A Feasibility Study of Cell Injection Therapy in Human Intervertebral Discs Cultured under Dynamic Load

Abstract

Introduction Painful IVD degeneration affects the quality of life for millions of people, and it presents a major problem for direct and indirect global health care costs. A preclinical ex vivo screening platform is needed to study the relationship between mechanobiology, disc matrix composition, and metabolism in the context of degenerative disease. We therefore developed an ex vivo organ culture model that facilitates culture of intact human discs in a controlled dynamically loaded environment. The bioreactor is used in combination with a previously reported harvesting method, which maintains the integrity of the intervertebral discs by preserving the noncalcified part of the cartilage end plate and the entire annulus. Here, we determine IVD tissue integrity and cell viability in the system, and investigate the suitability toward cell supplementation for tissue repair. Methods A total of nine lumbar IVDs from seven individual donors were obtained through organ donations via Transplant Québec. The spines were assessed by X-ray to evaluate degree of degeneration and six discs were isolated from four donors as previously described. Overall, 106 primary fluorescently labeled allogeneic NP cells from three separate donors were seeded per 1 mL of thermogelling hyaluronic acid hydrogel (HA-pNIPAM). Approximately, 250 µL of cell/gel mixtures were injected laterally into the NP region of isolated discs. IVDs with the newly transplanted cells were transferred to the bioreactors and were loaded with 0.1 to 0.6 MPa cyclic loads for two periods of 2 hours/day. The dynamic compressive load periods were interrupted by recovery periods of 6 hours and 14 hours, respectively, under 0.1 MPa static loads. The scheme was repeated for 3 or 7 or 14 consecutive days. After loading, portions of nucleus pulposus and annulus fibrosus were excised and analyzed via LIVE/DEAD assay, and smaller sections were visualized using a laser-scanning confocal microscope. The proportion of viable cells was quantified. Results Our culture system maintains > 80% cell viability of the resident IVD cells and shows no signs of adverse effects in the extracellular matrix after more than 14 days of dynamic culture. Allogeneic NP cells were successfully transplanted in freshly isolated human discs. The transplanted cells were found dispersed throughout the nuclear and annular regions and displayed maintained viability after up to 14 days of dynamic culture. Conclusion This study shows that it is feasible to culture human IVDs under dynamic loading for more than 14 days while maintaining cell viability, tissue homeostasis, and integrity. It also presents a preclinical ex vivo screening platform where cell-based therapies, utilizing isolated disc cells or stem cells can be tested in combination with various injectable hydrogels. In addition, the effect of load magnitude and frequency, growth factors, O2 tension, glucose levels, and bioactive therapeutics can be evaluated. The bioreactor can also be used to evaluate loss or gain of mechanical function as a result of biological changes. Such knowledge is important for determining the value of cell/hydrogel injections and patient advisement on physical activities following a biological repair procedure.