In cartilage tissue engineering (TE),new solutions are needed to effectively drive chondrogenic differentiation of mesenchymal stromal cells in both normal and inflammatory milieu. Ultrasound waves represent an interesting tool to facilitate chondrogenesis. In particular, low intensity pulsed ultrasound (LIPUS)has been shown to regulate the differentiation of adipose mesenchymal stromal cells. Hydrogels are promising biomaterials capable of encapsulating MSCs by providing an instructive biomimetic environment, graphene oxide (GO) has emerged as a promising nanomaterial for cartilage TE due to its chondroinductive properties when embedded in polymeric formulations, and piezoelectric nanomaterials, such as barium titanate nanoparticles (BTNPs),can be exploited as nanoscale transducers capable of inducing cell growth/differentiation. The aim of this study was to investigate the effect of dose-controlled LIPUS in counteracting inflammation and positively committing chondrogenesis of ASCs embedded in a 3D piezoelectric hydrogel. ASCs at 2*10. 6. cells/mL were embedded in a 3D VitroGel RGD. ®. hydrogel without nanoparticles (Control) or doped with 25 µg/ml of GO nanoflakes and 50 µg/ml BTNPs.The hydrogels were exposed to basal or inflammatory milieu (+IL1β 10ng/ml)and then to LIPUS stimulation every 2 days for 10 days of culture. Hydrogels were chondrogenic differentiated and analyzed after 2,10 and 28 days. At each time point cell viability, cytotoxicity, gene expression and immunohistochemistry (COL2, aggrecan, SOX9, COL1)and inflammatory cytokines were evaluated. Ultrasound stimulation significantly induced chondrogenic differentiation of ASCs loaded into 3D piezoelectric hydrogels under basal conditions: COL2, aggrecan and SOX9 were significantly overexpressed, while the fibrotic marker COL1 decreased compared to control samples. LIPUS also has potent anti-inflammatory effects by reducing IL6 and IL8 and maintaining its ability to boost chondrogenesis. These results suggest that the combination of LIPUS and piezoelectric hydrogels promotes the differentiation of ASCs encapsulated in a 3D hydrogel by reducing the inflammatory milieu, thus representing a promising tool in the field of cartilage TE. Acknowledgements: This work received funding from the European Union's Horizon 2020 research and innovation program, grant agreement No 814413, project ADMAIORA (AdvanceD nanocomposite MAterIals for in situ treatment and ultRAsound-mediated management of osteoarthritis)

Hydrogels have been widely used for articular tissue engineering application, due to their controllable biodegradability and high water content mimicking the biological extracellular matrix. However, they often lack the mechanical support and signaling cues needed to properly guide cells. Graphene and its derivatives have recently emerged as promising materials due to their unique mechanical, physical, chemical proprieties [1]. Although not yet widely used for medical applications, preliminary works suggest that both structural and functional properties of polymeric substrates may be enhanced when combined with graphene oxide (GO) [2]. In this work, reinforced 3D GO/alginate (Alg) hydrogels have been realized and the opportunity of tuning hydrogels mechanical properties in relation to the required physiological needs has been investigated. After preparing GO nanosheets (Sigma Aldrich) aqueous suspension (1 mg/ml) by ultrasonic treatment, alginate (Manugel GMB, FMC Biopolymer) composite solutions were produced (0, 0.5, 2 wt% GO/Alg). Moulds of agarose (1% w/v in CaCl 0,1M) were prepared to allocate GO/Alg solutions and chemically cross-link gels via diffusion (2 hr. at 37 °C). GO/Alg hydrogels were characterized through optical/ AFM and FTIR analysis. Biocompatibility tests were performed embedding 3T3 fibroblasts (8 millions/ml) in the GO/Alg hydrogels; cell viability was evaluated at different time points up to two weeks with Dead/alive kit. Gels mechanical proprieties were assessed via Dynamic Mechanical- Analysis (DMA) up to 28 days of culture (with and w/o cells) at different time points. All tests were performed in triplicate and statistical analyisis carried out (Mann–Whitney U test, n=9, p<0,005). 3D composite GO/alginate hydrogels were successfully realized (3 mm height, 5 mm diameter). Cell viability tests showed that the presence of GO does not decrease cell viability, confirming absence of toxicity, at least up to 2% wt GO/Alg. For all time points cell viability was statistically higher in presence of GO, while there was no significant difference between 0.5 wt% and 2 wt% GO/Alg. Hydrogels functionalized with GO exhibit an Elastic modulus about 3 fold higher than the Alg control at T0. After an initial decreasing of the Young Modulus for the all GO/Alg samples, possibly due to a partial degradation of alginate, a drastic recovery was observed up to 28 days of culture only for GO functionalized samples. The mechanical features improvement was neither mediated nor triggered by cells activity. We successfully realized a natural-based 3D hydrogel nano-functionalized with graphene, where both mechanical and biological properties were successfully improved. The delayed stabilization of GO/Alg mechanical proprieties may be due either to a chemical interplay between GO and alginate matrix or to GO self-assembling processes over time. Future developments will be carried out to decouple the chemical and topological role of GO on the results observed up to now. Moreover, functional tests will be performed to evaluate the GO effects on in vitro cell differentiation for possible articular clinical applications