Objectives. In order to ensure safety of the cell-based therapy for bone regeneration, we examined in vivo biodistribution of locally or systemically transplanted osteoblast-like cells generated from bone marrow (BM) derived mononuclear cells. Methods. BM cells obtained from a total of 13 Sprague-Dawley (SD) green fluorescent protein transgenic (GFP-Tg) rats were culture-expanded in an osteogenic differentiation medium for three weeks. Osteoblast-like cells were then locally transplanted with collagen scaffolds to the rat model of segmental bone defect. Donor cells were also intravenously infused to the normal Sprague-Dawley (SD) rats for systemic biodistribution. The flow cytometric and histological analyses were performed for cellular tracking after transplantation. Results. Locally transplanted donor cells remained within the vicinity of the transplantation site without migrating to other organs. Systemically administered large amounts of osteoblast-like cells were cleared from various organ tissues within three days of transplantation and did not show any adverse effects in the transplanted rats. Conclusions. We demonstrated a precise assessment of donor cell biodistribution that further augments prospective utility of regenerative cell therapy. Cite this article: Bone Joint Res 2014;3:76–81

The field of nanoparticle related research for the diagnosis and therapy of diseases evolves rapidly. Magnetic nanoparticles in combination with magnetizable implant materials for the treatment of implant related infections present a possible implementation in orthopedics. Magnetic nanoporous silica nanoparticles (MNPSNPs) were developed and equipped with fluorescent dyes. In vitro/in vivo biocompatibility and in vivo biodistribution were examined to appraise their potential applicability. Cell culture tests with NIH-3T3 and HepG2 cell lines indicated a good in vitro biocompatibility. Ferritic and titanium alloy (control) plates were implanted subcutaneously at the hind legs of Balb/c mice. Immediately after i.v. or s.c. injection of MNPSNPs, the caudal half of the mice was placed between the poles of an electro magnet. Exposure to the electromagnetic field of approx. 1.7 T was maintained for 10 minutes. 10 animals each were euthanized at days 0, 1, 7, 21 or 42, respectively. Quantity of MNPSNPs in liver, spleen, kidney, lung and skin/muscle samples was assessed by fluorescent microscopic methods. MNPSNP existence on the implant surface was also appraised after several steps of detachment. MNPSNPs showed a time-dependent accumulation in the organs after i.v. injection with initial accumulation in the lungs followed by redistribution to liver and spleen. After s.c. injection no systemic distribution but local appearance of MNPSNPs could be found. First histological evaluation showed no pathological changes after i.v. injection. With good in vivo biocompatibility, future focus will be laid on increasing circle life time of MNPSNPs and evaluation in an infection model

Introduction. Low back pain (LBP) is a worldwide leading cause of disability. This preclinical study evaluated the safety of a combined advanced therapy medicinal product developed during the European iPSpine project (#825925) consisting of mesendoderm progenitor cells (MEPC), derived from human induced pluripotent stem cells, in combination with a synthetic poly(N-isopropylacrylamide) hydrogel (NPgel) in an ovine intervertebral disc degeneration (IDD) model. Method. IDD was induced through nucleotomy in 4 adult sheep, 5 lumbar discs each (n=20). After 5 weeks, 3 alternating discs were treated with NPgel (n=6) or NPgel+MEPC (n=6). Before sacrifice, animals were subjected to: MRI of lumbar spines (disc height and Pfirmann grading); blood sampling (hematological, biochemical, metabolic and lymphocyte/monocytes immunological). After 3 months the sheep were sacrificed. The spines were processed for: macroscopic morphology (Thompson grading), microscopic morphology (Histological grading), and glycosaminoglycan content (GAG, DMMB Assay). Furthermore, at sacrifice biodistribution of human MEPC was assessed by Alu-sequences quantification (qPCR) from three tissue samples of heart, liver, spleen, brain, lungs, and kidneys, and PBMCs collected to assess activation of systemic immune cells. To each evaluation, appropriate statistical analysis was applied. Result. Flow cytometry showed no induction of systemic activation of T cells or monocytes. Alu quantification did not give detection of any cells in any organ. Disc height index was slightly increased in discs treated with NPgel+MEPC. Pfirmann's and Thompson's classification showed that treatment with NPgel or NPgel+MEPC gave no adverse reactions. Histological grading showed similar degeneration in vertebrae treated with NPgel+MEPC or with NPgel alone. The amount of GAG was significantly increased in the nucleus pulposus following treatment with NPgel+MEPC compared to NPgel alone, in which a decrease was observed compared to untreated discs in both nucleus pulposus and annulus fibrosus. Conclusion. This study showed the safety of both NPgel+MEPC and NPgel treatments

Tissue engineering and regenerative medicine (TERM) hold the promise to provide therapies for injured tendons despite the challenging cues of tendon niche and the lack of specific factors to guide regeneration. The emerging potential of magnetic responsiveness and magnetic nanoparticles (MNPs) functionalities offers new perspectives to tackle TERM challenges. Moreover, pulsed electromagnetic field (PEMF) is FDA approved for orthopaedics with potential to control inflammation upon injury. We previously demonstrated that magnetic cell-sheets assisted by PEMF trigger the inflammation resolution by modulating cytokine-enriched environments [1]. To further understand the potential of magnetically assisted living patches, we have recently conducted in vivo studies using a rat patellar defect model. After labeling of human adipose stem cells with iron oxide MNPs for 16h, magCSs were cultured up to 3 days in α-MEM medium under non-magnetic or PEMF conditions. MagCSs were evaluated by immunocytochemistry, and real time RT-PCR for tendon markers. Cell metabolic activity was also assessed by MTS and ECM proteins quantified by Sirius Red/Fast Green. The MagCSs effect in ameliorating healing was assessed after implantation in window defects created in the patellar tendon of rats. PEMF was externally applied (3mT, 70Hz) 3d/week for 1h (magnetotherapy). After 4 and 8w, tendons were histologically characterized for immune-detection of tendon and inflammatory markers, and for Perls van Gieson and HE stains. Blood and detoxification organs were screened for inflammatory mediators and biodistribution of MNPs, respectively. In vitro results suggest that PEMF stimulates cellular metabolic activity, influences protein synthesis and the deposition of collagen and non-collagenous proteins is significantly increased compared to non-magnetic conditions. No adverse reactions, as infection or swelling, were observed after surgery or during follow-up. After 8w, magCSs remained at the implantation site and no MNPs were detected on detoxification organs. Plasma levels of IL1α, β, IL6 and TNFα assessed by multiplex assay were below detectable values (<12.5pg/ml). Thus, the combination of cell sheets and magnetic technologies hold promise for the development of living tendon substitutes. Acknowledgement to ERC-COG MagTendon772817, H2020 Achilles 810850, FCT - 2020.01157.CEECIND

Adipose derived mesenchymal stromal cells (ASC) are adult stem cells exhibiting functional properties that have open the way for cell-based clinical therapies. Primarily, their capacity of multilineage differentiation has been explored in a number of strategies for skeletal tissue regeneration. More recently, MSCs have been reported to exhibit immunosuppressive as well as healing capacities, to improve angiogenesis and prevent apoptosis or fibrosis through the secretion of paracrine mediators. Among the degenerative diseases associated with aging, osteoarthritis is the most common pathology and affects 16% of the female population over 65 years. Up to now, no therapeutic option exists to obtain a sustainable improvement of joint function beside knee arthroplasty. This prompted us to propose adipose derived stem cells as a possible cell therapy. We performed pre-clinical models of osteoarthritis and showed that a local injection of ASC showed a reduction of synovitis, reduction of osteophytes, joint stabilization, reducing the score of cartilage lesions. This work was completed by toxicology data showing the excellent tolerance of the local injection of ADSC and biodistribution showing the persistence of cells after 6 months in murine models. The aim of the ADIPOA trial is to demonstrate the efficacy of adipose derived stem cells therapy in knee osteoarthritis (OA) in a phase 2/3 controlled multicenter study controlled against standard of care. Safety and feasibility as well as dose response was previously assessed in the ADIPOA FP7 project. The bi-centric phase I clinical trial in Montpellier (France) and Würzburg (Germany) included 18 patients with moderate to severe knee OA, each patient received a single injection of autologous ADSC, in a open scale up dose trial, starting form 2 10 6 cells to 50 106 cells. The 107 dose appears to be well tolerated and showed preliminary response in terms of decreasing local inflammation. This first study confirmed the feasibility and safety of local injection of ADSC in knee OA and suggested the most effective dose (107 autologous ADSC). This work constituted a significant step forward treating this disease with ADSC to demonstrate safety of the procedure. we conduct a prospective multicenter randomized Phase 2/3 study with 86 patients with moderate to severe knee OA to demonstrate superiority of stem cell-based therapy compared to standard of care (SOC) in terms in reduction in clinical symptoms (WOMAC score) and structural benefit (assessed by T1rhoMRI that allow quantification of cartilage proteoglycan content). This project will offer EU a unique leadership in OA with strong positions in EU and US due to patents and quality of the methodology to demonstrate efficiency of ADSC. ADIPOA brings together a unique combination of expertises and leaders in clinical rheumatology, MRI specialists, Stem cell Institutes, national GMP grade adipose derived stem cell production platform (ECELLFRANCE) and SME specialized in cell therapy trials in the EU. The production of the cells will be granted to EFS through ECELLFRANCE national platform, which have the GMP facility and will work as a contracting manufacturing organization. The expertise, leadership and critical mass achieved by this Consortium should enable breakthroughs in ASC engineering directly amenable for clinical applications in OA