Non-linear optical microscopy as a novel quantitative and label-free imaging modality to improve the assessment of tissue-engineered cartilage.

OBJECTIVE: Current systems to evaluate outcomes from tissue-engineered cartilage (TEC) are sub-optimal. The main purpose of our study was to demonstrate the use of second harmonic generation (SHG) microscopy as a novel quantitative approach to assess collagen deposition in laboratory made cartilage constructs.

METHODS: Scaffold-free cartilage constructs were obtained by condensation of in vitro expanded Hoffa's fat pad derived stromal cells (HFPSCs), incubated in the presence or absence of chondrogenic growth factors (GF) during a period of 21 d. Cartilage-like features in constructs were assessed by Alcian blue staining, transmission electron microscopy (TEM), SHG and two-photon excited fluorescence microscopy. A new scoring system, using second harmonic generation microscopy (SHGM) index for collagen density and distribution, was adapted to the existing "Bern score" in order to evaluate in vitro TEC.

RESULTS: Spheroids with GF gave a relative high Bern score value due to appropriate cell morphology, cell density, tissue-like features and proteoglycan content, whereas spheroids without GF did not. However, both TEM and SHGM revealed striking differences between the collagen framework in the spheroids and native cartilage. Spheroids required a four-fold increase in laser power to visualize the collagen matrix by SHGM compared to native cartilage. Additionally, collagen distribution, determined as the area of tissue generating SHG signal, was higher in spheroids with GF than without GF, but lower than in native cartilage.

CONCLUSION: SHG represents a reliable quantitative approach to assess collagen deposition in laboratory engineered cartilage, and may be applied to improve currently established scoring systems.