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Graduate Student Seminar Series – Campbell Thom

April 25 @ 1:25 pm - 1:40 pm EDT

Graduate Student Seminar Series
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Location: TRI-UC (KITE) Basement Auditorium, 550 University Avenue
Presentation Title: Development of an In Vitro Model of the Outer Annulus Fibrosus-Cartilage Endplate Interface within a Perfusion Bioreactor
Abstract: BACKGROUND: Lower back pain is a leading cause of disability worldwide and is often associated with intervertebral disc (IVD) degeneration. IVDs are fibrocartilaginous tissues between the vertebral bodies of the spine which facilitate load transmission and enable spinal flexibility. Once damaged there is a lack of treatment options that permanently restore functionality. Tissue engineering aims to address the limitations of existing therapeutics by generating living tissue constructs that could replace the diseased disc. Given the complex disc architecture coupled with the limitations of conventional static culturing methods, the production of tissue constructs that recapitulate the structural features and mechanical strength of the native disc has yet to be achieved. The current study aims to develop an in vitro-formed model of the outer annulus fibrosus (OAF)-cartilage endplate interface, which is often overlooked in current research despite its critical role in maintaining structural and biochemical integrity of the IVD. It is hypothesized that in vitro-formed OAF can be integrated with in vitro-grown cartilage tissue, such that the interfacial strength of the tissue interface is significantly enhanced when cultured under perfusion flow within a bioreactor. ,,METHODS: OAF cells are isolated from bovine caudal discs and seeded onto polyurethane scaffolds, as described previously1, in a spinning bioreactor to generate in vitro-formed OAF tissue. These tissues are then co-cultured with deep zone articular chondrocytes that have been isolated from bovine caudal metacarpal-phalangeal joints and cultured on PTFE membranes. OAF-chondrocyte constructs are co-cultured statically for 7 days in mineralizing media containing β-Glycerophosphate. Co-cultures are either maintained in static culture or transferred to a perfusion bioreactor system in which they are dynamically cultured using a flow rate of 5mL/min, for an additional 7 days before performing histological or mechanical assessment. All experiments, except for mechanical testing, were repeated with 3 biological sets, with each condition done in quadruplicate. Statistical analysis was performed using a T test and significance assigned at p<0.05.,,,RESULTS:,Co-culture of in vitro-formed OAF tissue and chondrocytes generated an integrated tissue interface composed of cartilage and AF tissue. More tissue appeared to be present, histologically (Figure 1), in constructs grown in the perfusion bioreactor versus static conditions. The interface was also stronger in the perfused constructs as determined using a pull-apart testing, where the average interfacial strength of perfused and static constructs was 53.3kPa and 18.9kPa, respectively (preliminary data; N=1). Immunostaining of the OAF-cartilage constructs demonstrate collagen type I, collagen type II, and aggrecan distribution for both conditions appear similar to that of the native OAF-CEP interface (Figure 1). Von Kossa staining demonstrates cartilage mineralization under both perfusion and static culture conditions. However, OAF-cartilage constructs that undergo perfusion appear to have more mineral deposition.,,CONCLUSIONS: This study provides a first-generation in vitro OAF-CEP model that incorporates key structural properties of the native interface, and demonstrates that the use of a perfusion bioreactor enhances development of in vitro-grown IVD tissue interfaces. These findings fill a translation gap in the field of IVD tissue engineering and will support the scale-up of such tissues to physiological sizes.
Supervisor Name: Rita Kandel
Year of Study: 2
Program of Study: MASc
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April 25
1:25 pm - 1:40 pm EDT
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