title: Noninvasive Monitoring of Three-Dimensional Chondrogenic Constructs Using Molecular Beacon Nanosensors.
abstract: Chondrogenic differentiation of human mesenchymal stem cells (MSCs) in three-dimensional hydrogel holds promise as a method for repairing injured articular cartilage. Given MSC plasticity (its potential to mature into alternative lineages), nondestructive monitoring is critical for the optimization of chondrogenic differentiation conditions and the evaluation of the final product. However, conventional validation/assessments of the differentiation process (i.e., quantitative reverse transcription polymerase chain reaction [qRT-PCR] and histology) are end-point assays requiring disruption of the sample. This report introduces molecular beacon (MB)-based nanosensors to achieve noninvasive monitoring of chondrogenic differentiation. These nanosensors consist of biodegradable poly(lactic-co-glycolic acid) nanoparticles (PLGA NPs) encapsulating MBs to detect Type II Collagen (Col2) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNAs that serve as lineage-specific and housekeeping biomarkers, respectively. The sustainable release of MBs from MB-NPs allows longitudinal monitoring of MSCs undergoing chondrogenic differentiation over a period of 28 days. Dual-colored MB loading ensures accurate assessment of Col2 mRNA expression level, where potential heterogeneity in nanosensor uptake and retention by MSCs are taken into account. When normalized nanosensor signal was compared against qRT-PCR result, a tight correlation was observed (R2= 0.9301). Finally, nanosensor usage was compatible with MSC potency with minimal influence on chondrogenic, adipogenic, and osteogenic differentiation.
authors.affiliation: 1 School of Chemical and Biomedical Engineering, Nanyang Technological University , Singapore, Singapore .|2 Nanyang Institute of Technology in Health & Medicine, Interdisciplinary Graduate School, Nanyang Technological University , Singapore, Singapore .
authors.forename: Li Min
authors.initials: LM
authors.lastname: Tay
authors.affiliation: 1 School of Chemical and Biomedical Engineering, Nanyang Technological University , Singapore, Singapore .
authors.forename: Christian
authors.initials: C
authors.lastname: Wiraja
authors.affiliation: 1 School of Chemical and Biomedical Engineering, Nanyang Technological University , Singapore, Singapore .
authors.forename: David C
authors.initials: DC
authors.lastname: Yeo
authors.affiliation: 3 Tissue Engineering Program, Life Sciences Institute, National University of Singapore , Singapore, Singapore .|4 Department of Orthopaedic Surgery, Yong Loo Lin School of Medicine, National University of Singapore , Singapore, Singapore .
authors.forename: Yingnan
authors.initials: Y
authors.lastname: Wu
authors.affiliation: 3 Tissue Engineering Program, Life Sciences Institute, National University of Singapore , Singapore, Singapore .|4 Department of Orthopaedic Surgery, Yong Loo Lin School of Medicine, National University of Singapore , Singapore, Singapore .
authors.forename: Zheng
authors.initials: Z
authors.lastname: Yang
authors.affiliation: 1 School of Chemical and Biomedical Engineering, Nanyang Technological University , Singapore, Singapore .
authors.forename: Yon Jin
authors.initials: YJ
authors.lastname: Chuah
authors.affiliation: 3 Tissue Engineering Program, Life Sciences Institute, National University of Singapore , Singapore, Singapore .|4 Department of Orthopaedic Surgery, Yong Loo Lin School of Medicine, National University of Singapore , Singapore, Singapore .
authors.forename: Eng Hin
authors.initials: EH
authors.lastname: Lee
authors.affiliation: 5 Faculty of Materials and Energy, Institute for Clean Energy and Advanced Materials, Southwest University , Chongqing, People's Republic of China .
authors.forename: Yuejun
authors.initials: Y
authors.lastname: Kang
authors.affiliation: 1 School of Chemical and Biomedical Engineering, Nanyang Technological University , Singapore, Singapore .|6 NTU-Northwestern Institute for Nanomedicine, Nanyang Technological University , Singapore, Singapore .
authors.forename: Chenjie
authors.initials: C
authors.lastname: Xu
mesh_terms: D015374:Biosensing Techniques / Q000379:methods*; D002454:Cell Differentiation; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology*; D020219:Chondrogenesis / Q000502:physiology; D006801:Humans; D020100:Hydrogels / Q000737:chemistry*; D021621:Imaging, Three-Dimensional / Q000379:methods*; D059630:Mesenchymal Stem Cells / Q000166:cytology*; D018613:Microscopy, Confocal; D036103:Nanotechnology
chemical_list: D020100:Hydrogels
keywords: agarose hydrogel; chondrogenic differentiation; mesenchymal stem cells; molecular beacon; nanosensor