The Effect of Intervertech 1 on Proteoglycan and Collagen Expression in Human Intervertebral Disk Cells

Abstract

Introduction Back pain is one the most frequent complaints and has an impact on the quality of life. Intervertebral disk (IVD) 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. Intervertech 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 purpose of the present study is to determine the effect of IVT 1 on human disk cell survival and extracellular matrix synthesis. Materials and Methods Isolation and Culture of Human Intervertebral Disk Cells Human lumbar IVDs, from a 54-year-old donor (5 discs) without spinal pathology, were obtained through organ donations via Transplant Quebec within 24 hours after death. With the procedure approved by Local Research Ethics Committee, cells were isolated from the nucleus pulposus (NP) and annulus fibrosus (AF) regions of the disks by sequential digestion with Pronase followed by Collagenase IA digestion for NP and Collagenase II digestion for AF. About 1 million NP cells at passage 3 were cultured in 20 mL DMEM/high glucose supplemented with 10% FBS, 100 U/mL penicillin, 100 µg/mL streptomycin in 100 × 20 mm dishes. Cells were then incubated in serum-free DMEM in the absence (control) or the presence of 50, 100, or 150 nmol/ L IVT 1 for 3 to 72 hours. Real Time PCR After the cells were cultured for 3 to 72 hours, they were washed with PBS and then lysed in 1 mL Trizol (Invitrogen, Burlington ON). Total RNA was isolated from the cells in Trizol according to the instruction of the supplier. After digestion with DNase I, 1 µg RNA was employed for reverse transcription. Real time PCR was applied to quantitatively analyze message levels of aggrecan and type II collagen using gene-specific primers. The relative ratio of gene expression was calculated using GAPDH as the reference gene. Data were presented as the mean ± SD of three experiments. Statistical differences were calculated by student t test with p < 0.05 as the level of significance. Receptor Expression Supernatants from Trizol extractions were collected and the protein was precipitated with isopropyl alcohol. Total protein was quantified by the Bio-Rad DC protein assay. Expression of intervertech 1 receptor (IVT 1R) was determined by immunoblotting using a rabbit polyclonal antibody raised against intervertech 1-receptor of human origin. Results The response of cells from the IVD to varying IVT 1 levels from 50 nM to 150 nM was examined when cultured in monolayer. Human IVD cells express the IVT 1R and are sensitive to its ligand. In both cells from the NP and AF, the receptor increased with 100 nM and 150 nM IVT 1 but not at 50 nM after 3 hours stimulation when compared to the control, then tended to decrease at 24 hours, but increased after 24 hours at all concentrations. Finally, after 72 hours, the IVT 1R was only stimulated at 50 nM. When human NP cells were exposed to 100 nM IVT 1 for 48 hours, aggrecan (ACAN) and COL2A1 message increased significantly compared with control cells (Fig. 1). Conclusion The role of IVT 1 in disk metabolism is not understood. IVT 1 is a small peptide involved in a wide variety of physiological functions. The present data indicates that IVT 1 receptors are present in human disk cells. The existence of these receptors means that IVT 1 can affect disk cell metabolism through them. IVT 1 represents a novel growth factor for enhancing the expression of aggrecan and type II collagen in human IVD. It holds promise as a potential therapeutic agent for IVD repair. One major advantage of IVT 1 over recombinant growth factors for therapeutic use is the large saving in cost. IVT 1 therefore represents a potential economical growth factor with beneficial effects on disk repair. I confirm having declared any potential conflict of interest for all authors listed on this abstract Yes Disclosure of Interest None declared