Bone Abstracts

Multiphoton microscopy (MPM) is a powerful approach to visualize live cells and tissues, including bone, in three dimensions and over time. To enable preclinical longitudinal monitoring of bone by intravital MPM we developed an animal window model of bone using existing tissue engineering strategies. Electrospun polycaprolactone scaffolds and human mesenchymal stem cells after osteoblastic differentiation, in vitro, were implanted into the mouse skin in the presence of BMP7 to generate a tissue engineered bone construct (TEBC) and characterized by μCT, bone histomorphometry, and histology. After a phase of massive bone deposition and primary remodelling (weeks 1–4), TEBC stabilizes, showing i) consistent cortical thickness, trabecular bone, and bone resident cells and ii) the best cortical thickness/bone cavity ratio compared to other mouse bones, providing optimal optical accessibility.

For intravital multiparameter 3D visualization of the neobone through the body window, MPM excitation was used to co-register the collagenous and mineralized bone matrix (second harmonic, fluorescent-labeled bisphosphonates), adipocytes and bone surface (third harmonics), blood vessels and stromal phagocytes (fluorescence-labeled dextran) and osteoclasts (cathepsin K). Whereas the cortical bone was well-preserved and intact in native bone, co-implantated human prostate cancer cells (PC3) induced reactive remodeling of bone (osteolysis and osteoplastic apposition) together with expansive growth of the lesion.

In conclusion, by combining innovative tissue engineering with optical windows, state-of-the-art fluorescence reporter technology and intravital MPM, integrated analysis of bone formation and remodeling will enhance mechanistic insight into bone physiology and disease.

Disclosure: The authors declared no competing interests.