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

Area of Science:

• Biomedical Engineering
• Regenerative Medicine
• Microscopy

Background:

• Label-free imaging techniques like CARS and SHG are crucial for non-invasive cellular and tissue analysis.
• Previous live-imaging studies raised concerns about potential molecular alterations in sensitive biological samples.
• Validating the non-invasive nature of these techniques is essential for applications in stem cell differentiation and tissue engineering.

Purpose of the Study:

• To assess the impact of Coherent anti-Stokes Raman scattering (CARS) and second harmonic generation (SHG) live imaging on human skeletal cell differentiation.
• To demonstrate the live-imaging capability of CARS and SHG microscopy in engineered neo-cartilage.
• To establish the non-invasive nature of label-free nonlinear microscopy for tissue engineering applications.

Main Methods:

• Monitoring human fetal-femur derived skeletal cell differentiation into cartilage in 3D cultures.
• Utilizing CARS and SHG microscopy for live-imaging of developing neo-tissue over time.
• Analyzing cell phenotype, gene expression, growth, and behavior post-imaging.

Main Results:

• Conclusively established that non-linear label-free imaging does not alter cell phenotype or gene expression during differentiation.
• Demonstrated no adverse effects on human skeletal cell growth and behavior.
• Showcased CARS microscopy's ability to image lipids, proteins, and glycosaminoglycans in bioengineered neo-cartilage.

Conclusions:

• Live CARS and SHG imaging are truly non-invasive, label-free techniques.
• These methods do not induce molecular or phenotypic changes in differentiating skeletal cells.
• CARS/SHG microscopy holds significant value and translation potential for skeletal research, regeneration medicine, and tissue engineering.