Addressing bone defects is a complex medical challenge that involves dealing with various skeletal conditions, including fractures, osteoporosis (OP), bone tumours, and bone infection defects. Despite the availability of multiple conventional treatments for these skeletal conditions, numerous limitations and unresolved issues persist. As a solution, advancements in biomedical materials have recently resulted in novel therapeutic concepts. As an emerging biomaterial for bone defect treatment, graphene oxide (GO) in particular has gained substantial attention from researchers due to its potential applications and prospects. In other words, GO scaffolds have demonstrated remarkable potential for bone defect treatment. Furthermore, GO-loaded biomaterials can promote osteoblast adhesion, proliferation, and differentiation while stimulating bone matrix deposition and formation. Given their favourable biocompatibility and osteoinductive capabilities, these materials offer a novel therapeutic avenue for bone tissue regeneration and repair. This comprehensive review systematically outlines GO scaffolds’ diverse roles and potential applications in bone defect treatment. Cite this article: Bone Joint Res 2024;13(12):725–740

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)

Introduction. Intervertebral disc degeneration (IVDD) associated with low back pain is a major contributor to global disability. Current treatments are poorly efficient in the long-term resulting in medical complications. Therefore, minimally invasive injectable therapies are required to repopulate damaged tissues and aid regeneration. Among injectable biomaterials, self-assembling peptide hydrogels (SAPHs) represent potential candidates as 3D cell carriers. Moreover, the advent of graphene-related materials has opened the route for the fabrication of graphene-containing hydrogel nanocomposites to direct cellular fate. Here, we incorporated graphene oxide (GO) within a SAPH to develop a biocompatible and injectable hydrogel to be used as cell carrier to treat IVDD. Methods and results. Hydrogel morphology and mechanical properties have been investigated showing high mechanical properties (G'=12kPa) comparable with human native nucleus pulposus (NP) tissue (G'=10kPa), along with ease of handling and injectability in dry and body fluid conditions. Hydrogel nanocomposites resulted biocompatible for the encapsulation of bovine NP cells, showing higher viability (>80%) and metabolic activity in 3D cell culture over 7 days, compared to GO-free hydrogels. Moreover, GO has demonstrated to bind TGF-β3 biomolecules with high efficiency, suggesting the use of GO as local reservoir of growth factors within the injected hydrogel to promote extracellular matrix deposition and tissue repair. Conclusions. Our results show that incorporation of GO within the SAPH improves cell viability and metabolic activity. Furthermore, its tissue-mimicking mechanical properties and chemical tunability make it a promising candidate as injectable carrier of NP cells for the treatment of IVDD. Part of this work has been published (DOI: 10.1016/j.actbio.2019.05.004). Conflicts of interests: No conflicts of interest. Sources of funding: The authors thank the EPSRC & MRC CDT in Regenerative Medicine for its financial support (EP/L014904/1)

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