"Introduction: Intervertebral disc (IVD) disease is a common condition characterized by degeneration of one or more discs that separate the vertebrae causing back and neck pain. Current treatments focus entirely on symptom management rather than causal cure. This research focuses on using nasal chondrocytes (NCs) to ameliorate degenerative disc disease (DDD). NCs originate from multipotent cranial neural crest cells (CNCCs) during embryonic development and possess high regenerative capacity. However, NCs would face several challenges such as low oxygen, reduced glucose, increased matrix acidity and elevated levels of proinflammatory cytokines within the DDD microenvironment. The research focuses on identifying genes or markers that enable selection of high performing cell sub-populations within a nasal cartilage biopsy and priming NCs to survive the DDD environment.
Methodology: 200 clones from a fresh nasal biopsy were isolated and differentiation potetential into chondrogenic tissue was assessed. The most and least chondrogenic clones will be subjected to RNA-sequencing to identify a genetic basis for intra-tissue variability. For the priming experiment, NCs were subjected to different “starvation” conditions and then tested for clonogenicity in inflammatory conditions.
Results: When clones from nasal biopsy were subjected to chondro-pellet differentiation, Safranin-O staining showed a wide variety of chondrogenic capacities.This indicates that there exists intra-tissue variability in human nasal cartilage. Chondrogenic and non-chondrogenic clones were isolated based on histological staining intensities, cell distance and morphology. Furthermore, the speed of proliferation of clones was independent of chondrogenicity.
In order to observe the behaviour of good and bad clones under different environmental conditions as compared to standard (4.5g/L glucose in normoxia), NCs were cultured in physiological blood-glucose levels (1g/L glucose) in hypoxia. After 5 days in culture, the rate of proliferation of both chondrogenic and non-chondrogenic clones slowed down in hypoxia in both conditions. Interestingly, while there was little to no difference in cells from the chondrogenic clone in 4.5g/L glucose and 1g/L glucose conditions in hypoxia, the non-chondrogenic clone could not cope in 1g/L glucose in hypoxia. The validity of this phenomenon was confirmed by exposing more non-chondrogenic clones to these experimental conditions.
NCs were also ‘primed’ by glucose and lipids (FBS) deprivation. Since the IVD microenvironment is characterised by low oxygen, NCs were cultured in hypoxia. NCs, when deprived of both glucose and lipids, performed the best in normal as well as DDD-mimicking CFU assays as compared to control conditions.
Conclusion: It is clear there exists intra-tissue variability in human nasal cartilage. RNA-sequencing and ATAC sequencing might provide clues to what causes this variability. It is also apparent that non-chondrogenic clones differ in terms of coping with hypoxia and lower levels of glucose. Since the normal environment of nasal chondrocytes is not as harsh and deprived as the IVD, it will be important to find a way to prepare these cells in vitro before injecting them into the IVD. This research will address the selection of only chondrogenic cells from a nasal biopsy and priming these cells under harsh conditions in vitro to achieve best possible results in vivo."