Markus T Berninger1,2, Pouyan Mohajerani3, Melanie Kimm4, Stephan Masius5, Xiaopeng Ma3, Moritz Wildgruber4,6, Bernhard Haller7, Martina Anton8, Andreas B Imhoff5, Vasilis Ntziachristos3, Tobias D Henning9,10, Reinhard Meier4,11. 1. Department of Orthopaedic Sports Medicine, Klinikum rechts der Isar, Technische Universität München, Munich, Germany. Markus.Berninger@bgu-murnau.de. 2. Department of Trauma and Orthopaedic Surgery, BG Unfallklinik Murnau, Prof.-Küntscher-Strasse 8, 82418, Murnau, Germany. Markus.Berninger@bgu-murnau.de. 3. Institute for Biological and Medical Imaging, Technische Universität München und Helmholtz Zentrum München, Neuherberg, Germany. 4. Department of Radiology, Klinikum rechts der Isar, Technische Universität München, Munich, Germany. 5. Department of Orthopaedic Sports Medicine, Klinikum rechts der Isar, Technische Universität München, Munich, Germany. 6. Department of Clinical Radiology, Universitätsklinikum Münster, Münster, Germany. 7. Institute for Medical Statistics and Epidemiology, Klinikum rechts der Isar, Technische Universität München, Munich, Germany. 8. Institute for Experimental Oncology and Therapy Research, Klinikum rechts der Isar, Technische Universität München, Munich, Germany. 9. Section of Neuroradiology, Uniklinik Köln, Cologne, Germany. 10. Section of Neuroradiology, Krankenhaus der Barmherzigen Brüder, Trier, Germany. 11. Department of Diagnostic and Interventional Radiology, Universitätsklinikum Ulm, Ulm, Germany.
Abstract
OBJECTIVES: To assess labelling efficiency of rabbit mesenchymal stem cells (MSCs) using the near-infrared dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide (DiR) and detection of labelled MSCs for osteochondral defect repair in a rabbit model using fluorescence molecular tomography-X-ray computed tomography (FMT-XCT). METHODS: MSCs were isolated from New Zealand White rabbits and labelled with DiR (1.25-20 μg/mL). Viability and induction of apoptosis were assessed by XTT- and Caspase-3/-7-testing. Chondrogenic potential was evaluated by measurement of glycosaminoglycans. Labelled cells and unlabeled controls (n = 3) underwent FMT-XCT imaging before and after chondrogenic differentiation. Osteochondral defects were created surgically in rabbit knees (n = 6). Unlabeled and labelled MSCs were implanted in fibrin-clots and imaged by FMT-XCT. Statistical analyses were performed using multiple regression models. RESULTS: DiR-labelling of MSCs resulted in a dose-dependent fluorescence signal on planar images in trans-illumination mode. No significant reduction in viability or induction of apoptosis was detected at concentrations below 10 μg DiR/mL (p > .05); the chondrogenic potential of MSCs was not affected (p > .05). FMT-XCT of labelled MSCs in osteochondral defects showed a significant signal of the transplant (p < .05) with additional high-resolution anatomical information about its osteochondral integration. CONCLUSIONS: FMT-XCT allows for detection of stem cell implantation within osteochondral regeneration processes. KEY POINTS: • DiR-labelling of MSCs shows no toxic side effects or impairment of chondrogenesis. • Fluorescence molecular tomography allows for detection of MSCs for osteochondral defect repair. • FMT-XCT helps to improve evaluation of cell implantation and osteochondral regeneration processes.
OBJECTIVES: To assess labelling efficiency of rabbit mesenchymal stem cells (MSCs) using the near-infrared dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide (DiR) and detection of labelled MSCs for osteochondral defect repair in a rabbit model using fluorescence molecular tomography-X-ray computed tomography (FMT-XCT). METHODS: MSCs were isolated from New Zealand White rabbits and labelled with DiR (1.25-20 μg/mL). Viability and induction of apoptosis were assessed by XTT- and Caspase-3/-7-testing. Chondrogenic potential was evaluated by measurement of glycosaminoglycans. Labelled cells and unlabeled controls (n = 3) underwent FMT-XCT imaging before and after chondrogenic differentiation. Osteochondral defects were created surgically in rabbit knees (n = 6). Unlabeled and labelled MSCs were implanted in fibrin-clots and imaged by FMT-XCT. Statistical analyses were performed using multiple regression models. RESULTS:DiR-labelling of MSCs resulted in a dose-dependent fluorescence signal on planar images in trans-illumination mode. No significant reduction in viability or induction of apoptosis was detected at concentrations below 10 μg DiR/mL (p > .05); the chondrogenic potential of MSCs was not affected (p > .05). FMT-XCT of labelled MSCs in osteochondral defects showed a significant signal of the transplant (p < .05) with additional high-resolution anatomical information about its osteochondral integration. CONCLUSIONS: FMT-XCT allows for detection of stem cell implantation within osteochondral regeneration processes. KEY POINTS: • DiR-labelling of MSCs shows no toxic side effects or impairment of chondrogenesis. • Fluorescence molecular tomography allows for detection of MSCs for osteochondral defect repair. • FMT-XCT helps to improve evaluation of cell implantation and osteochondral regeneration processes.
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