Oral Citrate Supplementation Mitigates Age-Associated Pathological Intervertebral Disc Calcification in LG/J Mice

Despite the high prevalence of age-dependent intervertebral disc calcification, there is a glaring lack of treatment options for this debilitating pathology. Here, we investigate the efficacy of long-term oral K3Citrate supplementation in ameliorating disc calcification in LG/J mice, a model of spontaneous age-associated disc calcification. K3Citrate successfully reduced the incidence of disc calcification in LG/J mice without deleterious effects on vertebral bone structure, plasma chemistry, and locomotion. Notably, a positive effect on grip strength, a marker of frailty, was evident in treated mice. Spectroscopic investigation of the persisting calcified nodules indicated K3Citrate did not alter the mineral composition, and collagen 10 and aggrecan staining revealed that reactivation of an endochondral differentiation program in endplates may drive LG/J disc calcification. Importantly, K3Citrate reduced calcification incidence without altering the abundance of endplate hypertrophic chondrocytes, suggesting mitigation of disc calcification through Ca2+ chelation. This was further supported by the inability of K3Citrate to inhibit hypertrophic differentiation of chondrogenic ATDC5 cells and minimal effects on their metabolic status. Overall, this study sheds light on the pathogenesis of intervertebral disc calcification in LG/J mice and underscores the therapeutic potential of K3Citrate as a systemic intervention strategy for disc calcification. To investigate the impact of the presence (or absence) of intervertebral disc calcification on nucleus pulposus (NP) tissues, caudal NP tissue was collected from 23-month-old LG/J mice from two treatment cohorts: untreated controls, which received regular drinking water; or K3Citrate mice which received water continuously supplemented with 80mM K3Citrate from the time mice were 17 months of age (prior to developing disc calcification) until euthanasia. RNA-sequencing was then conducted on RNA isolated from NP tissues from control and K3Citrate cohorts. Each cohort contains four samples, each corresponding to a different mouse.