The Effect of Link N on the Differentiation of Human Mesenchymal Stem Cells
Authors: Mwale F. , Wang H. T. , Haglund L. , Roughley P. J. , Antoniou J. 
Published: June 2012

Introduction The degeneration of intervertebral disk (IVD) can cause low back pain, and invoking the regeneration of degenerated IVD can relieve the pain.1,2 Mesenchymal stem cells (MSCs) are multipotent cells that can differentiate into chondrocytes and can be employed in cartilage regeneration.3,4 Link N (DHLSDNYTLDHDRAIH) is the amino terminal peptide of link protein in the extracellular matrix of cartilage. It can stimulate the biosynthesis of collagens II and IX and aggrecan in IVD cells in vitro as well as in vivo.5–7 We hypothesized that Link N can enhance the chondrogenic differentiation of MSCs, and can be applied with MSCs in bio-regeneration of IVDs. The specific aim of this research is to determine the effect of Link N on gene expression of MSCs. Materials and Methods MSCs were isolated from the bone marrow of osteoarthritis (OA) patients. The cells were cultured in 24-well plates (3000 cells/well) in chondrogenesis differentiation medium (Invitrogen, Canada) according to the manufacturer's instructions. Link N was dissolved in the media with a final concentration of 0.1 µg/mL and 1 µg/mL, respectively. Medium without Link N was applied as a control. The media were changed every 3 days, and the used media were collected and stored at −20°C for GAG analysis. For gene expression analysis, the cells were cultured for 7, 14, and 21 days, then were washed twice with PBS and dissolved in Trizol (Invitrogen, Canada). Total RNA was isolated and cDNA was synthesized by reverse transcription. Gene expression was analyzed by real-time PCR and relative expression was calculated using GAPDH as a reference gene. MSCs from three OA patients were analyzed and statistical significance was calculated using analysis of variance followed by Fisher protected least significant difference (PLSD) comparison test. Results With the concentrations of 0.1 and 1.0 µg/mL, no toxic effect of Link N on MSCs was observed. After MSCs were cultured for 7 days and 14 days, the expression of aggrecan (ACAN) (Fig. 1), collagen II (COL2A1) (Fig. 2) and the transcription regulator SOX9 increased significantly in the media with 0.1 µg/mL or 1.0 µg/mL Link N. No significant differences were observed at day 21 (Figs. 1 and 2). When cells were cultured with 0.1 or 1.0 µg/mL Link N, the quantity of GAG in the media increased significantly at day 9, 12, and 15, but no major difference was observed at day 3 and 6 and at day 18 and 21. When MSCs were cultured for 7, 14, and 21 days, no significant effect of Link N on the expression of alkaline phosphatase (ALP) was observed. Furthermore, Link N significantly inhibited osteocalcin (OC) gene expression after culturing MSCs for 21 days. Conclusion Link N can improve the chondrogenic differentiation of MSCs and inhibit their osteogenic differentiation. It can therefore be used in conjunction with MSCs in the repair of IVD degeneration. I confirm having declared any potential conflict of interest for all authors listed on this abstract Yes Disclosure of Interest None declared Luoma K, Riihimaki H, Luukkonen R, Raininko R, Viikari-Juntura E, Lamminen A. Low back pain in relation to lumbar disk degeneration. Spine 2000;25:487–492 Roughley PJ. Biology of intervertebral disk aging and degeneration: involvement of the extracellular matrix. Spine 2004;29:2691–2699 Xian CJ, Foster BK. Repair of injured articular and growth plate cartilage using mesenchymal stem cells and chondrogenic gene therapy. Current Stem Cell Research Therapy 2006;1:213–229 Koga H, Engebretsen L, Brinchmann JE, Muneta T, Sekiya I. Mesenchymal stem cell-based therapy for cartilage repair: a review. Knee Surgery Sports Traumatol Arthroscopy 2009;7:1289–1297 Mwale F, Demers CN, Petit A et al. A synthetic peptide of link protein stimulates the biosynthesis of collagens II, IX and proteoglycan by cells of the intervertebral disc. Journal of Cell Biochemistry 2003;88:1202–1213 Petit A, Yao G, Rowas SA, et al. Effect of synthetic Link N peptide on the type I and type II collagens in human intervertebral discs cells. Tissue Engeneering Part A 2011;17:899–904