For the treatment of ununited fractures, we developed a system of delivering magnetic labelled mesenchymal stromal cells (MSCs) using an extracorporeal magnetic device. In this study, we transplanted ferucarbotran-labelled and luciferase-positive bone marrow-derived MSCs into a non-healing femoral fracture rat model in the presence of a magnetic field. The biological fate of the transplanted MSCs was observed using luciferase-based bioluminescence imaging and we found that the number of MSC derived photons increased from day one to day three and thereafter decreased over time. The magnetic cell delivery system induced the accumulation of photons at the fracture site, while also retaining higher photon intensity from day three to week four. Furthermore, radiological and histological findings suggested improved callus formation and endochondral ossification. We therefore believe that this delivery system may be a promising option for bone regeneration.

Summary Statement. A single, locally-delivered injection of a human placental product containing multipotent stromal cells reduced severity of infection in an immunosuppressed murine osteomyelitis model and eliminated infection in 25% of animals compared with 0% of controls without the use of antibiotics. Introduction. Implant–associated osteomyelitis is a serious orthopaedic condition and is particularly difficult to treat in immunosuppressed individuals. Despite great advancement in the field of biomaterials and pharmaceuticals, emerging patterns of antibiotic resistance, complex biofilm production and penetration of therapeutic concentrations of effective antibiotics into bone continue to represent unmet clinical challenges. The promise of adult multipotent stromal cells (MSCs) for tissue regeneration has been of intense interest in recent years. Among their many potential therapeutic uses, MSCs have also been shown to have direct antimicrobial properties. The objective of this study was to evaluate the efficacy of a locally–delivered human placental-based tissue product containing multipotent stromal cells (hAmSC) to reduce the severity of implant-associated Staphylococcus aureus osteomyelitis in an immunosuppressed murine model. We hypothesised that athymic mice with implant-associated osteomyelitis would have diminished infection following treatment with hAmSC as evidenced by decreased bioluminescence intensity and lower histologic scores for infection and bacterial load when compared to saline-treated controls. Methods. An athymic murine model of chronic implant-associated osteomyelitis was developed using luciferase-transfected Staphylococcus aureus to study the antimicrobial effects of a human placental-based product containing multi-potent stromal cells (hAmSC). Sixteen athymic mice had osteomyelitis established in the right femoral diaphysis. Fifteen days after inducing luc S. aureus osteomyelitis, the mice were randomised to receive a single 0.5 cc injection of hAmSC (n=8) or vehicle (0.9% saline) (n=8) into the soft tissues immediately adjacent to the infected bone. No antibiotics were administered throughout the duration of the study. Mice were imaged with an In Vivo Imaging System (IVIS 1000, PerkinElmer) twice weekly for 30 days to assess change in bioluminescence intensity from baseline immediately prior to treatment with either hAmSC or saline. Radiographs were obtained at days −10, 0, 10, 20 and 30 days post-injection and scored for bone changes secondary to osteomyelitis by a reviewer blinded to treatment group. Mice were sacrificed 30 days after treatment and femurs were examined histologically and scored for bacterial load and degree of inflammation by a pathologist blinded to treatment group. Results. Osteomyelitis was successfully established in all mice as evidenced by baseline bioluminescence imaging and radiographs. Mean bioluminescence intensity decreased from baseline in animals receiving hAmSC and remained below baseline for 28 days, whereas vehicle-treated animals showed an increase in mean bioluminescence intensity throughout the study period. Osteomyelitis resolved in 2/8 hAmSC-treated animals and 0/8 vehicle-treated animals as evidenced by bioluminescence imaging and histological examination for bacteria/inflammation at sacrifice. Radiograph scores for secondary bone changes were lower in mice treated with hAmSC than vehicle at 10, 20 and 30 days post injection. Median inflammatory score was lower in the hAmSC-treated mice than vehicle treated controls. Conclusions. A single injection of hAmSC was effective at reducing the severity of S. aureus infection without the use of antibiotics in this chronic implant associated osteomyelitis immunosuppressed murine model. In addition to reduced bioluminescence intensity below baseline for 28 days during the study period, infection was eliminated in 25% of animals in the hAmSC-treated group

Background. Large bone defects still challenge the orthopaedic surgeon. Local vascularity at the site of the fracture has an important influence on the healing procedure. Vascular endothelial growth factor (VEGF) and it's receptor (VEGFR2) are potent inducer of angiogenesis during the fracture healing. Aim of the present study was the investigation of critical size fracture (CSF) healing in VEGFR2-luc mice using tailored scaffolds. Methods. CSFs were performed and stabilised in mouse femur using an external fixator. The fracture was bridged using a synthetic 3D printed scaffold with a defined porosity to promote regeneration. The ß-tricalciumphosphate (ßTCP) and strontium doped ß-tricalciumphosphate (ßTCP+Sr) scaffolds were investigated for their regenerative potential. The expression levels of VEGFR2 could be monitored non-invasively via in vivo bioluminescence imaging for 2 months. After the longitudinal measurements the animals were euthanised for an in depth histological endpoint analysis. The different scaffold induced tissue regeneration was quantified for both, the ßTCP and the ßTCP+Sr group. Results. Expression levels of VEGFR2 were significantly higher in the ßTCP+Sr group when compared to the ßTCP, control and sham group. Both types of scaffolds significantly enhanced new bone formation when compared to the sham group. The ßTCP+Sr scaffolds showed a significantly greater regenerative potential. Conclusions. This standardised defect model mimics a clinically relevant situation to study the regenerative effects of biomaterials on bone. Moreover, the rate of regeneration correlates with the VEGFR2 expression levels, what affirms the usability of our method for longitudinal fracture healing studies. As in line with relevant literature, it could be shown that strontium does have an enhancing effect on bone regeneration. Consequently, strontium doped scaffolds might be a useful addition in the surgeon's spectrum of methods. Level of Evidence. Experimental

Summary. Despite similar, early and massive death, hMSCs promote bone formation which was higher in orthotopic than ectopic site suggesting a trophic effect of hMSCs. Ectopic implantation is suitable to evaluate cell survival, but assessment of bone formation requires orthotopic implantation. Introduction. Tissue constructs containing mesenchymal stem cells (MSCs) are appealing strategies for repairing large segmental bone defects but they do not allow consistent bone healing and early and massive MSCs death was identified as a cause of failure. However, little is known about cell survival in the clinical micro-environment encountered during bone healing process, whereas ectopic evaluation is well documented. In vivo, luciferase-labelled human MSCs survival, within osteoconductive scaffold, was compared in orthotopic and ectopic locations, and bone formation ability of LF-hMSCs-Acropora constructs was evaluated. Interest and limits of each model were highlighted. Methods. Osteoconductive scaffold with or without LF-hMSCs were implanted either in a critical-segmental-femoral-bone defect stabilised by plate or subcutaneously in 44 mice. Cells survival was evaluated by serial bioluminescence imaging (BLI) and osteogenic capabilities by histology and microCT. Twenty mice were sacrificied 15 days after surgery for “short term” evaluation. The other mice were kept for 10 weeks after surgery for “long term” evaluation. Results. BLI provided evidence of fast and continuous cell death: 85% decrease of the BLI signal over the first 15 days in both location; less than 2% of the initial cell number were present in all constructs analyzed 30 days post-implantation and less than 1% of the initial cell number was present in all constructs analyzed 55 days post-implantation. By 2 weeks post implantation, the amount of newly formed bone was self-limited and was similar between ectopic and orhtotopic group, with or without cell. By 10 weeks post implantation, bone formation was significantly enhanced in the presence of LF-hMSC. The amount of newly formed bone in the cell-containing constructs groups was significantly higher than that observed in the scaffold alone groups. Most importantly, the amount of newly formed bone in cell-containing constructs implanted in orthotopic locations was significantly higher than that observed in the ectopic, cell-containing construct group. Conclusion. Corroborating previous ectopic studies, our results indicated that hMSCs promote bone formation despite early and massive cell death when loaded on ceramic scaffold. Interestingly, bone formation was higher in orthotopic than ectopic site despite a same survival pattern and a massive and early cell death, suggesting a trophic effect of hMSCs. Ectopic implantation of cell-containing constructs is suitable to evaluate cell survival, but assessment of bone formation ability requires orthotopic implantation

Summary. In this study, we challenged the current paradigm of human Mesenchymal Stem Cells survival, which assigned a pivotal role to oxygen, by testing the hypothesis that exogenous glucose may be key to their survival. Introduction. The survival of human mesenchymal stem cells (hMSCs) has elicited a great deal of interest, because it is relevant to the efficacy of engineered tissues. However, to date, hMSCs have not met this promise, in part due to the high death rate of cells upon transplantation. In this study, we challenged the current paradigm of hMSC survival, which assigned a pivotal role to oxygen, by testing the hypothesis that exogenous glucose may be key to hMSC survival. Materials and methods. In vitro model of ischemia 2.10. 4. hMSCs from five donors, were seeded into individual wells of a 24-well plate, cultured overnight, washed twice with PBS and then maintained in hypoxia (0.1% oxygen) under serum (FBS) free αMEM medium in either the absence or in the presence (1 or 5 g/L) of glucose for 21 days. In vitro Cell viability: To assess the role of glucose on hMSCs viability, cells were cultured under hypoxia in the absence or in the presence of glucose (1 and 5g/L), At days 0, 3, 7, 14 and 21, cell viability was evaluated by flow cytometry and ATP content per cell quantified. In vivo effect of glucose supply on hMSCs viability. 3.10. 5. eGFG-luc hMSCs were seeded on a cylindrical AN-69 scaffolds. At the time of implantation, 100 µl of hyaluronic acide (HA) (2%) containing either 0g/L (negative control) or 10g/L of glucose was gently injected inside the construct. Cell- constructs were implanted subcutaneously in eight week-old mice (2 per animal) and were imaged by bioluminescence imaging (BLI) at day 1, 4, 7 and 14 until sacrifice. Results. hMSCs were able to survive and to maintain their ATP content 21 days under sustained hypoxia providing that they were cultured in the presence of a sufficient glucose supply (i.e. 5g/L). In contrast, hMSCs cultured without or with 1g/L of glucose failed to survive. These results established that glucose depletion but not sustained hypoxia affected cell survival. In vivo results showed a striking increase of cell viability in cell constructs loaded with glucose. At day 14, a five-fold increase in cell number was observed in cell constructs loaded with glucose when compared to the control cell constructs without glucose. Discussion. The present study challenge the current paradigm that gives a pivotal role to oxygen on hMSCs massive cell death. By using an in vitro model of hypoxia/ischemia, we demonstrated that in the presence of sufficient glucose, hMSCs were able to survive 21 days under sustained hypoxia. Most importantly, an appropriate glucose supply strongly increases cell viability of hMSCs implanted subcutaneously in a mice model. This study provides evidences that glucose depletion but not hypoxia affects hMSCs viability. Further investigations need to be performed to develop hydrogels that ensure continuous glucose delivery to the implanted cells. Theses findings are particularly relevant because they pave the way to the development of new delivery systems to ensure hMSCs viability in order to increase their therapeutical potential after implantation