Joint tissues consist of trabecular and cortical bone as well as calcified and hyaline cartilage, which presents a challenge for hard X-ray-based visualization on the sub-cellular level due to the wide range of local X-ray absorption values. The density of the calcified tissues requires rather high photon energy, which often leads to insufficient contrast within the cartilage and impedes the visualization of individual biological cells. Decalcification of the tissues reduces the total and local X-ray absorption values and allows for selecting a lower photon energy. Further contrast enhancement can be achieved by ethanol fixation and paraffin tissue embedding. In this study, we (i) searched for an appropriate visualization method to investigate lesions generated by a laser osteotome and (ii) visualized a decalcified porcine joint after ethanol fixation and subsequent paraffin embedding using laboratory- and synchrotron radiation-based microtomography. The experiments at the ANATOMIX beamline of Synchrotron SOLEIL were performed in off-axis scan mode with a pixel size of 1.3 µm. Individual cells in all layers of the joint could be made visible and the effect of ethanol fixation and paraffin embedding demonstrated.
We are engineering cartilage from autologous nasal chondrocytes and a collagen scaffold in chondrogenic conditions to treat knee cartilage defects in an ongoing phase II clinical trial. To comply with regulatory requirements, we are developing quality controls to characterize and ensure the safety and quality of the engineered cartilage products. Our preliminary results show that we can measure the Raman spectra of engineered cartilage. Here we propose a standardized procedure for collecting and preprocessing the Raman spectral data. We currently have experienced, trained technicians manufacturing the engineered cartilage, but in the future, these grafts will be made by various labs, therefore ensuring the standardization of the manufacturing process is a challenge that could be addressed with Raman spectroscopy-based quality controls. In this manuscript we discuss how Raman spectroscopy-based quality controls could be incorporated into the Good Manufacturing Practice (GMP) compliant process for our engineered cartilage.
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