Abu Owida, Hamza (2018) Investigation of cellular and scaffold strategies for engineering articular cartilage. Doctoral thesis, Keele University.

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Abstract

Articular cartilage is a thin hydrated tissue, which covers articulating surfaces. In the native articular cartilage tissue, the extracellular matrix (ECM) is a fibrillar mesh of interacting proteoglycans, collagens, and other non-collagenous proteins residing in a highly aqueous environment. Chondrocytes are surrounded by a pericellular matrix (PCM) forming chondrons. The PCM, exclusively rich in collagen VI, is more easily destroyed during their extraction and subsequent in vitro culture than ECM. The retention of the PCM has a significant influence on the metabolic activity of the chondrocytes in addition to the mechanical signalling from and to the ECM via cell-matrix interactions. The ECM and the residing chondrocytes are organised into three distinct zones: superficial, middle and deep. The complex and organised structure of cartilage allows it to resist the tensile stress ‘superficial zone’, sheer stress ‘middle zone’, and compressive stress ‘middle and deep zones’ imposed by articulation.

This study initially focused on the morphology and chondrogenic capacity of chondrocytes, chondrons (bovine) and mesenchymal stromal cells (MSCs, rat) alone in monolayer cultures to establish a baseline of PCM preservation and regeneration approaches. Co-culture monolayer models of cartilage cells with MSCs (20%, 50% and 80%) was established to assess the effect of MSC on PCM maintenance and ECM production by biochemical assays, immunofluorescence and histological staining. Co-culture of MSCs with chondrons enhanced ECM production, as compared to chondrocyte or chondron monocultures. The co-culture of MSCs with chondrons appeared to decelerate the loss of the PCM as determined by collagen VI expression, whilst the expression of a high temperature requirement family of serine proteases, HtrA1, demonstrated an inverse relationship to that of the collagen VI. The 50:50 ratio of MSCs: chondrons in co-culture presented the highest potential for better cartilage regeneration. For the first time, it is confirmed that MSCs directly or indirectly inhibited HtrA1 activity in the co-culture, which played a role in enhancement of ECM synthesis and the preservation of the PCM. However, PCM could not be fully preserved or regenerated in 2D culture up to 7 day culture even starting from chondron, and co-culture with MSCs.

Next 3D model systems using hydrogels to improve PCM formation and maintenance were developed. Four culture conditions were compared; hyaluronic acid (HA) versus agarose hydrogel; basal medium versus chondrogenic medium; chondron or chondrocytes versus co-culture with 50% MSC. Up to 21 day culture, chondron samples in both mono- and co-culture maintained PCM at all culture conditions. The quantity and quality of regenerated PCM in chondrocyte samples were culture condition dependent. HA in combination with chondrogenic media and co-culture with MSC was the best support for PCM generation and ECM deposition. Basal and chondrogenic culture mediums influenced the expression of cartilage-specific ECM markers but did not affect collagen VI synthesis. Synchrotron microFTIR measurements assisting with PCA analysis of spectra in fingerprint and lipid regions on the 3D cultured samples have cross-validated that culturing chondrocytes in HA hydrogel up to 21 days might generate chondron-like cell morphology and composition because day 14 and day 21 samples clustered to chondron spectra, whilst day 7 samples to chondrocyte spectra.

Zonal-specific 3D hybrid scaffolds have been fabricated using a combination of polylactic acid and HA to induce the generation of near-native cartilage. For the superficial and middle zones, specifically orientated or randomly arranged polylactic acid nanofibre meshes were embedded in HA. For the deep zone, vertical channels in HA were created. The aligned nanofiber mesh used in the superficial zone induced an elongated cell morphology, lower GAG and collagen II production, than the middle zone scaffold. Within the middle zone scaffold, which comprised of a randomly orientated nanofiber mesh, the cells were clustered and expressed more collagen II. The deep zone scaffold induced the highest GAG production, the lowest cell proliferation and the lowest collagen I expression of the three zones. Overall a convenient and reproducible model system which mimics the zonal organisation of articular cartilage has been developed.

Item Type: Thesis (Doctoral)
Subjects: R Medicine > R Medicine (General)
Divisions: Faculty of Medicine and Health Sciences > Institute for Science and Technology in Medicine
Depositing User: Lisa Bailey
Date Deposited: 22 Oct 2018 10:47
Last Modified: 22 Oct 2018 10:47
URI: http://eprints.keele.ac.uk/id/eprint/5440

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