Characterising Type I Collagen Architecture in Human Bone and Osteosarcoma with Second Harmonic Generation Microscopy

Aberrant collagen matrix production is a hallmark of many cancers, including osteosarcoma (OS), the most common primary cancer of bone which remains poorly diagnosed. Here, the phenotyping potential of second harmonic generation (SHG) microscopy has been demonstrated through label-free imaging of pathologically altered collagen matrices within clinical biopsies. This work examined SHG imaging in quantifying OS-specific collagen (type I) signatures – focused on fibre length-based parameters – across clinical biopsies. We describe a novel SHG analysis workflow, following systematic optimisation of image resolution and field of view (FOV) size to enable robust quantification of collagen fibre-length metrics in human bone and OS. This included analyses of SHG images with a FOV spanning 350 µm × 350 µm to 1400 µm × 1400 µm, with the latter enabling whole-biopsy visualisation via stitching of up to 64 regions. In normal cortical (rib) bone biopsies, average fibre lengths did not significantly differ across increasing FOV size (26.44 ± 1.75 µm, 26.34 ± 2.01 µm, 27.25 ± 2.47 µm, 28.78 ± 3.58 µm). However, analysis of the top 50th percentile of collagen fibres revealed significant differences between the smallest FOVs; 350 µm × 350 µm and 466 µm × 466 µm, versus the largest 1400 µm × 1400 µm (p = 0.03 and p = 0.04 respectively), highlighting the requirement for larger FOVs for optimal characterisation of human bone collagen. Comparison of normal bone and OS (stage IIB) biopsies, the largest FOV revealed the largest differences, with OS exhibiting significantly reduced collagen fibre lengths (female OS: 23.18 ± 0.56 µm vs. bone: 29.39 ± 2.17 µm, p < 0.001; male OS: 24.39 ± 1.32 µm vs. bone: 27.87 ± 1.05 µm, p = 0.02) versus normal bone. Subsequent analyses across OS stages (IB – IVB) showed further evidence of pathological collagen signatures amplified with OS progression. These findings demonstrate that SHG microscopy enables robust, quantitative phenotyping of human bone and osteosarcoma collagen matrices, offering new potential for improved diagnosis and disease staging.