Nanotherapy In Temporomandibular Joint:Increasing Endogenous Hyaluronan Production By Delivering Hyaluronan Synthase 2

Osteoarthritis of temporomandibular joints(TMJ OA)is a common joint degenerative disease with the high mobility,that often causes pain,joint damage and dysfunction,deteriorating patient’s quality of life.When TMJ OA occurs,high molecular weight of HA(hyaluronic acid)is depolymerized and decreased compared to normal situation,resulting impaired viscoelasticity of synovial fluid.Clinically,in order to lubricating the joints and promote the bone repair,external HA supplement is a common method for the management of osteoarthritis which requires multi-injections.In addition,exogenous HA degrades very fast,thus multiple injections with one week interval are usually needed,which are inconvenient and uncomfortable for patients,increasing the physical and mental pain,as well as psychological burden of patients.In the joint,HA with high molecular weight is mainly synthesized inside synoviocytes by hyaluronan synthase type 2(HAS2).It is always a big challenge to find an alternative approach to improve the level of HAS2 in synoviocytes.With the development of advanced biomaterials and nanotechnology,mesoporous silica nanoparticle(MSN)is regarded as an ideal nanocarrier for protein drug delivery due to its high protein loading capacity,good biocompatibility and stability,adjustable pore size and abundant surface chemistry.In this project,MSN will be synthesized with surface modification to explore the HAS2 protein nanotherapy and its mechanism in the treatment of TMJ OA.Part 1.Preparation of MSN-CC-PEI,biocompatibility analysis,construction and evaluation of HAS2 delivery system(MSN-CC-PEI+HAS2)Objective:The advantage of mesoporous silicon nanoparticles as multifunctional nanomaterials is their abundant surface functional modification.In this part of the study,in order for the application of nanotherapy in TMJ by appropriate encapsulation and transport of macromolecule HAS2,mesoporous silicon nanoparticles were constructed,characterized and the surface was modified for optimization.MSN-CC-PEI was used as a nanocarrier for the delivery of the macromolecular protein HAS2 in the subsequent nanotherapy study for TMJ OA.The purpose is to construct delivery system(MSN-CC-PEI+HAS2),study the biodegradation ability,the behavior of loading and releasing efficiency of HAS2 by MSN-CC-PEI as a transport vector in vitro.By using RITC-labeled MSN-CC-PEI,this part aims to evaluate the cell growth,proliferation,cycle,apoptosis and other biological activities in synociocytes and negative control cells both in vitro and in vivo.Methods:The MSN-CC was synthesized in a chlorobenzene-water system,using cetyltrimethylammonium chloride(CTAC)as a structure-directing agent.During preparation,tetraethyl orthosilicate(TEOS)was introduced as a silica source,and triethanolamine(TEA)as a catalyst.MSN-CC-PEI was modified with polyethylenimine(PEI).The products were characterized by detection methods such as field emission scanning electron microscope(FE-SEM),transmission electron microscope(TEM),electron tomography(ET),pore size distribution analysis(BJH),specific surface area analysis(BET),and dynamic light scattering(DLS).Rhodamine B isothiocyanate(RITC)was used for fluorescent labeling for subsequent experiments.Synoviocytes were collected from TMJ OA and normal synovial tissues and cultured,respectively.In vitro,the influence of MSN-CC-PEI on biological characteristics such as cellular activity,proliferation capacity,cell cycle and apoptosis were detected by using MTT,EdU proliferation assays,cell cycles,Annexin V-FITC/PI double-staining flow cytometry,and TUNEL staining assays.HAS2 loaded delivery system(MSN-CC-PEI+HAS2)was established and the protein release curve is detected and drawn.The effects of nanoparticles toward the proliferation of synoviocytes and biodegradable ability under in vivo environment were also examined by subcutaneously injecting the MSN-CC-PEI with/without HAS2 treated synoviocytes in nude mice.Results:Monodispersed surface-modified MSN-CC-PEI nanoparticles were characterized with spherical core-coned shape,highly porous structures,and a uniform size of about 175nm.The surface zeta potential was+43 mV,and the radial pore size was up to 15 nm × 40nm.The polydispersity index in PBS,0.9%NaCl,and cell culture medium varied from 0.23 to 0.32.MSN-CC-PEI showed a high loading capacity of 420 mg/g and a 42%loading efficacy for HAS2,successfully constructing the HAS delivery system(MSN-CC-PEI+HAS2).MSN-CC-PEI can be internalized by HL-7702 human normal liver cell line(LO2)through endocytosis and no cytotoxicity was observed.Compared to control group without adding nanoparticles,the proliferation ability was retained well at MSN-CC-PEI with concentrations of 100 and 200 μg/mL,while less than 10%reduction of the EdU incorporated capability was observed at the concentration of 400 μg/mL.The TUNEL and Annexin V-FITC/PI double staining assays show that MSN-CC-PEI did not increase the apoptosis of synoviocytes at the concentration of 100 and 200 μg/mL,with only marginal difference in the phases of cell cycle.At 400 μg/mL,G1 phase was slightly increased.The silicon internalized rate of the synoviocytes was up to 82.1%after 24 hours of treatment.The presence of the inflammatory factor IL-1β had no obvious effect on the internalization of MSN-CC-PEI.H&E histological examinations showed similar cell morphology and tissue structure from subcutaneous mass harvested from the mice treated with the MSN-CC-PEI or MSN-CC-PEI+HAS2 and untreated or HAS2 treated group.The growth curve of xenograft mass as well as the weight of mice showed no obvious difference between groups.MSN-CC-PEI can be gradually degraded in stimulated body fluid(SBF)environment in vitro and in injection site in vivo.Conclusion:In this experiment,MSN-CC-PEI with large pore volume,pore size and a specific surface area was successfully synthesized.It exhibited a certain degree of aggregation in various mediums.MSN-CC-PEI can be easily internalized by HL-7702 human normal liver cell line(LO2)and TMJ OA synoviocytes.No cytotoxicity was observed.MSN-CC-PEI provided a good nanocarrier for loading and transportation of large molecular proteins.It showed a relatively high protein load capacity of HAS2 and could maintain the protein bioactivity,with good releasing behavior.The HAS2 delivery system(MSN-CC-PEI+HAS2)has good material biosafety,biocompatibility and biodegradability for TMJ OA synoviocytes with minor cytotoxicity for cell endocytosis.It is safe and degradable in vitro and in nude mice in vivo,which could get access to study the subsequent MSN-CC-PEI+HAS2 delivery system on TMJ OA therapy.Part 2.The performance and mechanism investigation of HAS2 delivery system(MSN-CC-PEI+HAS2)in TMJ OA synoviocytesObjective:In order to investigate the process of delivery,releasing,expression of active HAS2 and to evaluate the enzymatic function of delivered HAS2,the amount and molecular weight of HA from the OA synoviocytes culture medium with different treatments were measured.This part aims to clarify its process mechanism and select the appropriate concentration of HAS2 delivery system for subsequent in vivo studies of the treatment of TMJ OA.Methods:The different concentrations of the delivery system(MSN-CC-PEI+HAS2)were delivered into the TMJ OA synoviocytes,and the fluorescence-labeled HAS2 in synoviocytes was detected by the Confocal immunofluorescent experiment,and the endosomal escape process of the delivery system was examined through the labeled HAS2 protein and endosome/lysosome fluorescence staining.WB analysis was examined to detect HAS2,CD44,HMGB1 and other related protein expression level with quantitative statistics.Immunohistochemistry was used to detect the expression of HAS2,CD44 in subcutaneous cell mass of nude mice.With HL-7702 normal liver cell line(LO2)without HA secretion capacity as negative controls,HA secretion and high molecular weight(HMw)HA concentration were detected by ELISA and high-performance liquid chromatography(HPLC)analysis.Results:The results of confocal laser scanning showed that after TMJ OA synoviocytes treated with the delivery system(MSN-CC-PEI+HAS2),fluorescent labeled HAS2,can be detected in the cell at 4h,most HAS2 is encapsulated in MSN-CC-PEI.At 12 h,the HAS2 had released outside of the endosome/lysosome region.The relative intracellular HAS2 concentration of OA synoviocytes was increased to the level of normal synoviocytes.The intracellular delivery of HAS2 has no effect on the level of HA receptor CD44.Both CD44 and HAS2 were not detected in LO2 cells.HMGB1 in OA synoviocytes was decreased by MSN-CC-PEI+HAS2,and HA concentration was upregulated,in which the isolated high molecular weight HA was significantly increased through HPLC detection.Conclusion:These data confirmed that in TMJ OA synoviocytes,the delivery system is based on the PEI modification of the "proton-sponge" mechanism to enter the cells and successfully deliver HAS2.After entering the cell,MSN-CC-PEI+HAS2 can successfully escape the endosome/lysosome encapsulation,release HAS2 into the cytoplasm,and maintain the biological activity of HAS2 protein.Biodegradable MSN-CC-PEI effectively engaged in its function of loading HAS2 in synoviocytes,increasing endogenous secretion of high molecular weight HA,reducing the expression of inflammation associated factors.Part 3.Analysis of the treatment and long-term efficacy of MSN-CC-PEI+HAS2 system in TMJ OAObjective:This part of the study was designed to confirm that the HAS2 delivery system could successfully enter the synovial tissue of the joint cavity in animals in vivo,explore the HAS2 delivery ability of MSN-CC-PEI+HAS2 delivery system to deliver HAS2 in vivo and the therapeutic effect of TMJ OA.Construction of temporomandibular arthritis animal model,analysis of the therapeutic significance of delivery system therapy for temporomandibular joint synovitis.Construction of SD rat temporomandibular degenerative OA animal model and knee degenerative OA model,respectively,to make a preliminary therapeutic evaluation of delivery system on the repair of bone defects in TMJ OA.Methods:An TMJ osteoarthritic SD rat model and an long-term orthotopic OA model was established by injecting complete Freund’s adjuvant(CFA)and monoiodoacetic acid.Injecting MSN-CC-PEI at a certain concentration(200 μg/mL)into the joint cavity.The injection site was observed with fluorescent images with NIR bioimaging technology,and the silicon content inside the synovium tissues were measured with atomic absorption spectroscopy(AAS).The fresh samples were cryostat serial sectioned and the slides were stained to determine the distribution of the nanoparticles.After treatment in the TMJ osteoarthritic SD rat model,the general specimen of TMJ was observed and recorded,and the concentrations of HA,IL-1 beta and TNF-α in TMJ joint fluid were tested by ELISA assay.The histopathological changes and HAS2 expression of the synovial tissues after treatment were tested by H&E and IHC staining.The change of condyle cartilage thickness was measured.The condyle height,morphology,and repair of subchondral bone were determined by micro CT analysis,as well as the joint changes in the knee OA group after administration.Results:After the injection of MSN-CC-PEI into the joint cavity,fluorescence detection found that it entered the TMJ joint synovial tissues of the SD rats,and a significant distribution is detected.Around 71.59%of total silica was internalized in the synovium tissues of TMJ,indicating a majority part of MSN-CC-PEI entered into the cells of synovium tissues.In the treatment group of the temporomandibular joint synovitis animal model,the H&E and micro CT showed it presented a significant reduction in inflammation degree of the synovial tissues,with no significant thickening of the synovial layers.Those results showed MSN-CC-PEI+HAS2 can promote the HA production ability in vivo sustainably for 3 weeks,and the content is much higher than that in the exogenous HA injection treatment group over the same period.IHC results show increased expression of HAS2 in synovial lining layers.The concentrations of two typical proinflammatory cytokine,IL-1β and TNF-α,from the synovial fluid were also examined and ELISA results showed significantly decreased during the observation period after treatment.The condylar cartilage thickness recovered significantly after treatment,showing an obvious role in cartilage protection.For the animal model of osteoarthritis degeneration,micro CT results of the condyles,sagittal axis sections and three-dimensional reconstructions showed that the subchondral bone was reconstructed after treatment in the nanotherapy group.After 2 weeks,obvious bone defects and disordered trabecular arrangement appeared in the MIA group,and the condylar cortex was discontinuous due to bone destruction.The results were similar to those of the nanoparticle group after MIA injection,and the thickness of the condyles and cancellous bone was greatly reduced after 12 weeks.After 12 weeks,in both MIA and MIA with nanoparticles only group bone remodeling was observed,but both the height of condyle and cancellous bone layer reduced.It is noted that HAS2 loaded MSN-CC-PEI injection resulted in similar effects to the HA injection group,and the bone defects were repaired.The trabecular bones of the subchondral bone are arranged neatly,which can repair the condylar surface and bone defects,and the normal morphology surface of the condyle was kept complete and continuous.The micro CT results of the knee joint also showed that the delivery system could significantly improve the destruction of subchondral bone and inhibit the MIA-induced bone destruction process.In the MIA group at 1st week,there was a slight change in the bone tissue on the knee joint surface.At the second week,the knee joint femoral articular surface and the tibial plateau articular surface were uneven,while the treatment group had no obvious bone damage.In MIA group at 2nd,4th,8th and 12th week,obvious progressive bone destruction was observed,joint defects and deformation continued to increase,and this process could be greatly relieved after delivery system treatment.Conclusion:In a SD rat TMJ osteoarthritis inflammation model,this strategy could successfully delivers HAS2 into synoviocytes,promotes endogenous HA production,relieves and suppresses synovial inflammation of OA for more than 3 weeks with one-shot administration by this delivery system(MSN-CC-PEI+HAS2).Such nanotherapy could greatly optimize the traditional repeated injection of hyaluronic acid to treat OA,and it also helps repairing the osteochondral lesions in a SD rat OA bone defect model.At the same time,it has a significant reversal effect on the TMJ OA degenerative pathological changes in SD rats and this strategy is safely degradable.