Quantitative temporal interrogation in 3D of bioengineered human cartilage using multimodal label-free imaging

Abstract

The unique properties of skeletal stem cells have attracted significant attention in the development of strategies for skeletal regeneration. However, there remains a crucial unmet need to develop quantitative tools to elucidate skeletal cell development and monitor the formation of regenerated tissues using non-destructive techniques in 3D. Label-free methods such as coherent anti-Stokes Raman scattering (CARS), second harmonic generation (SHG) and two-photon excited auto-fluorescence (TPEAF) microscopy are minimally invasive, non-destructive, and present new powerful alternatives to conventional imaging techniques. Here we report a combination of these techniques in a single multimodal system for the temporal assessment of cartilage formation by human skeletal cells. The evaluation of bioengineered cartilage, with a new parameter measuring the amount of collagen per cell, collagen fibre structure and chondrocyte distribution, was performed using the 3D non-destructive platform. Such 3D label-free temporal quantification paves the way for tracking skeletal cell development in real-time and offers a paradigm shift in tissue engineering and regenerative medicine applications.

Live-imaging of Bioengineered Cartilage Tissue using Multimodal Non-linear Molecular Imaging

“…These encode the α-chain of hyaline cartilage-specific Type II collagen and the main proteoglycan in cartilage, respectively, in fetal skeletal cells following 21 days of chondrogenic culture (Fig. 3), as previously demonstrated 27,28 . COL10A1 , which encodes the α-chain of Type X collagen expressed by hypertrophic chondrocytes, and SOX9 , the chondrogenic transcription factor, were negligibly expressed after 21 days of culture (gene amplification only detected beyond 30 cycles).…”

“…Surrounding skeletal muscle and connective tissue were removed. Segments were cut from each sample, treated and cultured as in our previous publications 27 . Briefly, cells were harvested at 85% confluence using 0.025% (w/v) trypsin/EDTA with 0.05% glucose for 5 minutes at 37 °C and cryopreserved in 10% (v/v) dimethyl sulfoxide (DMSO) in fetal calf serum (FCS).…”

Characterization of Mesenchymal Stem Cell Differentiation within Miniaturized 3D Scaffolds through Advanced Microscopy Techniques

“…Similar to what we found by enlarging the distance among scaffold matrixes, Martinez demonstrated that collagen deposited in highly oriented manner in the whole volume of the channel without any external intervention except for the mechanical one. Furthermore, the presence of collagen observed by CARS microscopy resulted in a different signal localization with respect to the SHG one, as found in other works [52,53], giving a wider information content with respect to fluorescence microscopy of fixed and stained samples.…”

Nonlinear Optical Microscopy: From Fundamentals to Applications in Live Bioimaging

“…In the last decade, it has been demonstrated that non-invasive nonlinear microscopy represents a promising strategy for label-free live imaging of 3D engineered tissues. In fact, several studies had been performed to characterize morphology ( Hofemeier et al, 2016 ; Nguyen et al, 2017 ; Syverud et al, 2017 ; Costa Moura et al, 2018 ; Kaushik et al, 2019 ; Moura et al, 2019 ), functionality ( Hofemeier et al, 2016 ; Li et al, 2017 ; Syverud et al, 2017 ; Cong et al, 2019 ; Okkelman et al, 2020 ), composition and distribution of chemicals ( Hofemeier et al, 2016 ; Li et al, 2017 ; Syverud et al, 2017 ; Costa Moura et al, 2018 ; Moura et al, 2019 ; Sood et al, 2019 ), invasion, infiltration and mechano-regulation of cellular constructs ( Hofemeier et al, 2016 ; Nguyen et al, 2017 ; Syverud et al, 2017 ; Costa Moura et al, 2018 ; Kaushik et al, 2019 ; Moura et al, 2019 ; Sood et al, 2019 ), which have been collected in Table 4 . A representative study made by Hofemeier et al (2016) carried out a multi-spectral CARS and SHG imaging on an engineered bone tissue from stem cell differentiation toward osteogenic phenotype within 3 weeks of culture.…”

“…This work established label-free NLO microscopy as a valid alternative to collagen and myosin immunostaining, even if the tissue construct was fixed and sliced before the acquisitions, and only monolayered cells were imaged in their entirety and vitality. Contrarily, Moura et al ( Costa Moura et al, 2018 ; Moura et al, 2019 ) investigated the composition and the distribution of collagen, proteins and lipids with bioengineered cartilage constructs by the use of multi-modal TPEF, SHG and CARS microscopy both in the fingerprint than in the C-H stretch region of the Raman spectrum. To date, Moura provided a comparison between SHG and CARS imaging of collagen to highlight the difference in signal distribution and observed the synthetic constructs in their thickness.…”