Hyaluronate, Collagen, and Ionic Distribution: A Relation Study with Annulus Fibrosus Cells (Fibroblast-Like Cells)

Abstract

Introduction Degenerative disc disease has been associated with low back pain and many therapies have been proposed for its treatment. The correlation between disc degeneration and back pain is still to be determined, however, studies tend to analyze potential therapies including gene applications and molecular biology to deal with this complex public health problem. Precise methods to quantify disc components are mandatory for any type of proposed therapy. The aim of this study was to correlate glycosaminoglycan amounts with the quantitation of cellular ions and collagen fiber distributions in different intervertebral disc sections. Materials and Methods In total, 14 intervertebral discs were used from 14 tails of 24 to 30-month old cattle. All of the discs were dehydrated, separated in seven sections and digested in sodium-free papain buffer. Sulfated glycosaminoglycan measurements were performed in the samples according to DMMB and agarose electrophoresis methods; hyaluronate was determined by an ELISA-like fluorimetric method, total cells were measured using the PicoGreen technique, ions were quantified by an automated procedure, and collagen fiber birefringence was analyzed with polarized light microscopy. Results Significant differences were observed between electrophoresis and DMMB ( p < 0.01); GAGs were up to six times higher when using electrophoresis in annulus (mean/SD = 3.82/1.96) and were almost two times higher in the nucleus (mean/SD = 1.63/0.21). The correlation between HA and GAG ( p < 0.001), cell number, sodium ( p = 0.007), and hyaluronate ( p = 0.034) were significant. Thin collagen fibers were largest in the nucleus ( p < 0.001). Fibroblast-like cells were negatively correlated with hyaluronate and sodium concentrations. Collagen thin fibers (collagen II) were more related to hyaluronate. Conclusion In summary, this report demonstrated a positive correlation between hyaluronate and glycosaminoglycan; and a negative correlation between hyaluronate and cell number, suggesting that fibroblast-like cells are also sensitive to changes in hyaluronate and ionic concentrations. Hyaluronate could be closely related to thin collagen fibers (collagen type II). Acknowledgments This work was supported by INFAR/UNIFESP Confocal and Flow Cytometry Facility and by grants from the “Fundacao de Amparo à Pesquisa do Estado de São Paulo” (FAPESP: Grant number: 2009/54793–6). D.E.M. was supported by a PhD's fellowship grant from CAPES (Coordenacao de Aperfeicoamento de Pessoal de Nível Superior).