Construction And In Vitro Biological Evaluation Of Hydrogel Microspheres Dual Delivery System For Inducing Bone Marrow Mesenchymal Stem Cells To Differentiate Into Nucleus Pulposus Like Cells

Background: The unique microenvironment of ischemia,low nutrition,and low cell density makes intervertebral disc degeneration face problems such as incomplete treatment,easy recurrence,and high disability rate.In recent years,local injection based on biological agents such as stem cells,bioactive molecules,and nucleic acids is a new treatment strategy for intervertebral disc degeneration(IVDD).However,protecting biological agents from in situ degradation in harsh microenvironments and allowing them to be continuously delivered in situ for a long time still face significant challenges.This study aims to develop a sustainable slow-release transformation growth factor β3(TGFβ3)The poly(lactic co glycolic acid)(PLGA)microspheres of bone marrow mesenchymal stem cells(BMSCs)and the photocurable gelatin methylacryloyl(Gel MA)hydrogel that can encapsulate the natural microenvironment of the bionic Nucleus pulposus(NP)tissue were collected.Finally,a hydrogel microsphere dual delivery system supporting NP cells(NPC)like regeneration was constructed in vitro,which is expected to achieve in situ regeneration and repair of NP like tissue in clinical IVDD.Methods: This experiment consists of four parts.Part Ⅰ: TGFβ3@PLGA Preparation and process optimization of sustained-release microspheres: We prepared sustained-release microspheres using the common and efficient double emulsion solvent evaporation method(W/O/W method)used in the traditional preparation of PLGA microspheres.We used drug loading,encapsulation efficiency,and drug loading-encapsulation efficiency as outcome indicators.Among the many factors that affect the preparation of microspheres,we fixed the relatively mature influencing factors through literature review,and selected orthogonal experiments to explore the impact of three levels of four factors,namely,the ratio of V inner water phase to V oil phase,the ratio of V oil phase to V outer water phase,stirring rate,and different PLGA capping materials,on the outcome indicators.After integrating and analyzing the orthogonal experimental data,we obtained the optimal preparation parameters for drug loaded PLGA sustained-release microspheres.Finally,the characterization,particle size range,and drug distribution of microspheres prepared with the best parameters were also explored.Part Ⅱ: Study on the in vitro performance of polydopamine(PDA)coated microspheres: Based on the first part of the experiment,the optimal preparation parameters and process were selected,and batch production was conducted TGFβ3@PLGA Sustained release microspheres.In order to delay the initial explosive release of drug loaded microspheres,we used three different concentrations of PDA solutions(0.5 mg/m L,1.0 mg/m L,and 2.0 mg/m L)to modify the surface of drug loaded microspheres.The experiment was divided into four groups: 0 mg/m L PDA group,0.5 mg/m L PDA group,1.0 mg/m L PDA group,and 2.0 mg/m L PDA group.The PDA coating with different concentrations was modified by naked eye,light microscope,scanning electron microscope(SEM),hydrophilicity measurement,Fourier Transform Infrared Spectroscopy(FTIR)measurement,and in vitro TGFβ3 release performance TGFβ3@PLGA Biological properties of sustained-release microspheres were evaluated.Part Ⅲ: Preparation of Gel MA hydrogel and its performance test: Dissolve the Gel MA precursor through photoinitiator to prepare a photocurable Gel MA hydrogel solution with the concentration of 3%,5% and 7%(w/v).The strength is 25w/cm~2 at the wavelength of 405 nm The microstructure,pore size,swelling performance,and degradation performance of the adhesive were evaluated after being irradiated under a violet light source for 15-30 seconds.In addition,we used 3D cell culture technology to detect the proliferation and biological activity of 3%,5% and 7% light cured Gel MA hydrogels on BMSCs.Part Ⅳ: Research on the dual delivery system inducing BMSCs to differentiate into NPC like cells in vitro: The second and third part of the experiment showed that the best concentration of 1.0mg/m L PDA surface coated PLGA drug loaded sustained release microspheres and 5% Gel MA hydrogel were the best.After 10 mg drug loaded microspheres were compounded with 1m L hydrogel precursor fluid,a dual delivery system that could promote BMSCs to differentiate into NPC like cells was constructed.In the subsequent experiment,four groups were set up: Control group,PDA/PLGA group,Gel MA group,and PDA/PLGA-Gel MA-TGFβ3 group.We used a co-culture mode to test the characterization structure,migration recruitment,and biocompatibility of the composite scaffolds in the above groups.Finally,we explored the potential of dual delivery systems to induce differentiation of BMSCs into NPC like cells in vitro from both qualitative(immunofluorescence staining)and quantitative(real-time q-PCR)perspectives.Result:Part Ⅰ: The sustained-release microspheres of TGFβ3@PLGA prepared by double emulsion solvent evaporation method are ideal and have no obvious cracks.The orthogonal test showed that the best preparation parameters of microspheres were: V inner water phase: V oil phase: 1:10,V oil phase: V outer water phase: 1:2,rotational speed: 700rpm/min,ester(-COR)end capping.The final drug loading of the microspheres prepared by the optimum process was 83.11%±0.76%(ug/g),and the encapsulation efficiency was 91.21%±2.74%;The average particle size is107.78±0.53μm.Macroscopic observation showed that the drug-loaded microspheres were white powder,optical microscope and scanning electron microscope showed that the microspheres were round,without obvious adhesion,neat shape and uniform size.The fluorescence inversion microscope showed that the drug was evenly distributed in the microspheres prepared by the above process.Part Ⅱ: Through naked eyes,light microscopy,SEM,and FTIR,it was confirmed that PDA was successfully coated on the surface of TGFβ3@PLGA microspheres.The water contact angle experiment showed that the modified microspheres had certain hydrophilic properties.The drug loaded microspheres release experiment found that the surface modified microspheres in the 1.0 mg/m L PDA group delayed the initial burst release of the drug(p<0.001),which was superior to other groups and sustained slow release in vitro for more than 28 days.Part Ⅲ: Successful preparation of different concentrations of Gel MA precursor solutions using photoinitiator at a wavelength of 405 nm and an intensity of 25 w/cm~2 After being irradiated for 15 to 30 seconds under a violet light source,the adhesive is solidified.SEM showed that the cross section of Gel MA hydrogel was loose and porous,and the pore size of 3%,5% and 7% Gel MA hydrogel was 392.95±23.91μm、252.45±38.66μm 、 168.71±18.62μm 。 According to swelling ratio,simulated degradation performance in vivo and in vitro,3D biocompatibility and other experiments,Gel MA hydrogel has excellent water absorption performance,can produce relatively stable enzymatic degradation behavior,and the degradation products are water-soluble.In addition,Gel MA hydrogel can provide BMSCs with a3 D microenvironment for good attachment and proliferation,and maintain most cells in a long-term active state.Part Ⅳ: Successful Construction of TGFβ3@PDA/PLGA-Gel MA dual delivery system,SEM showed that PDA/PLGA microspheres were evenly distributed in the porous structure of Gel MA hydrogel.The migration results indicate that the TGFβ3released from PDA/PLGA microspheres has a significant recruitment ability for BMSCs.Cell proliferation and living/dead cell staining experiments showed that the biocompatibility of the four groups of biomaterials to BMSCs was basically the same,with no significant statistical difference(p>0.05).Finally,immunofluorescence qualitative experiment and real-time fluorescence q-PCR quantitative experiment showed that PDA/PLGA drug loaded microspheres could slowly release TGFβ3 and induce BMSCs in Gel MA hydrogel to differentiate into NPC like cells.Conclusion: In this study,we successfully incorporated TGFβ3@PDA/PLGA microspheres were fused with Gel MA hydrogel loaded with BMSCs to build a dual delivery system.The system implements TGFβ3 Slow release and Gel MA hydrogel are ideal scaffolds for cell 3D culture,and are endowed with the ability to recruit BMSCs to migrate and induce them to differentiate into NPC like cells,providing a research strategy and theoretical reference for preclinical research and treatment of IVDD.However,it is still necessary to conduct in vivo animal experiments and pre-clinical conversion studies to verify the feasibility.