Mesenchymal stem cells-derived extracellular vesicles (MSC-EVs) have great promise in the field of orthopaedic nanomedicine due to their regenerative, as well as immunomodulatory and anti-inflammatory properties. Researchers are interested in harnessing these biologically sourced nanovesicles as powerful therapeutic tools with intrinsic bioactivity to help treat various orthopaedic diseases and defects. Recently, a new class of EV mimetics has emerged known as nanoghosts (NGs). These vesicles are derived from the plasma membrane of ghost cells, thus inheriting the surface functionalities and characteristics of the parent cell while at the same time allowing for a more standardized and reproducible production and significantly greater yield when compared to EVs. This study aims to investigate and compare the osteoinductive potential of MSC-EVs and MSC-NGs in vitro as novel tools in the field of bone tissue engineering and nanomedicine. To carry out this investigation, MSC-EVs were isolated from serum-free MSC conditioned media through differential ultracentrifugation. The remaining cells were treated with hypotonic buffer to produce MSC-ghosts that were then homogenized and serially extruded through 400 and 200 nm polycarbonate membranes to form the MSC-NGs. The concentration, size distribution, zeta potential, and protein content of the isolated nanoparticles were assessed. Afterwards, MSCs were treated with either MSC-EVs or MSC-NGs under osteogenic conditions, and their differentiation was assessed through secreted ALP assay, qPCR, and Alizarin Red mineralization staining. Isolation of MSC-EVs and MSC-NGs was successful, with relatively similar mean diameter size and colloidal stability. No effect on MSC viability and metabolic activity was observed with either treatment. Both MSC-EV and MSC-NG groups had enhanced osteogenic outcomes compared to the control; however, a trend was observed that suggests MSC-NGs as better osteoinductive mediators compared to MSC-EVs.

Acknowledgements: The authors would like to acknowledge Canada Research Chair – Tier 1 in Regenerative Medicine and Nanomedicine, CHRP, and McGill's Faculty of Dental Medicine and Oral Health Sciences for their financial support.

Critical size bone defects deriving from large bone loss are an unmet clinical challenge1. To account for disadvantages with clinical treatments, researchers focus on designing biological substitutes, which mimic endogenous healing through osteogenic differentiation promotion. Some studies have however suggested that this notion fails to consider the full complexity of native bone with respect to the interplay between osteoclast and osteoblasts, thus leading to the regeneration of less functional tissue2. The objective of this research is to assess the ability of our laboratory's previously developed 6-Bromoindirubin-3’-Oxime (BIO) incorporated guanosine diphosphate crosslinked chitosan scaffold in promoting multilineage differentiation of myoblastic C2C12 cells and monocytes into osteoblasts and osteoclasts1, 3, 4. BIO addition has been previously demonstrated to promote osteogenic differentiation in cell cultures5, but implementation of a co-culture model here is expected to encourage crosstalk thus further supporting differentiation, as well as the secretion of regulatory molecules and cytokines2.

Biocompatibility testing of both cell types is performed using AlamarBlue for metabolic activity, and nucleic acid staining for distribution. Osteoblastic differentiation is assessed through quantification of ALP and osteopontin secretion, as well as osteocalcin and mineralization staining. Differentiation into osteoclasts is verified using SEM and TEM, qPCR, and TRAP staining.

Cellular viability of C2C12 cells and monocytes was maintained when cultured separately in scaffolds with and without BIO for 21 days. Both scaffold variations showed a characteristic increase in ALP secretion from day 1 to 7, indicating early differentiation but BIO-incorporated sponges yielded higher values compared to controls. SEM and TEM imaging confirmed initial aggregation and fusion of monocytes on the scaffold's surface, but BIO addition appeared to result in smoother cell surfaces indicating a change in morphology. Late-stage differentiation assessment and co-culture work in the scaffold are ongoing, but initial results show promise in the material's ability to support multilineage differentiation.

Acknowledgements: The authors would like to acknowledge the financial support of the Collaborative Health Research Program (CHRP) through CIHR and NSERC, as well as Canada Research Chair – Tier 1 in Regenerative Medicine and Nanomedicine, and the FRQ-S.

Purpose: Several studies have shown elevated levels of metal ions in the blood of patients with metal-on-metal (MM) total hip arthroplasty (THA). Even though there is no conclusive evidence that the elevated levels of ions have any detrimental effects on the patients, the presence of these ions is still a cause of concerns. The potential of metal ions released from MM implants for oxidative stress is unknown. In the present study, we measured the concentrations of oxidative stress markers in the plasma of patients with MM THAs.

Method: Blood from patients having MM THAs was collected up to 10 years post-operatively into Sarstedt Li-Heparin tubes. Plasma was prepared by centrifugation at 500 × g for 10 min. Plasma was chosen as opposed to whole blood because it is known that the assays for oxidative stress are not recommended for blood and can lead to erroneous data. Total antioxidant levels were measured by the Oxford Biomedical total antioxidant power assay to obtain an overview of the defense capacity of patients against oxidative stress. The activity of catalase and glutathione peroxidase, two antioxidant enzymes acting on specific reactive oxygen species, was measured by enzymatic assays. Peroxide concentrations were measured by the Biomedica Oxy-Stat assay to quantify damage to lipids in the systemic circulation. Nitrototyrosine levels were quantified using the BIOXYTECH® Nitrotyrosine-EIA assay to measure damage to proteins. Levels in patients without prostheses were used as control.

Results: There were no statistical differences in the concentrations of total antioxidants, lipid peroxides, and nitrotyrosines throughout the period of study. The activity of catalase and glutathione peroxidase was also stable over time. Moreover, there was no correlation between the concentrations of these markers and the concentrations of both cobalt and chromium ions.

Conclusion: Metal ions have the potential to induce the production of reactive oxygen species (free radicals) and cause oxidative stress in the plasma of patients with MM THAs. The present study showed that there were no changes in the levels of oxidative stress markers or antioxidant enzymes in these patients up to 10 years post-operatively. Taken together, the data strongly suggest that metal ions may not cause a significant oxidative stress in patients with MM THAs.

Purpose: One of the major concerns regarding metal-on-metal prostheses is the biological and biochemical activities of chromium (Cr) ions. Previous studies showed that Cr3+ ions form nanostructures in cell culture media and to date, there has been little attempt to understand the nature of implant-derived metal ions in adjacent tissues or in biofluid. The aim of this work was to determine the nature of proteins present in serum involved in the formation of Cr nanostuctures

Methods: RPMI 1640 and DMEM media supplemented with 5% human serum (HS) or 5% foetal bovine serum (FBS) were incubated for 1h at 37°C in the presence of 50 ppm of Cr3+ (CrCl3). Structures were then isolated and separated by SDS-PAGE. Proteins were stained by Coomassie blue and analyzed by liquid chromatography-quadrupole-time of flight-mass spectrometer (LC-Q-Tof-MS). Data were submitted to Mascot software for a search against the NCBI nonredundant database

Results: Results show that Cr-nanostructures can interact with proteins from both human and bovine serums. On SDS-PAGE, the molecular weights of the proteins were between 40 to 90 kDa. The LC-Q-Tof-MS results suggest that Cr-nanostructures are the result of the interaction with numerous proteins present in serum. However, the complete analysis of results demonstrates that only two proteins (in both RPMI and DMEM) are implicated in these nanostructures: albumin and trans-ferrin. For both proteins, at least 40 peptides matched to the complete sequence of the proteins. The ion scores (“peptide identity score”) were between 79 and 108. Ion scores > 45 indicate identity or extensive homology

Conclusions: Human serum contains more than 400 different proteins. Albumin, the major protein of human serum, has been shown to play a scavenger role by binding and transporting injected and ingested Cr. Albumin could also play an immunological role by addressing signals to defense cells, such as macrophages. Trans-ferrin, known as an iron-carrying protein, also plays a scavenger role for Cr. This suggests that the binding of Cr to these proteins may protect cells from the cytotoxic effect of Cr ions. However, the relation with Cr nano-structures in vivo remains to be determined