype, so as to enhance osteogenic differentiation and angiogenesis. Overall, the findings obtained indicate that PCLA regulates the immune microenvironment and enhances osseointegration of periosteal defects in diabetics. In addition, the present study provides an effective strategy for designing functionalized biomaterials for bone regeneration treatment in diabetics.
Description
Technical Field
The invention belongs to the field of bone regeneration, and particularly relates to a method for preparing a glucose responsive liposome, preparing ordered spinning, self-assembling type I collagen and the like, which comprises the following steps: a liposome material with glucose responsive group, an oriented electrospun membrane for space-time immunoregulation of periosteum regeneration and/or bone blood vessel regeneration in diabetic environment, and its preparation method are provided.
Background
Diabetes (DM) is a long-term metabolic disorder affecting 4.25 million people worldwide in 2017 and estimated to be more than 6.29 million by 2035. In addition to the well known macrovascular and microvascular complications, such as stroke, retinopathy, neuropathy and nephropathy, patients with diabetes mellitus are at a higher risk of fracture than healthy people, which is associated with their glucose metabolism disorder and increased basal inflammation levels in the body, diabetes mellitus can increase the risk of fracture by 20-300%. Typically periosteum contributes to over 70% of bone and cartilage formation in the early stages of fracture healing. However, in DM microenvironment, excessive accumulation of chronic inflammation and advanced glycation end-products and the presence of active oxygen may impair the activation and differentiation of mesenchymal stem cells, which will lead to reduced osteogenic differentiation, reduced angiogenesis, exacerbation of periosteal healing, and eventually bone defect healing. Therefore, the periosteum regeneration strategy of diabetics must be reviewed.
Currently, there are a number of methods available for improving bone healing, including drugs, growth factors, ultrasound, lasers, and pulsed electromagnetic fields; however, bone formation is insufficient and does not allow for accurate and controlled release of growth factors, limiting the application of these existing methods. In addition, bone repair materials can be used for adjuvant treatment, but traditional bone repair materials are mainly focused on optimizing bone formation, and inert materials are usually used for avoiding immune rejection, and the essential functions of the immune system in tissue repair and regeneration are ignored. Neglecting immune regulation, only focusing on osteoinduction, often cannot adapt to pathological microenvironment.
As a major defense in the innate system, macrophages have an anti-inflammatory and therapeutic phenotype (M2), which can alleviate inflammation and provide a beneficial immune microenvironment for bone regeneration. However, DM microenvironment is often accompanied by fluctuations in blood glucose levels, which further extend the local residence time of pro-inflammatory phenotype (M1) macrophages and significantly delay the production of M2 macrophages, further exacerbating inflammation and causing injury during bone repair. Thus, dynamic transition from the M1 to M2 phenotype is difficult to accomplish under chronic inflammatory conditions. In conclusion, the bone repair material capable of dynamically responding to the change of blood sugar level is reasonably designed, has multiple functions of immunoregulation, osteoinduction and the like, has important significance for promoting the bone healing of diabetics, and is expected to provide more effective treatment strategies and improve bone health and functional recovery.
Disclosure of Invention
In view of the above, the invention provides a sugar response liposome, an oriented electrospun membrane for bone regeneration in diabetes environment and a preparation method thereof, and the oriented electrospun membrane provided by the invention is a composite functional bracket material, can regulate immune function in a fluctuating high glucose inflammation microenvironment, realizes a relatively stable and favorable osteogenesis microenvironment, is favorable for effective design of functional biological materials, and is used for bone regeneration treatment of diabetes patients and the like.
The invention provides a sugar response liposome which is prepared from phospholipid substances, cholesterol and fluorobenzene boric acid grafts, wherein the fluorobenzene boric acid grafts are phospholipid p-fluorobenzene boric acid grafts or cholesterol p-fluorobenzene boric acid grafts.
In an embodiment of the present invention, the fluorobenzeneboronic acid graft is one or more of DSPE-PEG-FPBA, DPPE-PEG-FPBA and DOPE-PEG-FPBA, preferably DSPE-PEG-FPBA; the molecular weight of the PEG chain segment in the fluorobenzeneboronic acid graft is preferably 1000-3000.
In the embodiment of the invention, in the raw materials for preparing the sugar-responsive liposome, the mass ratio of the phospholipid substances to the cholesterol to the fluorobenzeneboronic acid grafts is 40:10:1-4.
The present invention provides a sugar-responsive liposome formulation comprising: the sugar-responsive liposomes as hereinbefore described, and their entrapped immunomodulatory agent, preferably APY29. Specifically, the release rate of the immunomodulator and the sugar concentration in the sugar-responsive liposome formulation are positively correlated.
The invention provides an oriented electrospun membrane for bone regeneration in a diabetes environment, which comprises an oriented nanofiber scaffold and a self-assembled substance compounded by the oriented nanofiber scaffold, wherein the self-assembled substance is formed by collagen and the glucose responsive liposome preparation; the collagen is preferably type I collagen.
In an embodiment of the present invention, the material of the oriented nanofiber scaffold is one or more of PLA, PCL and PCLA, preferably PLA.
In an embodiment of the present invention, the average fiber diameter of the oriented nanofiber scaffold is 0.8-1.3 μm, and the oriented electrospun film has hydrophilicity, and the water contact angle is preferably 32 ° -100 °, more preferably 35-80 °.
The invention provides a preparation method of an oriented electrospun membrane for bone regeneration in a diabetes environment, which comprises the following steps:
s1, preparing nanofibers with different orientations through an electrostatic spinning process, and collecting to obtain an oriented nanofiber membrane; preparing the glucose responsive liposome preparation by reverse evaporation;
s2, mixing the sugar response liposome preparation with a collagen solution, and then placing the mixture on the oriented nanofiber membrane, and obtaining the oriented electrospun membrane through self-assembly.
In the embodiment of the present invention, in step S2, the collagen solution is neutral, and the solute is type I collagen; the self-assembly temperature is 35-38 ℃ and the self-assembly time is more than 30 minutes.
Referring to fig. 1 and 2, in order to solve the key problems of adverse effects of chronic inflammation microenvironment on periosteum repair and dynamic adjustment of blood sugar fluctuation on osteogenesis microenvironment in a diabetes pathological environment, the embodiment of the invention designs and prepares a liposome surface modification material with glucose response groups, collagen self-assembly and electrostatic spinning to gradually construct a bionic microenvironment response fiber repair material. In a first aspect, fig. 1 illustrates a sugar-responsive liposome and preparation thereof, and construction of a time-sequence-controlled immune and osteogenic vascular composite system, which comprises a technical scheme of constructing by using a self-assembly strategy of a type-I collagen and a surface-modified sugar-responsive group of the liposome. In a second aspect, FIG. 2 illustrates functional characterization verification, and an embodiment of the present invention verifies the ability of the composite system to modulate immunity and osteogenesis and the therapeutic effect of the system in diabetic rat periosteal defects.
The pathological diabetic microenvironment consists of glucose fluctuations and chronic inflammation, resulting in delayed healing of periosteal defects. Therefore, there is a need to develop new materials that facilitate osseointegration and are suitable for use in the complex microenvironment of diabetes. The present application is directed to a novel glucose-reactive fibrous scaffold composite that can modulate the local immune microenvironment and promote osseointegration of the periosteal-implant interface.
Among these, micro/nanoparticle-based glucose responsive drug delivery systems are promising therapeutic drug delivery strategies. For example, liposomes have been given a stimulus response character that can trigger the complete release of encapsulated drug molecules. When the liposome surface membrane becomes porous, the contents of the liposome begin to leak, which in turn causes the membrane tension to further decrease and eventually rupture. In the DM microenvironment, high glucose concentrations may promote endogenous stimulation and alter the structural state of the liposomes. Phenylboronic acid (PBA) is a common glucose responsive component that has good structural stability and flexibility in addition to being easy to manufacture at low cost. In addition, PBA can combine with hydroxyl groups in the 1,2 or 1,3 positions of glucose to form a dynamic borate structure. This reaction results in a negatively charged PBA molecule. This negative charge imparts further chemical properties and reactivity to the PBA, giving it the potential to modify positively charged liposomes. However, the stability of borates formed by this modification method is poor due to the wide variety of boric acid structures formed at different acidity, with PBA having pKa ranging from 7.8 to 8.6. More specifically, in a physiological environment (pH 7.4< pka, neutral environment of the human body), PBA is converted to an uncharged planar structure, is difficult to react with hydroxyl groups, and is less likely to bind to glucose. This results in the glucose borates becoming unstable and readily hydrolyse, thereby causing low glucose reactivity.
In contrast, the present invention provides a liposome material with glucose responsive groups modified on the surface, wherein the liposome structure is modified with fluorobenzeneboronic acid grafts (containing-FPBA). In this application electrophilic groups are introduced in the PBA to produce 4-fluorophenylboronic acid (FPBA) with a pKa reduced to 7.2. Using this strategy, the negative charge created by the binding of glucose to FPBA can cause the FPBA to change from hydrophobic to hydrophilic. Thus, glucose should be bound to the FPBA unit on the liposome membrane, which will lead to structural transformation and swelling of the membrane. Eventually, the liposomes should release the loaded drug or other factors.
In the embodiment of the application, the substance of phospholipid or cholesterol grafted FPBA (namely phospholipid para-fluorobenzene boric acid graft or cholesterol para-fluorobenzene boric acid graft) is directly used for preparing the sugar-responsive liposome with liposome preparation raw materials, so that the FPBA group is used as a modification group to modify the liposome. Further, the application selects FPBA and phosphatidylethanolamine-polyethylene glycol to be combined into PE-PEG-FPBA as a modification group for modifying liposome; the modified liposome has the advantages that the modification technology is mature, the modified liposome material is stable, and the liposome loading of a pharmaceutical preparation and the like can not be influenced.
Liposomes (lipomes) are an artificial membrane; the hydrophilic head of the phospholipid molecule is inserted into water, the hydrophobic tail of the liposome extends to the air, and the spherical liposome of the bilayer lipid molecule is formed after stirring. Pharmacy definition of liposomes: refers to a miniature vesicle formed by encapsulating a drug within a lipid bilayer. The liposome preparation raw materials comprise phospholipid substances and cholesterol; cholesterol can strengthen the lipid bilayer membrane, reduce membrane flow, and reduce leakage rate. Wherein the phospholipid substance comprises natural phospholipid and synthetic phospholipid, natural phospholipid is mainly lecithin (phosphatidylcholine, PC), and is neutral, and synthetic phospholipid such as DSPE (distearoyl phosphatidylethanolamine), DPPE (dipalmitoyl phosphatidylethanolamine), DOPE (dioleoyl phosphatidylethanolamine), etc.
The examples herein employ phospholipids, cholesterol and fluorobenzeneboronic acid grafts (e.g., DSPE-PEG-FPBA), and optionally an immunomodulator or other agents, preferably in the form of glucose-responsive liposomes or pharmaceutical formulations thereof, prepared by reverse evaporation. More specifically, lecithin, cholesterol and DSPE-PEG2K-FPBA (commercially available) were dissolved in chloroform in a certain ratio, and the resulting solution was then mixed with trichloroethane containing octadecylamine (which may also contain a specific immunomodulator APY29, commercially available). The obtained solution is further mixed by ultrasonic to obtain uniform emulsion. Removing the organic solvent by using a rotary evaporator to obtain a colloid product; finally, hydration treatment is carried out to prepare liposome emulsion; the liposome emulsion was sonicated using 450nm and 220nm polycarbonate membranes and filtered to obtain the corresponding glucose-responsive liposome material. The liposome structure is required to have uniform particle size and be prepared into liposome with a single-chamber structure; liposomes of a multi-compartment structure may lead to decreased liposome responsiveness, which may affect therapeutic efficacy.
Wherein, the DSPE-PEG2K-FPBA can also be replaced by DPPE-PEG-FPBA and DOPE-PEG-FPBA; the molecular weight of the PEG chain segment in the fluorobenzeneboronic acid graft is preferably 1000-3000, more preferably 2000. The mass ratio of the phospholipid substance to the cholesterol to the fluorobenzeneboronic acid graft is preferably 40:10:1-4; blank liposome materials can be prepared from lecithin, cholesterol, octadecylamine and DSPE-PEG2K-FPBA, and if drugs such as immunomodulator APY29 or other factors are added, a sugar-responsive liposome preparation is formed.
In the embodiment of the application, the immunoregulator APY29 is filled in the membrane of the sugar response liposome preparation, the whole outside is in a nanosphere shape, and the surface is modified with an FPBA grafted structure; APY29 is a high-efficiency and selective IRE1 alpha small molecule regulator with the following structural formula.
In the examples of the present application, it is generally required that the drug encapsulation efficiency of the liposome is 80% or more. The release rate of the immunoregulator in the sugar response liposome preparation is positively correlated with the sugar concentration, the drug release amount can reach 80% in 72 hours under the condition of high sugar concentration, and the simultaneous low sugar content is only 20-30%.
Electrospinning is widely used to replicate the physiological microenvironment that directs cell fate and regulates tissue regeneration due to its similarity to the natural extracellular matrix (ECM). Electrospinning is considered a good candidate for periosteal regeneration, as functional electrospinning can autonomously respond to external stimuli and actively fine tune the delivery of therapeutic compounds. Regulating local immune microenvironment and in situ osteoblast differentiation of mesenchymal stem cells is critical for diabetic bone defects. The application contemplates loading liposomes with specific immunomodulators (APY 29) and self-assembly in combination with collagen type I (COLI). In order to achieve the aim, liposome surface modification with glucose response groups, collagen self-assembly and electrostatic spinning are adopted to gradually construct a bionic microenvironment response fiber composite system.
The embodiment of the application provides an oriented electrospun membrane for the time immunoregulation of periosteum regeneration and bone blood vessel regeneration in a diabetes environment, and the novel fibrous membrane comprises: an oriented nanofiber scaffold and its complex self-assembled substance formed from type I collagen and the previously described glycoresponsive liposome formulation. The liposome modified FPBA group on the surface of the spinning membrane is expected to respond to a high glucose environment, so that the hydrophilicity of the liposome membrane is changed, the outer membrane swelling is increased, and the release of encapsulated APY29 is caused by the destruction of a lipid bilayer; subsequently, released APY29 polarizes macrophages towards the M2 phenotype and induces secretion of osteogenesis and angiogenesis-related factors. The ordered electrospun scaffolds described herein will have a variety of functions, such as glucose response, bone immunomodulation, osteoinduction and angiogenesis.
According to the embodiment of the application, the oriented nanofiber membrane is used as a fiber bracket material, nanofibers with different orientations can be prepared through an electrostatic spinning process, and the oriented nanofiber membrane is obtained through collection. The spinning film is used as a platform and a carrier for cell growth, and particularly, the difficulty is that ordered spinning or unordered spinning is selected. Under the normal condition, the spinning membrane defaults to disordered spinning, but the disordered spinning has weak mechanical property as a cell carrier, the intervention on the growth morphology of cells is weak, and the cells are not easy to grow on the surface of the spinning membrane. Therefore, the ordered spinning with better mechanical property is selected, and the ordered spinning membrane can influence cell morphology, such as guiding the conversion of macrophages to long spindle-shaped M1 morphology, which is more beneficial to the growth of cells and the phenotype conversion of macrophages.
Specifically, in the embodiment of the invention, spinning polymers such as PLA and the like are dissolved in Dichloromethane (DCM) and N, N-Dimethylformamide (DMF); the resulting electrospinning solution was inserted into a 10 cm long, 0.9 mm diameter syringe using conventional electrospinning equipment for preparing nanofiber scaffold membrane materials of different orientations. Preferably, the speed of the propelling pump of the electrostatic spinning is 60-80 mu L.min -1 At 15-20kV, the distance between the needle tip and the parallel electrode receiver (Parallel Electrode Receiver) is 10-20cm, and parallel oriented nanofiber membrane scaffolds can be collected between electrode rods.
The polymer substance for preparing the electrospun membrane is polylactic acid (PLA-COOH) with carboxyl, and belongs to special polymer materials. The reason for choosing PLA-COOH was to increase the force between the spinning membrane and the collagen; the surface of the common PLA has no electron-withdrawing groups such as O, N, while the PLA-COOH has definite carboxyl, and the O group has stronger electron-withdrawing performance, so that the O group can be mutually attracted with H on the surface of the collagen type I to strengthen acting force between the two. The oriented nanofiber scaffold according to the embodiment of the invention is made of one or more of polylactic acid (PLA), polycaprolactone and caprolactone/lactide copolymer, preferably PLA. Polylactic acid is also called as polylactide, is polyester polymerized by taking lactic acid as a raw material, and is a non-toxic and non-irritating synthetic high polymer material. Polycaprolactone is a polymer formed by ring-opening polymerization of caprolactone monomers, and has good biocompatibility and the like. Illustratively, the Mw of the PLA can be 109-150kDa; the average fiber diameter of the prepared oriented nanofiber scaffold is preferably 0.8-1.3 μm. In the scheme, the fiber diameter of the spinning membrane is about 1 mu m, and compared with the common fiber membrane, the fiber membrane is coarser, and aims to improve the mechanical property of the spinning membrane, and the fiber membrane can be better mechanically supported after being implanted into an animal model body in the later period; the thickness of the spinning film is proper, and the curative effect is reduced when the thickness is too thick and too thin.
The embodiment of the invention carries out construction of the functional film: and mixing the sugar response liposome preparation with a collagen solution, and then placing the mixture on the oriented nanofiber membrane to obtain the oriented electrospun membrane through self-assembly. The embodiment of the invention mainly utilizes self-assembled hydrogel to load liposome preparation onto the surface of an electrostatic spinning membrane, and compared with the method for destroying the liposome structure by high-voltage electric injection, emulsion preparation and the like, the method has relatively simple technological conditions, and the structure and stability of the liposome can not be destroyed during the preparation.
The invention also includes sterilizing the oriented nanofiber membrane, such as with 75% ethanol, followed by rinsing with water (typically deionized water) to remove any residual ethanol, prior to functionalizing the membrane scaffold with the COLI and the prepared liposome formulation. Embodiments of the present invention may dissolve lyophilized Collagen type one (Collagen I) in aqueous acetic acid and store at 4 ℃ until further use is desired. Diluted collagen one solution was incubated in ice with PBS (10×) at 6:1 and the diluted solution pH was adjusted to 7.0 with NaOH (0.1M) in water. Then, the liposome preparation prepared above is added into the neutralized collagen solution, fully stirred and placed on an electrostatic spinning membrane. Finally, the sample can be placed in an incubator at 35-38 ℃ for more than 30 minutes for self-assembly, and then the sample is obtained after washing. The type I collagen is mainly distributed in tissues such as skin, tendons and the like, is protein with the highest content of waste (skin, bone and scale) in aquatic product processing, accounts for about 80-90% of the total collagen content, and is most widely applied in medicine. The type I collagen is in a rod shape and consists of three peptide chains, and amino groups in the molecule can be combined with hydroxyl groups in liposome molecules; the rat collagen type I of Sigma used in the examples of the present invention may be 300kD, but is not limited thereto, for example, 200kD to 350kD, etc. According to the embodiment of the invention, the liposome preparation is mixed with the type I collagen and then self-assembled, and the formed hydrogel state meets the rheological property and other requirements of modifying the liposome to the surface of the electrostatic spinning membrane to a certain extent. If the liquid collagen alone is dispersed around after being implanted into a body, the liquid collagen cannot be used for long-acting on the surface of bone defects, and the bone repair effect is affected. The oriented electrospun membrane provided by the embodiment of the invention has certain hydrophilicity, and the compounded self-assembled substance is uniformly distributed on the surface of the membrane.
In some embodiments of the invention, the mass to volume ratio of the liposome to collagen type one can be 15.6:100 (15.6. Mu.g of the liposome was dissolved in 100. Mu.l of a collagen-type solution) and self-assembled efficiently; the loading ratio to the oriented fiber film was 15.6:100 (w/v), at 15-16: 100 Between the (w/v) ranges, the added liposomes will slightly impair the assembly of collagen type one itself, but the overall effect is not great.
Through the research of the system for treating the periosteum defect of the diabetic rat shown in fig. 2, the PCLA isotactic electrospun membrane of the embodiment of the application can remodel the pathological diabetes microenvironment into a regeneration microenvironment by blocking IRE alpha/NOD-like/NF kappa B signal channels, and simultaneously reprogram the transition of macrophages from an M1 phenotype to an M2 phenotype, so as to enhance osteogenic differentiation and angiogenesis. Overall, the findings obtained indicate that PCLA regulates the immune microenvironment and enhances osseointegration of periosteal defects in diabetics. In addition, the present study provides an effective strategy for designing functionalized biomaterials for bone regeneration treatment in diabetics.
Drawings
FIG. 1 is a schematic illustration of a flow chart for preparing a sugar-responsive liposome and an oriented electrospun membrane according to some embodiments of the present invention;
FIG. 2 is a schematic representation of functional characterization of oriented electrospun films prepared according to some embodiments of the present invention;
FIG. 3 is an SEM and fiber diameter characterization of PLA and PC group composites of embodiments of the invention;
FIG. 4 is a TEM characterization of liposome materials in an embodiment of the present invention;
FIG. 5 is a PDI characterization of liposome materials in an embodiment of the present invention;
FIG. 6 is an SEM characterization of PCL group composites of an embodiment of the invention;
FIG. 7 is an XPS characterization of various sets of composites of embodiments of the invention;
FIG. 8 is an AFM characterization of various sets of composites of embodiments of the invention;
FIG. 9 is a graph depicting the adhesion capability, elastic modulus, water contact angle, and drug release rate of various groups of composites in accordance with embodiments of the present invention;
FIG. 10 shows cell compatibility of each experimental group in accordance with an embodiment of the present invention;
FIG. 11 is a graph showing the modulation of macrophage polarization phenotype in vitro for each experimental group in accordance with an embodiment of the present invention;
FIG. 12 is a graph showing that immunomodulation enhances in vitro osteogenic and angiogenic differentiation for each experimental group in accordance with an embodiment of the invention;
FIG. 13 is an in vivo modulation of bone remodeling by PCLA-oriented electrospun membranes in an embodiment of the present invention.
Detailed Description
The technical disclosure of the present invention will be clearly and fully described in connection with the following embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The invention constructs a composite material system for dynamically regulating and controlling immune microenvironment in diabetes mellitus environment, time-sequence osteogenesis and vascularization based on sugar response liposome, electrostatic spinning and the like, and performs characterization verification of functions and the like.
Examples
1. Materials and methods
1. Material
1.1 main medicines and reagents: PLA (mw=109-150 kda, iv=4.0-5.0) was obtained from the company of bioengineering, inc. Rat tail collagen one was purchased from Sigma Aldrich (united states), 300kD. Dulbecco's modified medium (DMEM), fetal Bovine Serum (FBS), trypsin and penicillin/streptomycin were purchased from Gibco (USA).
1.2 main instruments: spin-steaming (Qiu Zuo technology, china), high voltage electricity (wisdom electrospinning, china), push pumps (wisdom electrospinning, china), micro-CT (SkyScan 1176, belgium), paraffin microtomes (Leica 2135, germany), flaker (Leica 1120, germany), paraffin embedding machine (BMJ-ii, china, everstate), axiovert 40C optical microscope (Zeiss, germany), surgical suite, etc.
1.3 experimental animals: healthy SD rats, 40, male, weighing 300+ -20 g,12 weeks old, cleaning grade, were offered by the university of Suzhou animal experiment center. The feeding conditions were as follows: the room temperature is 18-20 ℃, the humidity is 50-60%, ventilation is good, and water is fed by free ingestion.
2. Experimental method
2.1 preparation of oriented nanofiber scaffolds: PLA (0.5 g) was dissolved in a solution of dichloromethane (4.0 g, DCM, national pharmaceutical agents Co., ltd., beijing, china) and N, N, DMF (2.0 g Shanghai Lingfeng, shanghai, china). The electrospinning solution was inserted into a syringe having a length of 10 cm and a diameter of 0.9 mm, and used to prepare fiber scaffolds of different orientations. The speed of the propelling pump of the electrostatic spinning is 70 mu L min -1 At 18kV the distance between the tip and the parallel electrode receiver was 15cm. Parallel oriented fibrous scaffolds were collected between electrode rods.
2.2 preparation of glucose-responsive liposomes: glucose-responsive liposomes were prepared by reverse evaporation. More specifically, lecithin (160 mg, shanghai Yuan Zhi, china), cholesterol (40 mg, belgium Alcos) and DSPE-PEG2K-FPBA (4 mg, shanxi Rui xi, china) were dissolved in chloroform (5 ml, shanghai Lingfeng, shanghai, china). The resulting solution was then mixed with trichloroethane (1 ml) containing octadecylamine (5 mg, shanghai aladine chemical Co., ltd., china) and APY29 (4 mg, shanghai MCE, china). The obtained solution is further mixed by ultrasonic to obtain uniform emulsion. Removing the organic solvent by using a rotary evaporator to obtain a colloid product, and finally, carrying out hydration treatment to prepare liposome emulsion; the sugar-responsive liposome preparation was obtained by using 450nm and 220nm polycarbonate membranes (Millex-GP, irish), sonication and filtration. Blank liposomes were prepared from lecithin, cholesterol, octadecylamine and DSPE-PEG2K-FPBA (40:10:5:4, w/w/w/w). All liposomes were stored after lyophilization.
2.3 construction of functional films: before functionalizing the pure PLA film with COLI and the prepared liposomes, the film was first sterilized with 75% ethanol for 30 minutes, then rinsed three times with deionized water to remove any residual ethanol. The lyophilized collagen type I was dissolved in an aqueous acetic acid solution (collagen solution concentration 0.1M,3mg ml) -1 ) And stored at 4 ℃ until further use is required. Diluted collagen one solution was incubated in ice with PBS (10×) at 6:1 and the diluted solution pH was adjusted to 7.0 with NaOH (0.1M) in water. The prepared liposome (15.6. Mu.g) was then added to the neutralized collagen solution (100. Mu.L), stirred well and placed on an oriented electrospun membrane. Finally, the samples were placed in an incubator at 37 ℃ for 30 minutes for self-assembly, and the self-assembled samples were washed three times with deionized water to obtain composite PCLA, PCL, and PC films, respectively, which were immediately used for further material characterization or cell experiments.
The pure PLA film is a pure polylactic acid spinning group, PC is short for PLA/Collagen I, and pure Collagen is assembled on the surface of the polylactic acid spinning film; PCL is PLA/Collagen I/liponame, i.e. blank liposomes are added on the basis of PC; these three groups were used as control groups, and PCLA was PCL, and the liposomes were loaded with the drug APY29, and the test groups were designated. In addition, the preparation processes are simple, convenient and smooth to operate.
2.4 characterization of materials and results:
SEM was performed using a Hitachi S-4800 scanning electron microscope (Japan). Average fiber diameter of 200 random fibers was measured with ImageJ. The orientation of the fibrous scaffold was determined using the orientation insert of ImageJ, while the structure of the sample was imaged using a confocal microscope (Axio image M1, zeiss, germany). FTIR spectra were obtained on a Frontier TM Fourier transform infrared spectrometer (perkin elmer). Rheological testing and water contact angle measurements were performed using a HAAKE RheoStress 6000 instrument (american thermal science company) and a contact angle meter (data physical company). The mechanical properties and nanofiber surface elements were studied using the universal kinetic test system (Shanghai Heng Yi, china) and XPS system (250 Xi, american science Etskara). The topology of the fiber surface was observed using AFM (Dimension ICON, bruke, usa), while the surface morphology of the liposomes was observed using TEM (HT 7700, japan). Particle size, PDI and electromotive potential of each mixture were measured using a dynamic light scattering particle size analyzer (Nano-ZS 90Zeta sizer, malva, uk). To measure the release of APY29 in liposomes at different glucose concentrations (25, 5.6 and 0 mM), the liposomes were immersed in 50mL centrifuge tubes containing the corresponding glucose solutions (10 mL) for 3, 6, 9, 12, 15, 18, 21 and 24 hours. The extent of release of APY29 was determined using HPLC (HPLC, agiline system using Kromasil 100-5C18 chromatography column) and the corresponding cumulative release curve was plotted.
The ordered spinning membrane was constructed by electrospinning techniques using poly (DL-lactide) COOH solution on a parallel electrode receiving device. More specifically, liposomes were mixed with DSPE-PEG2K-FPBA and APY29 to produce glucose-responsive drug-carrying nanospheres, and then self-assembled after mixing with COLI on aligned spun nanofibers. The composite material has uniform distribution, stable plasticity and mechanical property and reactivity to glucose.
Scanning Electron Microscope (SEM) imaging confirmed the regular, directional and smooth fiber distribution in the PLA group collected using parallel electrode receivers (fig. 3b 1). After the PLA nanofiber membrane was assembled with COLI, the membrane collagen was tightly bound by electrospinning and uniformly laid on the spun film to produce a micro/nanofiber layered structure (fig. 3c 1). Quantitative analysis showed that the average spinning diameters of PLA and PC oriented electrospun films were 1.04±0.23 μm and 1.09±0.20 μm, respectively, with a regular distribution (fig. 3b2 and c 2).
Liposomes (blank liposomes) were dissolved in Phosphate Buffered Saline (PBS), hypoglycemic (LG) medium and Hyperglycemic (HG) medium for 2 hours to verify their glucose reactivity. For the PBS group, transmission Electron Microscopy (TEM) imaging showed typical phospholipid bilayer membrane structures in most liposomes. However, in LG and HG groups, the bilayer structure of some liposomes is broken, increasing in diameter; this trend is more pronounced in the HG group (fig. 4). To verify the change in liposome core-shell structure at different glucose concentrations, the size dispersion of the liposome microgel particles was assessed using dynamic light scattering. The polydispersity index (PDI) is used to describe the degree of heterogeneity of a particle size distribution, with relatively small PDI values meaning relatively uniform particle dispersion. It was found experimentally that upon increasing the glucose concentration in the solution, the liposome particle size increased while the surface charge and PDI decreased (fig. 5 e-g). More specifically, the average diameter and PDI of the HG group microsol particles were determined to be 544.42.+ -. 24.90nm and 0.64.+ -. 0.071, respectively; however, the PDI was found to be low, indicating a uniform dispersion of particle size. The average diameters of the LG group and PBS group microsol particles were 341.52.00 + -19.95 nm and 266.42 + -21.89 nm, respectively, while the corresponding PDIs were 0.84+ -0.065 and 0.81+ -0.038, respectively. However, the particle distribution was not as uniform as the HG group. The data indicate that liposomes without structural bilayer disruption are more stable and more uniform in size in the HG group. The decrease in surface charge in the HG group may be due to changes in FPBA structure and negative charge resulting from binding of FPBA to glucose in high glucose solutions. Since the immunomodulatory ability of electrospun fibers is largely dependent on their film-like surface structure, TEM was used to study the assembly and structure of the spun-surface films. Compared with the PC group, the liposome is uniformly distributed among the spinning of the PCL group, which proves that the liposome transplantation is successful, and the liposome and the collagen are tightly combined (figure 6).
X-ray photoelectron spectroscopy (XPS) was used to determine changes in the surface chemistry of fibrous scaffolds and to verify the construction of collagen type one and liposomes. The results indicate that PLA group elements are mainly C and O, whereas no signal is observed for group B elements. Further, as shown in fig. 7a, the C1s signal corresponds to polylactic acid. For the PC group, the presence of C-P, C-N, N-c=o and other components was confirmed in the C1s spectrum, thus confirming the successful introduction of COLI (fig. 7 b). For the PCL group, C, O, N and H were present, and signals were also observed in the B1s spectrum. These results indicate that the introduction of liposome/FPBA was successful, as further demonstrated by the presence of a signal corresponding to the C-B bond in the C1s spectrum (fig. 7C). The PCLA group contained more N-c=n bonds than the PCL group, demonstrating the presence of APY29 (fig. 7 d).
To further investigate the binding pattern between fibrous scaffolds, collagen and liposomes, four chemical bonds of electrospun fibers (PLA, PC, PCL and PCLA) were identified using Fourier Transform Infrared (FTIR) spectroscopy. At 1751cm -1 The characteristic absorption peak observed for PLA corresponds to the typical c=o stretching vibration of PLA. In contrast to the spectrum observed for the electrospun sample, the COLI-bound composite product was at 3333cm -1 A weak broad peak appears, mainly due to the hydrogen bonding between electrospun fibres and collagen. After liposome binding, 3287cm -1 The shift in the-OH absorption peak at the site confirms that the addition of the drug molecule alters the hydrogen bonding forces present in the polymerization system. C-H absorption peak at 2944cm due to weak hydrogen bond interaction formed between drug related group and liposome/electrospun collagen -1 This was confirmed by the significant change in the position. Thus, the disappearance of several characteristic peaks and the appearance of new characteristic peaks confirm that the combination of electrospinning with collagen/drug-loaded liposome complexes was successful.
It should be noted that the formation of such complexes is primarily dependent on hydrogen bonding, particularly strong hydrogen bonding between the collagen H-N groups and the O-C portion of the electrospun fiber. The hierarchical micro/nanofibrous structures made with collagen and electrospun fibres are likely to guide the behaviour of bone marrow mesenchymal stem cells (BMSCs), including their adhesion, proliferation and further differentiation, which are necessary for inducing intra-ossification. To further investigate this, atomic Force Microscopy (AFM) was used to examine the change in surface roughness values (Ra, roughness) of the four groups of materials (fig. 8).
The obtained AFM phase diagram shows that annular protruding liposome exists on the surface of PCLA membrane, which indicates that the consolidation of the liposome on the surface of fiber is stable. The adhesion capacity of the 4 groups of materials was measured and the adhesion strength of the PC, PCL and PCLA films was found to be greater than that of the pure PLA films (24.66.+ -. 4.34, 43.82.+ -. 4.33, 49.52.+ -. 3.53 and 52.42.+ -. 3.74nN, FIG. 9g, respectively). This is probably due to the fact that the loading of the COLI hydrogel enhances the tissue adhesion of the material compared to PLA electrospinning. Furthermore, the enhanced adhesion observed in the presence of liposomes can be attributed to their H-bond interactions with the ColI hydrogel. As an important basis for bone tissue engineering, scaffolds not only provide a growth platform for cells, but also guide the bone remodeling process. Thus, the modulus of elasticity of the prepared test specimen was evaluated. More specifically, PC (36.64.+ -. 1.26N mm) -2 )、PCL(39.49±1.88N mm -2 ) And PCLA (40.31.+ -. 0.81N mm) -2 ) Is higher than PLA (23.41 + -0.81N mm) -2 ) (fig. 9 h), these results are due to self-assembly of COLI and the presence of hydrogen bonds between collagen and electrospun surface, between liposomes and collagen. Subsequently, the water contact angles of PLA, PC, PCL and PCLA fibers were determined to be 122.30 ±4.49, 59.19 ±7.27, 42.65±4.62 and 38.59 ±6.56°, respectively (fig. 9 i). From these values, it is clear that the water contact angle is reduced by about 60 ° after collagen assembly, indicating that the COLI nanofibers have significant hydrophilicity. Thus, the hydrophilicity of PCL and PCLA membranes is slightly higher than that of PC membranes, and the water contact angle of PCL and PCLA fibers is significantly reduced as the hydrophilicity of the liposome surface phospholipids extends into the aqueous phase to enhance hydrophilicity.
The bone microenvironment of diabetes results in a local area forming a high glucose environment. Based on the pathological characteristics, in-vitro simulation is adopted to detect the release of the reactive medicine in the high-sugar environment. More specifically, by electrospinning a membrane and drug-loaded liposomesSoaking in a mixture containing different glucose concentrations (high glucose: 25.0mmol L) -1 The method comprises the steps of carrying out a first treatment on the surface of the Low glucose: 5.6mmol L -1 ) Three groups of liposomes (PBS, LG and HG) were determined for drug (apt 29) release rates at different time points (2, 4, 6, 12, 24, 48 and 72 hours) (fig. 9 j). In all three groups, a gradual increase in the degree of drug release from the liposomes over time was observed. However, in the high sugar group, the release rate of APY29 at 72h reached 86.19±0.68%, which is significantly higher than that of LG group (65.09±1.71%) and PBS group (12.18±0.38%). Thus, the rapid response of these reactive fiber scaffolds to high glucose environments was validated in vitro, providing the basis for further research.
2.5 preparation of cell cultures: RAW264.7 cells were cultured in DMEM (Hyclon) at 37℃and BMSC was cultured in 5% CO 2 In alpha-MEM of (A), each containing 10% FBS (Gibco) and 1% penicillin/streptomycin. Prior to cell culture, the hybrids were sterilized with ethanol under uv light overnight and then washed with sterilized PBS.
2.6 cell viability and proliferation: after 3d incubation, viability of BMSC and RAW264.7 cells on different nanofiber membranes was assessed by staining with live dead cell staining kit (Invitrogen, usa). Stained cells (Axio image M1, zeiss, germany) were observed under a fluorescence microscope and semi-quantitative fluorescence analysis was performed using ImageJ. Cell counting kit-8 (NCMBio, su zhou, china) was used to study the proliferation rate of BMSCs on cell membranes 3 and 7 days after transplantation. More specifically, BMSC (5X 10) 3 Individual cells/well) were implanted into 96-well plates (100 μg per well). The absorbance of each plate was measured with a microplate reader at an optical density of 450nm on days 3 and 7 (Biotek, usa).
2.7 cell adhesion: BMSC (1X 10) 4 Individual cells/wells) were seeded on a cell crawler in the hybrid material extract. After one day of co-incubation in 24-well dishes, cells were fixed with 4% paraformaldehyde on ice for 30 minutes and membranes were perforated with a solution containing 0.1% triton X-100 for 10 minutes (Sigma Aldrich, usa). To avoid nonspecific staining, cells were blocked overnight at 4 ℃ using bovine serum albumin. Cells in the medium were then fixed with 4% paraformaldehyde,and incubated at 4℃with protein antibodies (ab 130007, abcam) at night. Subsequently, cells were washed with PBS containing 0.1% tween to remove any unconjugated primary antibody, and then incubated with fluorescent secondary antibody (ab 150115, abcam) for 1 hour. F-actin was subsequently stained with FITC-phalloidin, while nuclei were stained with 4',6' -diamidino-2-phenylindole (DAPI). Fluorescence images (Axio image M1, zeiss, germany) were obtained under laser excitation using a fluorescence microscope.
2.8 cell morphology: using BMSC (2X 10) 4 Individual cells/well) were cultured for 3d, and paraformaldehyde of 4% paraformaldehyde was fixed on ice for 1 hour. Subsequently, the cells were washed with ethanol at different concentrations (30, 50, 70, 80, 90, 95, 100 and 100%) for 15 minutes. By CO 2 After the critical point dryer dried for about 2 hours, the sample surface was gold-plated for 45 seconds, washed 3 times with PBS, and fixed with paraformaldehyde for 30 minutes to obtain an SEM image.
2.9 in vitro osteogenesis: the osteogenic capacity of the prepared material was evaluated using an alkaline phosphatase assay kit (Beyotime Biotechnology, shanghai, china). More specifically, BMSCs (1X 10) were isolated using mixtures extracted from different populations 4 Individual cells/well) were seeded in 24-well plates. After 7d, cells were fixed with 4% paraformaldehyde. Subsequently, alkaline phosphatase staining solution (300. Mu.L/well) was added to each well without illumination and allowed to stand for 30 minutes. Stained (positive) cells were visible under light microscopy. After 2 days of incubation, the cells were fixed in 4% paraformaldehyde solution for 30 minutes. Subsequently, the cells were cleaned several times with PBS to remove excess dye, and the calcium nodules were observed with an optical microscope. To quantify ARS staining results, 5% perchloric acid was added to each well and uptake was measured at 490 nm.
2.10 immunofluorescence: initially, BMSCs were washed three times with PBS and incubated in cold paraformaldehyde for 15 minutes. BMSCs were then blocked with QuickBlockTM IF blocking solution (Beyotime Biotech) and incubated at 4℃for 1 hour, followed by overnight primary antibody at 4 ℃. After the subsequent two PBS washes, igG H & L antibodies (Alexa Fluor 647; abcam, ab150079; alexacksal 488; abcam, ab 150165) were used for 1 hour. Finally, BMSCs were stained with DAPI and intracellular protein expression was observed by confocal fluorescence microscopy.
2.11 in vitro angiogenesis: the angiogenic capacity of the different material groups was observed using a growth factor reducing agent substrate (matrigel 100. Mu.g/well, conning USA). HUVEC (3.5X10) 4 Individual cells/wells) were inoculated into each well of the mixture. In the presence of 5% CO 2 After incubation at 37 ℃, each plate was observed with an optical microscope after 0,3 and 6 hours. Migration ability was verified with a Transwell plate (corning). Specifically, PLA, PC, PCL and PCLA extracts were placed in the lower chamber while HUVEC suspension (200. Mu.L, 2.5X10) 5 Individual cells ml -1 ) Added to the upper chamber and containing 5% CO 2 Is incubated at 37 ℃. After 16 hours of incubation, the incubator was removed, the upper membrane carefully removed with a cotton swab, and fixed with 4% paraformaldehyde for 20 minutes. Cells were then stained with 0.1% crystal violet solution (Solarbio, beijing, china) and then observed under an optical microscope. Wound healing was assessed to assess angiogenesis and 4 x 10 5 Individual cells/well were seeded in 6-well plates and in the presence of 5% co 2 Is incubated at 37 ℃. When the cell flow reached 90% fluency, a 200 μl pipette tip was used to scratch the cell layer. All images were captured with an inverted microscope after 0, 12 and 24 hours.
2.12 identification of macrophage phenotype:
polarized RAW264.7 cells change at the gene expression level under the influence of reactive fibrillar membranes. Then, primary antibodies iNOS (M1-labeled) and Arg-1 (M2-labeled) containing F-actin were incubated with the cells and stored overnight at 4 ℃. IgG H & L (Alexa Fluor 647, red; acarm; alexagram Safford 488, green; abcam) and DAPI solutions. All images were captured with a fluorescence microscope. On day 7 of co-culture, M1 and M2 macrophages were treated with iNOS (Abcam, ab49999, 1:100) and Arg-1 (Abcam, ab239731, 1:100) and then incubated with goat anti-rabbits (Abcam, ab155079, 1:200) and goat anti-mice (Abcam, ab150113, 1:400). Semi-quantitative analysis was then performed using ImageJ. The different sets of cell suspensions were centrifuged at 300g for 10 min, antibody was added for 30 min, and then further incubated at 4 ℃. For this purpose, the following mouse conjugated antibodies were used: mouse anti-CD 11b-APC (562102,1:20;BD Pharmingen), mouse anti-CD 86-FITC (561961,1:20;BD Pharmingen) and mouse anti-CD 206-PE (sc-58986, 1:50; st. Kruese Biotechnology). Cells were then analyzed using a flow cytometer (merck, usa) and the results were analyzed using FlowJo 7.6. To this end, cells were initially CD11b gated to ensure that only RAW264.7 cells were selected, and then M1 (CD 86) and M2 (CD 206) macrophages were identified using a combination of specific markers. Three samples (n=6) were randomly selected from each group.
2.13RT-PCR: total RNA was isolated from BMSCs according to standard protocols. mRNA concentration and purity were assessed using NanoDrop-2000 (Siemens technology, vol. Of Massachusetts, U.S.A.). The expression was calculated using the 2- ΔΔCq method.
2.14 preparation of conditioned Medium: the combination of conditioned media was collected to induce osteogenesis and angiogenesis. For this, RAW264.7 cells (4×10 5 Cells/well) were cultured in the presence of 5% CO 2 Is placed on a 6-well plate in the atmosphere. After incubation with LPS (100 ng mL) -1 ) Each medium was treated for 12 hours and then the supernatants of the different groups were collected and filtered to remove cell debris in a sterile environment.
2.15 animal model: female Sprague-Dawley rates (average body weight: 200-250 g) purchased from Suzhou university laboratory animal center (ethical approval number: 20220925A 01). Intraperitoneal injection of STZ (35 mg kg) -1 ). Blood was withdrawn from the tail vein and stored weekly for analysis of blood glucose levels,>a value of 16.7 mm was considered a successful model of diabetes. The rats were then anesthetized intraperitoneally with 2% sodium pentobarbital. After complete shaving and sterilization, a longitudinal incision is made in the middle of the surgical field, carefully separating the soft tissue to expose the skull. Periosteum was removed and two bilateral defects of 5 mm diameter were carefully created on the skull using a toothed trephine to simulate poor bone conditions and periosteal defects. The defective area is then covered with a thin film. Penicillin is injected once daily for 3 days.
2.16Micro-CT evaluation: SD rats were euthanized 4 and 8 weeks post-surgery, and skull specimens were collected and fixed in 10% formalin for further characterization. Micro-CT (SkyScan 1176, skyScan, belgium) was initially used to set the regeneration conditions for evaluating defective areas using 65kV,385 mM A, 1mm Al filters, etc. The skull was reconstructed in 3D using the chemicals software. For the calculation of BV/TV, BMD, tb.N and Tb.Sp, a cylindrical space representing the region of interest was designated to evaluate bone and tissue volumes. These values were obtained using CT analyzer software (SkyScan, belgium). 3D reconstructed images were performed using chemicals (version 21.0).
2.17 histological analysis: each skull was removed 2, 4, 8 weeks post-surgery, fixed/decalcified with 10% formic acid for 1 week at room temperature, followed by alcohol gradient dehydration and paraffin retardation. The embedded specimens were cut into 6 μm thick histological sections in the center of the defect area and observed for morphological changes under an optical microscope using conventional H & E and Masson staining (Carl Zeiss). The levels of iNOS, CD31, runx2 and periostin markers were also identified by immunohistochemical staining. More specifically, the sections were dewaxed and gradient hydrated for 30 minutes at 37 ℃ prior to treatment with trypsin. After 30 minutes, diluted horse serum was added to block any nonspecific sites, followed by three PBS purges. Subsequently, the primary antibody was added and incubated overnight at 4 ℃. The sections were then incubated with the corresponding secondary and tertiary antibodies for 30 minutes.
2.18 statistical analysis: data were taken as standard deviation ± average. Statistical analysis (Origin 9.1 or GraphPad Prism 7.0 software) evaluates differences between groups by one-way or two-way analysis of variance using Tukey multiple comparison test, unless otherwise specified. p-values <0.05 were considered statistically significant.
2. Functional test results
(1) In vitro biocompatibility: the in vitro biocompatibility of PLA, PC, PCL and PCLA materials was subsequently assessed by seeding mouse macrophages (RAW 264.7 cells) and BMSCs on the spinning membrane surface. For this purpose, a staining of live/dead cells was initially performed. The number of dead cells in the PC, PCL and PCLA groups was significantly reduced compared to the surface of bare lactic acid spun fibers (fig. 10a and 10 b), as confirmed by quantitative analysis of live/dead cells (fig. 10 c). To further understand proliferation of RAW264.7 cells, a cell count kit-8 method was performed after 3 and 7 days of co-culturing the cells with four fiber groups (fig. 10 d). The results indicate that RAW264.7 cells grown on the surface of PC, PCL and PCLA have good viability, probably due to the synergistic effect of collagen type one and liposomes in promoting cell proliferation.
(2) PCLA-based modulation of immune response effects in simulated DM conditions:
Bone immune cells are essential for bone formation and angiogenesis, especially in the early immune setting. To investigate the ability of PCLA to reprogram macrophages in the diabetic inflammatory microenvironment, the diabetic metabolic inflammatory environment was initially simulated using high glucose media and Lipopolysaccharide (LPS), followed by examination of the phenotypic changes of the media macrophages seeded on the PCLA surface (fig. 11 a). In the PLA, PC and PCL groups, macrophages were mainly in pancake-like morphology, whereas in the PCLA group, macrophages were elongated (fig. 11 b). In addition, in the PCLA surface of the elongated cells, M2 polarization-related proteins fluorescence intensity significantly increased, indicating macrophage from pro-inflammatory M1 state polarization to anti-inflammatory M2 state. As shown in FIGS. 11c and 11d, M2 macrophages (Arg-1+, green) were significantly increased in the PCLA group compared to the other three groups. In contrast, M1 macrophages (inos+, green) predominate in the PLA group. It is worth mentioning that Arg-1 was slightly up-regulated in the PC group and PCL group, whereas iNOS was slightly down-regulated in comparison with the PLA group. Flow cytometry was used to further explore the M1 and M2 phenotype switches at the cellular level (fig. 11e and 11 f). The results showed that the CD11b+/CD206 ratio of the APY29 loaded scaffolds was significantly higher than that of the APY29 free scaffolds, and the corresponding ratios of PC, PCL and PLA groups increased significantly after COLI addition, 161% and 211% higher than that of the PLA group, respectively. Likewise, the expression of CD11b+/CD86 was significantly lower in the PCLA group than in the PLA, PC and PCL groups. These significant differences suggest that PCLA is able to manipulate macrophage behavior and strongly influence the immune environment. Next, we detected a number of inflammatory and anti-inflammatory factors. The results show that the expression of pro-inflammatory factors such as interleukin-1 beta (IL-1 beta) in the PLA group is higher, while the expression of anti-inflammatory factor IL-10 in the PCLA group is higher. Thus, PCLA appears to significantly inhibit the secretion of pro-inflammatory cytokines and promote the expression of anti-inflammatory cytokines. Furthermore, the key role of the osteogenesis-related genes in regulating osteogenesis and angiogenesis is well elucidated, and the results indicate that the expression of osteogenesis-and angiogenesis-related markers (BMP-2 and VEGF-Sub>A) on pclSub>A is significantly enhanced (fig. 11 g).
In summary, PCLA down regulates the inflammatory response, up regulates anti-inflammatory factors, activates macrophages to convert to the M2 phenotype, and releases a large number of osteogenic/angiogenic mediators; thus, it can be considered as an effective and beneficial bone immunomodulating material.
(3) Bone immunomodulation of electrospun scaffolds BMSC and HUVEC under DM conditions was simulated:
periosteal regulation is the manipulation of immune cells by biological materials to create a beneficial bone immune environment, thereby regulating the formation of new bone. Following early immune responses by macrophages, HUVEC and BMSC cells are recruited to the implant surface, while EC and BMSC function is largely affected by the immune microenvironment regulated by macrophages. To investigate the effect of immunomodulation on angiogenesis and osteogenesis, RAW264.7 cells pre-treated with PL, PC, PCL and PCLA were stimulated with LPS and collected in Conditioned Medium (CM) (fig. 12 a). Then, bone immunoregulatory function of PCLA was assessed by collecting macrophage CM for culturing BMSC and HUVEC. ALP staining and ALP activity clearly showed that PC, PCL and PCLA groups were significantly higher than the PLA group (FIGS. 12b and 12 c). Furthermore, ARS staining for calbindin in mineralized matrix showed that the number and size of mineralized nodes in PCLA group was significantly larger than in other groups on day 21 (fig. 12b and 12 d). Osteocalcin (OCN) staining also demonstrated that the relative expression of OCN was significantly higher in the PCLA group than in the PLA, PC and PCL groups, with significantly enhanced osteogenic differentiation in the PCLA group (fig. 12 e). The expression levels of other genes related to bone formation (Runx 2, osterix and BMP-2) were also examined (FIG. 12 f-h), and their expression in PCLA was significantly higher than in the other groups. These results indicate that PCLA has a greater capacity for osteogenic differentiation than the other groups, which may be related to the immunomodulatory properties of APY29, promoting secretion of the osteogenic growth factor BMP-2 by macrophages. The results of migration of PCLA-stimulated HUVECs are shown in FIG. 12i, where it can be seen that the exposed gap gradually closes due to directional migration of cells. HUVECs co-cultured with macrophages showed more pronounced migration capacity compared to the PLA group, especially in the case of the PCLA group. Semi-quantitative analysis showed that the wound closure area was significantly smaller after 24h in PCLA group 12 than in the other 3 groups (FIG. 12 l), and the wound healed completely after 24 h. As shown by the optical image of the capillary network in FIG. 12j, there is no evidence of capillary network formation in the PLA group, whereas the formation of capillary-like structures (i.e., tubes, nodes and branches) in the PCLA group was significantly elevated at all predefined time points compared to other specimens (FIGS. 12m and 12 n). Furthermore, HUVEC migration was analyzed with crystal violet staining, and it was found that more cells in the PCLA group were switched to the other side of the Transwell membrane; indeed, few metastatic cells were observed in the other groups (fig. 12 k). Subsequently, expression of VEGF was determined after 72 hours of incubation (FIG. 12 o). Transcriptional expression of VEGF-A was found to be significantly higher in the PCLA group than in all other groups, probably due to the contribution of APY29 to the regulation of the immune microenvironment and thus to the secretion of VEGF-A by macrophages. Overall, these results indicate that the favorable bone microenvironment results in strong osteogenic and angiogenic bioactivity under the action of PCLA.
(4) Evaluation of in vivo performance: to determine whether PCLA promotes bone fusion in diabetic rats, different material sets were implanted into the skull defect surface according to the grouping arrangement described above. New bone formation in the skull defects was assessed 8 weeks and 12 weeks post implantation. After obtaining the rat skull, a three-dimensional (3D) reconstruction of the micro CT image and a histological analysis were performed. More specifically, reconstructed 3D micro-CT images showed that the PCLA group had a large amount of newly formed bone tissue after 6, 8 and 12 weeks, whereas little new bone tissue was found in the composite implanted PLA group (fig. 13 a). This result was further confirmed by quantitative analysis, in which PCLA group was found to have the highest Bone Mineral Density (BMD), bone volume/tissue volume (BV/TV) and small Liang Shu (tb.n), but the lowest trabecular separation (tb.sp) (fig. 13 b). At the same volume of interest, the new trabecular bone exhibits superior structural characteristics. As expected, PCLA showed the strongest bone formation capacity, probably due to the synergistic effect of APY29 and COLI. In contrast, collagen alone may not provide an optimal immunomodulatory microenvironment for bone regeneration. The newly formed bone was marked with calcitonin (green) and alizarin red (red) in sequence, with similar results as described above (fig. 13 c). More specifically, a large area of new bone mineralization was observed in the PCLA (18.80%), PCL (11.10%), PC (8.22%) and PLA (3.74%) groups (FIG. 13 d).
To further assess the effect of various treatments on rat skull defects, harvested samples were histologically stained 12 weeks after surgery. H & E and Masson stained images of different post-treatment skull defects showed that the new bone formation was higher for PCLA, PCLA and PC groups than for PLA groups; the largest bone value was observed in the PCLA group after 12 weeks, indicating a higher degree of bone formation (FIG. 13 e). Furthermore, masson staining showed a blue staining of new bone tissue and a red staining of cancellous bone in the PCLA group, indicating increased calcification, maturation and remodeling of new bone tissue (fig. 13 f). Immunofluorescence showed high expression of OCN and CD31 in PCLA, PCL and PC-implanted cells, with PCLA groups exhibiting the highest levels of OCN and CD31 expression (fig. 13 g). Overall, PCLA group promoted osteogenesis and angiogenesis in vivo compared to other groups, suggesting its potential for post-operative treatment of rat skull defects. These results clearly demonstrate that bone immunomodulation and regeneration can proceed simultaneously and mutually. The use of aligned spun fibers as a carrier in combination with collagen type one and PCLA loaded lipids, wherein PCLA is modified with a glucose responsive group, results in a biomaterial that enhances osteoinduction and immunomodulation. The dual-effect biomaterial induces not only the phenotype switch from M1 to M2 at an early stage, but also direct osteogenesis, synergistically creating an in vivo microenvironment conducive to bone regeneration (fig. 13 h). To date, glucose-reactive immunomodulatory liposomes have not yet bound to osteoblastic collagen and their synergy with osteogenic differentiation has not been clarified.
In summary, the pathological diabetic microenvironment consists of glucose fluctuations and chronic inflammation, leading to delayed healing of periosteal defects. Therefore, there is a need to develop new materials that facilitate osseointegration and are suitable for use in the complex microenvironment of diabetes. The present invention preferably develops a novel glucose-reactive PCLA composite that modulates the local immune microenvironment and promotes osseointegration of the periosteal-implant interface. PCLA was found to remodel the pathological diabetic microenvironment into a regenerative microenvironment by blocking IRE alpha/NOD-like/NF- κB signaling pathway, while reprogrammed macrophages transformed from the M1 to M2 phenotype, enhancing osteogenic differentiation and angiogenesis. Overall, the findings obtained indicate that PCLA regulates the immune microenvironment and enhances osseointegration of periosteal defects in diabetics. In addition, the present study provides an effective strategy for designing functionalized biomaterials for bone regeneration treatment in diabetics.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (10)
1. The sugar response liposome is characterized by being prepared from phospholipid substances, cholesterol and fluorobenzene boric acid grafts, wherein the fluorobenzene boric acid grafts are phospholipid p-fluorobenzene boric acid grafts or cholesterol p-fluorobenzene boric acid grafts.
2. The sugar-responsive liposome according to claim 1, wherein the fluorobenzeneboronic acid graft is one or more of DSPE-PEG-FPBA, DPPE-PEG-FPBA and DOPE-PEG-FPBA, preferably DSPE-PEG-FPBA; the molecular weight of the PEG chain segment in the fluorobenzeneboronic acid graft is preferably 1000-3000.
3. The sugar-responsive liposome according to claim 2, wherein the mass ratio of the phospholipid, the cholesterol and the fluorobenzeneboronic acid graft is 40:10:1-4 in the raw materials for preparing the sugar-responsive liposome.
4. A sugar-responsive liposomal formulation comprising: a sugar-responsive liposome according to any one of claims 1 to 3, and an immunomodulatory agent entrapped therein, preferably APY29.
5. The sugar-responsive liposome formulation of claim 4, wherein the release rate of the immunomodulator and sugar concentration in the sugar-responsive liposome formulation are positively correlated.
6. An oriented electrospun membrane for bone regeneration in a diabetic environment comprising an oriented nanofiber scaffold and a composite self-assembled substance thereof formed from collagen and the glycoresponsive liposome formulation of claim 4; the collagen is preferably type I collagen.
7. The oriented electrospun film according to claim 6, wherein the oriented nanofiber scaffold is made of one or more of PLA, PCL and PCLA, preferably PLA.
8. An oriented electrospun film according to claim 6, characterized in that the oriented nanofiber scaffold has an average fiber diameter of 0.8-1.3 μm, the oriented electrospun film having a hydrophilic character, the water contact angle preferably being 32 ° -100 °.
9. A method for preparing an oriented electrospun membrane for bone regeneration in a diabetic environment, comprising the following steps:
s1, preparing nanofibers with different orientations through an electrostatic spinning process, and collecting to obtain an oriented nanofiber membrane;
preparing the glucose-responsive liposome preparation of claim 4 by reverse evaporation;
s2, mixing the sugar response liposome preparation with a collagen solution, and then placing the mixture on the oriented nanofiber membrane, and obtaining the oriented electrospun membrane through self-assembly.
10. The method of producing an oriented electrospun membrane according to claim 9, wherein in step S2, the collagen solution is neutral and the solute is type I collagen; the self-assembly temperature is 35-38 ℃ and the self-assembly time is more than 30 minutes.
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