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Vibrational spectroscopic monitoring and biochemical analysis of pericellular matrix formation and maturation in a 3-dimensional chondrocyte culture model

DOI: 10.1039/C8AN01272E DOI Help

Authors: Hamza Owida (University of Keele) , Abigail V. Rutter (University of Keele) , Gianfelice Cinque (Diamond Light Source) , Nicola J. Kuiper (University of Keele) , Josep Sulé-suso (Keele University; University Hospital of North Midlands) , Ying Yang (University of Keele)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Analyst

State: Published (Approved)
Published: September 2018
Diamond Proposal Number(s): 17530

Abstract: Isolated and monolayer expanded chondrocytes are not the ideal cell form to produce cartilage matrix. In articular cartilage, each chondrocyte is surrounded by a 2-4 µm thick collagen VI-rich pericellular matrix (PCM) forming a chondron. Freshly extracted chondrons form a more cartilage-like extracellular matrix (ECM) than chondrocytes and their surrounding PCM is thought to maintain chondrocyte phenotype. To regenerate articular cartilage, preserving and/or regenerating a functional PCM is essential. In this study, a highly biomimic hyaluronic acid (HA) hydrogel was used as a 3-dimensional system to culture freshly isolated bovine chondrons (with an intact PCM) and chondrocytes (without a PCM) for up to 21 days. We assessed the HA hydrogels capacity to maintain and potentially re-generate PCM formation by both biochemical and immunological analyses for key components of the PCM. For the first time, Synchrotron based Fourier Transform Infrared microspectroscopy was utilised to reveal the dynamic process of PCM re-generation. At day 1, highly specific collagen VI staining was visible within chondron containing HA hydrogels. By contrast, collagen VI was absent at day 1 but punctate, focal staining increased during the culture period of chondrocyte containing HA hydrogels. Chondron containing HA hydrogels produced more collagen II and GAG than the chondrocyte containing HA hydrogels. Principal component analysis (PCA) of spectra in fingerprint regions on the chondrocyte-containing constructs at day 7, 14 and 21 culturing showed clear spectral differences. The clusters of day 14 and day 21 samples were closer to chondron samples, whilst day 7 samples were closer to chondrocytes. PCA scores in the lipid region revealed no major differences between chondrocytes and chondron samples, but differences between samples at day 7, day 14 and day 21. These data would indicate that SR-FTIR micro-spectroscopy can help to better understand the PCMs formation and maturation in tissue engineered models, which involves subtle changes in collagen and aggrecan.

Subject Areas: Chemistry

Instruments: B22-Multimode InfraRed imaging And Microspectroscopy

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