The Effects And Mechanism Of Inorganic Polyphosphate On Matrix Metabolism In Human Intervertebral Disc Cells

Intervertebral disc degeneration(IDD)is considered to be the most common form of orthopedic disease and a leading cause of significant neck or lower back pain.It is a major public health problem with great socioeconomic impact worldwide.The cause of IDD is complex and multifactorial etiology,including genetic inheritance,aging,nutritional compromise,loading history and so on.And the factors that lead to degeneration have not been entirely elucidated.Although,the mechanism(s)of initiation of the disease is controversial,there is general agreement that the process of IDD is significantly related to the result of imbalance between anabolism and catabolism of extracellular matrix of disc in favor of catabolism.Currently,Clinical treatments for IDD consisting of conservative therapies and spinal surgeries are only able to relieve the symptoms,but cannot prevent the degeneration and development of intervertebral disc degeneration.And there is no ideal treatment to reasonably intervene the degeneration of intervertebral disc.Recently,biological treatments of IDD have been developed,including cell therapies,tissue engineering and growth factors,and/or bioactive compounds.As a bioactive compounds,Inorganic Polyphosphate(PolyP)is a polymer consisting of ten to hundreds of phosphate residues linked by high-energy phosphoanhydride bonds,which is abundantly found in all organisms and nature.PolyP is essential for survival in lower organisms providing an alternate source of energy and maintaining virulence.Its role in mammalian cells and tissue is poorly understood although recent studies have shown polyP to regulate many essential processes in mammalian cell systems.It is assumed that PolyP stores high amount of energy within their covalent bonds which could be released for cellular production of the major molecular building blocks,including amino acids,nucleotides,sugar,and lipids.PolyP has been reported to enhance matrix anabolism in chondrocytes and cartilage tissue formation invitro.Additionally,It has been recently reported that PolyP at 0.5-1 mM was detected in bovine disc tissue which can enhance NP tissue formation by upregulating the expression of matrix genes.Hence,we hypothesized that polyphosphate can be utilized as a source of energy to enhance matrix anabolism in human intervertebral disc cells in favor of preventing or reversing IDD.Part 1 Effects of PolyP on Intervertebral Disc Cell Proliferation and Matrix MetabolismObjective.To test the effects of PolyP on intervertebral disc cell proliferation and matrix metabolismMethods:Human intervertebral disc anulus fibrosus cells were isolated and cultured in vitro,then treatment with PolyP(22 phosphate unit length)at different concentrations(0,0.5,and 1.0mM).After treatment,Cell morphology were observed under a phase contrast microscopy at different time.And the changes of cell proliferation activity were test by The CyQUANT cell proliferation reagent assay.when anulus fibrosus cells treated with different concentration PolyP for 48 hours,1,9-Dimethylmethylene Blue(DMMB)assay was used for analysis the cells glycosaminoglycan content;Cell proteoglycans synthesis was examined by 35S isotope tracing rapid microfiltration assay and collagen synthesis was examined by 3H-L-proline isotope tracing assay.Results:PolyP can improve the proliferation of human intervertebral disc anulus fibrosus cells;The results of DMMB assay and 35S and 3H-L-proline isotope tracing assays showed that PolyP increased glycosaminoglycan content,proteoglycans synthesis and collagen synthesis.Conclusion:PolyP can improve the proliferation of human intervertebral disc anulus fibrosus cells and increase matrix synthesis.Part 2 The Mechanism of Inorganic Polyphosphate on Matrix Metabolism in Intervertebral Disc CellsObjective:To investigate the effects of PolyP on the overall matrix homeostasis of anabolic and catabolism in intervertebral disc cells;To test effects of PolyP on ATP production in intervertebral disc cells.Methods:After treatment on intervertebral disc anulus fibrosus cells with PolyP-22 at different concentrations(0,0.5,and 1.0mM),The mRNA expression was measured by quantitative real-time reverse transcriptase polymerase chain reaction qRT-PCR for mediators of catabolic activity(matrix metalloprotease-1(MMP-1),MMP-3,A disintegrin and metalloproteinase with thrombospondin motifs-4(ADAMTS-4)and ADAMTS-5),and mediators of anabolism(Collagen I and Aggrecan).Protein-level changes in anabolic markers(aggrecan and collagen I)were measured by Western Blot and Cell Immunofluorescence Analysis.Protein-level of aggrecan-fragment expression was measured by Western Blot.Finally,ATP generation was measured using ATPlite Luminescence Assay at different time points.Results:PolyP increases cell growth in human anulus fibrosus cells.qRT-PCR analysis demonstrated increased mRNA anabolism expression of aggrecan and collagen I in anulus fibrosus cells treated with PolyP compared to an untreated control.Howerver,in matrix catabolism of human anulus fibrosus cells,PolyP increases MMP-3 and ADAMTS-4 mRNA expression but decreases MMP-1 and ADAMTS-5 mRNA expression.Immunofluorescence and Western blot demonstrated increased aggrecan and collagen I protein expression in PolyP-treated anulus fibrosus cells.These results demonstrated PolyP increased anabolic effects on anulus fibrosus cells with increasing PolyP concentrations.The effect of PolyP on disc matrix catabolism is more complex.PolyP increased ATP production in human anulus fibrosus cells.Conclusion:In disc anulus fibrosus cells,PolyP can increase anabolism mRNA expression and ATP production and promote matrix anabolism,but more confirmatory studies are required to determine how inorganic polyphosphates modulate matrix catabolism and the overall matrix homeostasis in disc anulus fibrosus and nucleus pulposus cells.