Abstract:

Objective To evaluate the role of a thermo- and photo-responsive dual-stimuli hydrogel based on Pluronic F127 diacrylate （F127-DA） as an exosomes （Exos） delivery system in articular cartilage repair， and to clarify its mechanism of action. Methods The Exos were isolated from bone marrow mesenchymal stem cells （BMSCs） by ultracentrifugation and characterized. Transmission electron microscope （TEM） was used to observe the morphology of Exos in various groups； nanoparticle tracking analysis （NTA） was used to detect the particle size distribution of Exos in various groups； Western blotting method was used to detect the expression levels of the marker proteins CD9， CD81， and TSG101 in the Exos in various groups； Exos uptake assay was used to detect the distribution of Exos taken up by the BMSCs in various groups； Western blotting method was used to detect the protein expression levels of type Ⅱ collagen （COL Ⅱ） and aggrecan （ACAN） in the BMSCs in two groups； scratch wound healing assay was used to detect the wound healing ability of the BMSCs in two groups； Transwell chamber assay was used to detect the migration ability of the BMSCs in various groups； scanning electron microscope （SEM） was used to observe the pore structure of the hydrogel. Twenty-four 6-week-old rats were randomly divided into control group， F127-DA group， Exos group， and F127-DA+Exos group， with 6 rats in each group. A cartilage defect （2 mm in diameter， 1 mm in depth） was created in trochlear groove of the knee joint； the rats in control group received no treatment， while the rats in the other three groups were treated with F127-DA hydrogel， Exos， or F127-DA hydrogel +Exos filling the cartilage defect site， respectively.At 8 weeks after operation， the rat knee joint tissues were collected for HE staining and safranin O and fast green （SOFG） staining to evaluate cartilage repair at the defect site in trochlear groove of the knee joint of the rats in various groups. Results The TEM and NTA results showed that the Exos were spherical in shape with a particle size concentrated around 80 nm. The Western blotting results showed that the exosomal surface biomarkers CD9， CD81， and Tsg101 were all highly expressed， while Calnexin was not expressed， confirming that the isolated vesicles were Exos. Compared with control group， the expression levels of COL Ⅱ and ACAN proteins in the BMSCs in Exos group were increased （P<0.05）. The Exos uptake assay results showed that the BMSCs efficiently internalized Exos. The scratch wound healing assay and Transwell chamber assay results showed that compared with control group， the migration ability of the BMSCs in Exos group was increased. The SEM results showed that the F127-DA hydrogel exhibited a loose and porous structure. The HE and SOFG staining results showed that in control group， fibrous tissue formed at the defect site in the trochlear groove； in F127-DA group， mild repair was observed at the defect site； in Exos group， neocartilage-like tissue was visible at the defect site； while in F127-DA+Exos group， the defect site was filled with abundant hyaline cartilage-like tissue， with cell arrangement and matrix formation most closely resembling normal cartilage， showing the optimal repair effect. Conclusion The F127-DA hydrogel loaded with Exos can achieve sustained and controlled release of Exos， and significantly promote cartilage regeneration； the results of this study provide an effective strategy for precise delivery of Exos therapy and multi-stage repair of cartilage injury.

Key words: Exosomes, Hydrogel, Cartilage repair, Osteoarthritis, Controlled release