Preparation Of Glucose Sensitive Nanofiber Scaffolds And Study On Osteogenic Properties In Vitro

Objective:Patients with alveolar bone defects with diabetes mellitus are a major challenge to clinical management,and the high blood glucose state can severely impede the current local bone tissue repair.In recent years,the rise of nanofibers scaffolds has provided a new approach to the repair of alveolar bone defects,but conventional drug-carrying scaffolds are a slow-release approach that does not meet the increasing demand for therapeutic agents in the high-glucose microenvironment and has a number of shortcomings.The combination of a glucose-sensitive system with nanofibers scaffolds is a promising therapeutic approach.The principle of this system is that glucose is catalyzed by glucose oxidase(GOD)to produce gluconic acid,causing a change in the local internal and external osmotic pressure of the material,which in turn produces a difference in the volume of the material,leading to a change in the release of the active molecule drug.In order to exert glucose sensitivity,the immobilization of GOD onto the scaffold matrix is a critical step.However,our previous studies have shown that glutaraldehyde(GA),a commonly used cross-linking agent,causes adverse toxic effects on cells.We hypothesized that covalent linkage plus cross-linking might be an effective way to improve the stability and quantity of GOD.In this study,we chose the natural bio-cross-linker Genipin(GnP)to immobilize GOD,explored its effectiveness through enzyme-related performance evaluation and biocompatibility,and finally evaluated the effect of this glucose-sensitive nanofiber in promoting osteogenesis by establishing an in vitro high-glucose environment.Methods:In this experiment,polycaprolactone/chitosan(PCL/CS)nanofibers were firstly prepared by electrostatic spinning,on the basis of which GOD was immobilized in nanofibers loaded with dexamethasone(DEX)using GnP to produce glucose-sensitive nanofibers(GnP-NF).The interaction between GOD and the nanofibers scaffolds was then analyzed by Fourier Transform Infrared Spectroscopy(FTIR)to show whether it was immobilized in the nanofibers,and the porosity and drug loading rate of the nanofibers were also determined.The mechanical properties of the nanofibers were also determined after drug loading and GnP treatment.The activity,binding capacity and stability of the enzyme after immobilization by direct immersion(P-NF),glutaraldehyde treatment(GA-NF)and GnP immobilization were then compared in order to evaluate the effectiveness of GOD immobilization.The glucose-sensitive nanofibers were then immersed in different concentrations of glucose solution(0 m M,5 m M,12 m M and 15 m M)and their pore size changes and drug release rates were analyzed by Scanning Electron Microscope(SEM)and drug release methods to evaluate the glucose sensitivity of the glucose-sensitive nanofibers.Mouse pre-osteoblasts(MC3T3-E1)were inoculated on the nanofibers for a period of time and their cell viability was analyzed by Cell Counting Kit-8(CCK-8),and the growth of MC3T3-E1 was observed by laser confocal microscopy(CLSM)to comprehensively evaluate the biocompatibility of the nanofibers.Finally,nanofibers with good glucose sensitivity were selected by the above experiments and co-cultured with MC3T3-E1 in vitro with a high glucose environment,and their effect on promoting osteogenic differentiation was evaluated by alkaline phosphatase activity(ALP)assay,alizarin red staining(ARS)and semi-quantitative assay.Results:1.FTIR characterization revealed that PCL/CS was successfully prepared,DEX was also loaded onto the nanofibers and GOD was successfully immobilized on the nanofibers,which showed high porosity(>90%)and high drug loading efficiency(85.51±1.21%)as measured by porosity and drug loading efficiency.2.In the mechanical property tests,the mechanical properties of both DEX-loaded nanofibers and GnP-treated nanofibers were improved and the latter was higher than the former.In addition,the GOD activity,binding capacity and stability tests showed that the enzyme activity of GnP-NF was higher than that of GA-NF and P-NF and more enzymes were immobilized than GA-NF and P-NF,but the stability performance of GnP and GA-NF were not significantly different and both were more stable than P-NF.In the glucose sensitivity test,the diameters of different glucose-sensitive nanofibers(GnP-NF,GA-NF,PNF)increased with the increase of glucose concentration after immersed in different concentrations of glucose solution(0 m M,5 m M,12 m M,15 m M)by SEM observation,and the diameter of GnP-NF increased with the increase of glucose concentration in 15 m M glucose solution by Image J analysis.Further,the drug release rate was measured and found that the release of DEX increased with the increase of glucose concentration,showing glucose concentration dependence,indicating that GnP-NF could swell under high glucose concentration conditions and increase drug release,exhibiting a significant glucose sensitivity.3.The CCK-8 results in the in vitro biocompatibility test showed that the treatment of GA reduced cell viability and decreased cell survival,while the GnP-treated group still had high cell viability.Further,MC3T3-E1 was further observed by CLSM to have altered growth morphology and reduced viability on GA-NF,while it grew well on GnP-NF with good biocompatibility.The osteogenic assay showed that the ALP activity in all groups was reduced but the GnP-NF group was higher than the DEX-only and PCL/CS groups,and more and wider calcium nodules were produced by GnP-NF in alizarin red staining.Conclusion:In this study,nanofibers were prepared from PCL and CS by electrospinning technique,loaded with DEX and then GOD was immobilized in the nanofibers by GnP-mediated onestep enzyme immobilization method to obtain glucose-sensitive nanofibers(GnP-NF).The correlation results showed that GnP-NF can respond to high glucose concentration by rapidly undergoing swelling to increase the diameter and internal pore size,which in turn controls the rapid release of DEX and exhibits good glucose sensitivity.The nanofibers scaffolds have suitable mechanical properties and good biocompatibility can allow cell adhesion growth.It showed good ability to promote osteogenic differentiation under high glucose environment in vitro.The results suggest that the GnP treatment method is an effective and low cytotoxic way to immobilized GOD,while endowing good glucose sensitivity to the nanofibers scaffolds has great potential for the repair of diabetic alveolar bone defects.