Effect of Intervertech-1 Levels on Proteoglycan Synthesis by Intervertebral Disk Cells

Abstract

IntroductionBack pain is a fairly common problem, which affects a large portion of the population across all ages and has an impact on quality of life. It has a major financial impact on the health care system and industry due to loss of productive labor hours. Intervertebral disk degeneration is the single most common implicated cause of back pain. Presently, there is no medical treatment or therapeutic agent to address this problem and surgery is the only offered option. Intevertech 1(IVT-1), currently being patented by Intervertech Inc, represents a novel peptide produced in mammals. It has the ability to heal injured tissues and increase cell resistance to hypoxic episodes. The aim of this study was to determine how nucleus pulposus (NP) and annulus fibrosus (AF) cells respond to different Intervertech 1 concentrations when cultured in a three dimensional system consisting of an alginate scaffold.Materials and MethodsIsolation and culture of Bovine intervertebral disk cells: Adult bovine tails (2 to 3 years old) were obtained 2 to 3 hours after slaughter. The IVDs were dissected from their adjacent vertebral bodies and separated into NP and AF. AF and NP cells were isolated by digestion with collagenase, and were resuspended in 1.2% alginate in 0.15M sodium chloride at a concentration of 2 million cells per mL. Droplets of cell suspension were released through a 18-gauge needle into 102 mM calcium chloride solution and left to polymerize for 5 minutes.1 The beads were placed in 48 well plates at a density of 5 beads/well (in 1 mL of DMEM high glucose supplemented with 10% fetal bovine serum and antibiotics). Analysis of proteoglycan (aggrecan) synthesis:Alginate beads were stabilized for 5 days in 1 mL of DMEM and then exposed to 5 nM-400 nM of IVT-1 in DMEM supplemented with 25µCi of 35S-sulfate/mL for 48 hours to determine aggrecan synthesis. At the end of the culture period, media was collected and dialyzed exhaustively against RO water (Microdialysis chamber, Life Technologies with Spectr/Por 3, 3-kDa molecular weight cut-off dialysis membrane) followed by cold chase with 1M MgSO4 for 2 hours to remove unincorporated 35S-sulfate. Counts per minutes (CPM) reads were normalized to control and expressed as an arbitrary value.All experiments were performed in triplicates. Results were considered significant with a p < 0.05.ResultsIn both cell types proteoglycan synthesis increased with dosage in culture up to 10 nM, then tended to plateau between 25 nM and 50 nM but increased at 100 n M and 200 nM (Fig. 1). Maximal response was at 200 nM but declining thereafter. At all time points, the levels of proteoglycan synthesis by AF cells was greater than by NP cells.ConclusionThe role of Intevertech 1 in disk metabolism is not understood. Intevertech 1 is a small peptide involved in a wide variety of physiological functions. This study indicates that IVT-1 is able to stimulate proteoglycan synthesis in bovine disk cells cultured in a 3D system. Perhaps surprisingly, intervertech 1 stimulates AF cells to produce more proteoglycan than NP cells. This difference may relate to receptor usage on AF and NP cells by intervertech 1. The most significant biochemical change to occur in disk degeneration is loss of proteoglycan. Promotion of proteoglycan synthesis is the main strategy for disk regeneration therapy and this peptide shows promise in this direction and can be established as a potential therapeutic agent for annular repair especially in later stages of disk degeneration when the disc's load bearing behavior shifts to the annulus. One major advantage of Intevertech 1 over recombinant growth factors for therapeutic use is the large saving in cost.I confirm having declared any potential conflict of interest for all authors listed on this abstractYesDisclosure of InterestNone declaredMwale, F, Ciobanu, I, Giannitsios, D, Roughley, P, Steffen, T, and Antoniou, J. Effect of oxygen levels on proteoglycan synthesis by intervertebral disk cells. Spine 2011;36:E131-E138