The Role of Parathyroid Hormone-related Peptide and Indian Hedgehog in Intervertebral Disc Degeneration

Abstract

Introduction - The vertebral column consists of vertebrae interconnected by intervertebral discs (IVDs). Each IVD consists of a central nucleus pulposus (NP) and an outer annulus fibrosus (AF). IVDs provide movement, flexibility and stability of the vertebral column. IVD degeneration is common in humans and dogs, and is associated with low back pain. Current treatments reduce the pain rather than repair the IVD. Therefore, regenerative treatments need to be developed, but further knowledge about the pathogenesis of IVD degeneration and the pathways involved is required. The Parathyroid hormone-related peptide (PTHrP) and Indian Hedgehog (IHH) growth-restraining feedback loop plays an essential role in endochondral ossification. IHH may be positively correlated with osteoarthritis, which resembles IVD degeneration. Additionally, IHH promotes chondrocyte hypertrophy and mineralization, while PTHrP suppresses these processes which also occur during IVD degeneration. The aim of this study was to determine the expression and possible role of PTHrP and IHH in canine and human healthy and degenerated IVDs.

Methods – Human and canine IVDs with different Thompson grades were immunohistochemically stained for PTHrP, IHH, PTHR1, Ptc and Smo. The mean ratio of positive cells per Thompson grade was assessed in the NP. Additionally, nucleus pulposus cells (NPCs) from early degenerated human and canine IVDs (Thompson grade III) were cultured in 3D micro-aggregates in chondrogenic differentiation culture medium (10 ng/mL TGF-ß1), or hypertrophic induction medium (1 nM triiodothyronine, 10 mM ß-glycerol phosphate and 10-9 M dexamethasone), or hypertrophic induction medium supplemented with PTHrP (10-8 M or 10-7M) or IHH (0.1 μg/mL or 1 μg/mL) in hypoxia and normoxia. Read out parameters were ECM production (RT-qPCR, glycosaminoglycan (GAG) production, Safranin O/Fast Green staining, immunohistochemistry) and cell proliferation (RT-qPCR, DNA content).

Results – Generally, in canines and humans, PTHrP and IHH (and its receptors Ptc and Smo) protein expression decreased from healthy to early degenerated IVDs and increased from early to severely degenerated IVDs. Collagen type X protein expression or calcifications, which are hypertrophic markers, were not present in the NPCs that were treated with hypertrophic induction medium. However, PTHR1 protein expression, which is a prehypertrophic marker, was present. The protein PTHR1 was more strongly expressed in the canine NPCs that were cultured in normoxia than it was in hypoxia. In normoxia, PTHR1 was most strongly expressed in canine NPCs that were treated with hypertrophic induction medium for 21 days. Supplementation with PTHrP or IHH did not augment GAG production in the hypertrophic NPCs. Calcifications were visible in one canine and one human donor that were treated with hypertrophic induction medium supplemented with 1 μg/mL IHH.

Conclusions – This study demonstrated that both PTHrP and IHH positively correlate with the IVD degeneration grade in humans. This indicates that PTHrP-IHH signaling is active in healthy and degenerated IVDs and might play a role in IVD degeneration or regeneration. Furthermore, the hypertrophic induction medium did not induce hypertrophy but did induce prehypertrophy. In addition, normoxia seems to be more beneficial for prehypertrophic differentiation of NPCs than hypoxia. PTHrP and IHH did not induce cell proliferation and ECM production or counteracted apoptosis in canine and human NPCs and thus did not have an anabolic effect. Lastly, IHH induced calcifications and these calcifications occur independently from collagen type X expression. More in vitro studies are needed to further elucidate the role of PTHrP and IHH in healthy and degenerated IVDs and to determine if PTHrP or IHH could be useful for the development of regenerative treatments.