Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.798816
Title: Design, development, and assessment of novel 3-dimensional co-culture systems to model musculoskeletal interfaces
Author: Alsaykhan, Hamad Mohammed H.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2019
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Abstract:
The interface between fibrous tissues (ligaments, tendons, cartilages and joint capsules) and bones is known as the enthesis. It has a unique anatomical structure transitioning from pure fibrous tissue to pure bone tissue. This unique structure gives the enthesis its ability to smoothly transfer mechanical power. However, when the enthesis is injured (e.g. in sports, automobile, or falls accidents), this unique structure is replaced by a weak scar tissue that is prone to re-injury and can cause chronic pain. The current gold standard management for enthesis repair is to re-attach the avulsed tendon directly to the bone, which results in the loss of the unique enthesis structure. To explore alternative treatments for enthesis repair, it is important to understand the normal development of the enthesis and its natural healing process. Therefore, developing a reproducible and standardised enthesis model is of great importance. The aim of this study was to design, develop and assess novel 3-dimensional (3D) co-culture systems to model the enthesis in vitro. Assessments of the 3D co-culture were intended to study two aspects: system suitability for cell culture and the effect of co-culture on extracellular matrix (ECM) formation. System suitability was determined by proving interface formation, cell viability and structural integrity using chick tendon fibroblasts (CTF) and mouse osteoblasts (MC3T3). The effect of co-culture on ECM formation was assessed by measuring the content of collagen and glycosaminoglycans (GAGs) using rat tendon fibroblasts (RTF) and bone cells (dROb). Two 3D interface co-culture methods were designed and developed: a hydrogelbased scaffold-dependent method and a scaffold-less method. The scaffolddependent 3D co-culture system was used to create an artificial 3D interface by encapsulating two populations of cells in agarose, gellan, fibrin and collagen hydrogels. A confocal fluorescent microscope was used to assess the interface presence and integrity over time. Moreover, cell viability was assessed by live-dead fluorescent staining and DNA quantification. These investigations were performed to assess hydrogel suitability for the system, which resulted in choosing fibrin hydrogel as the most suitable candidate to assess co-culture effect on ECM formation. ECM formation was assessed for bone and tendon cells encapsulated in fibrin hydrogel separately, then the summation of their results was compared to the co-culture of both. The results showed no significant effect of co-culture on ECM formation. This was followed by comparison of ECM formation in separately cultured bone and tendon cells when cultured in 3D cell-encapsulated hydrogels in standard 2D culture. Surprisingly, the ECM formation assays were significantly greater in 2D culture than 3D. Spheroids of tendon and bone cells were used as a second method of 3D co-culture interface. The interface formation between bone and tendon spheroids was observed by confocal fluorescent microscopy (CFM) and light microscopy, showing successful spheroid formation and integrity over time. ECM formation studies showed a decrease in collagen and GAGs due to co-culture. In summary, this study has evaluated two novel methodologies to create 3D tissue interfaces in vitro. These techniques will be valuable for future work to further enhance these models to study ECM formation, cell-cell interactions and responses at the enthesis as well as a number of other interfacial tissue sites (e.g., muscle-tendon, cartilage-bone, nerve-muscle).
Supervisor: Paxton, Jennifer ; Davies, Jamie Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.798816  DOI: Not available
Keywords: enthesis ; tissue engineering ; artificial 3D enthesis models ; fibrin gel ; ECM formation
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