Matrix stiffness determines the fate of nucleus pulposusederived stem cells Yosi Navaro a , Nadav Bleich-Kimelman a , Lena Hazanov b , Iris Mironi-Harpaz b , Yonatan Shachaf b , Shai Garty c, g , Yoav Smith d , Gadi Pelled a, e, f , Dan Gazit a, e, f , Dror Seliktar b , Zulma Gazit a, e, f, * a Skeletal Biotech Laboratory, The Hebrew UniversityeHadassah Faculty of Dental Medicine, Ein Kerem, Jerusalem 91120, Israel b Department of Biomedical Engineering, TechnioneIsrael Institute of Technology, Haifa 32000, Israel c Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot 76100, Israel d Genomic Data Analysis Unit, The Hebrew UniversityeHadassah Medical School, The Hebrew University of Jerusalem, Jerusalem 91120, Israel e Department of Surgery, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA f Board of Governors Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA g Max Planck Institute for Intelligent Systems, Stuttgart, Germany article info Article history: Received 17 September 2014 Accepted 20 January 2015 Available online Keywords: Elasticity Matrix stiffness Fibrinogen Hydrogel Stem cells Intervertebral disc abstract Intervertebral disc (IVD) degeneration and consequent low-back pain present a major medical challenge. Nucleus pulposusederived stem cells (NPeSCs) may lead to a novel therapy for this severe disease. It was recently shown that survival and function of mature NP cells are regulated in part by tissue stiffness. We hypothesized that modification of matrix stiffness will influence the ability of cultured NP-SCs to proliferate, survive, and differentiate into mature NP cells. NP-SCs were subcultured in three- dimensional matrices of varying degrees of stiffness as measured by the material's shear storage modulus. Cell survival, activity, and rate of differentiation toward the chondrogenic or osteogenic lineage were analyzed. NP-SCs were found to proliferate and differentiate in all matrices, irrespective of matrix stiffness. However, matrices with a low shear storage modulus (G 0 ¼ 1 kPa) promoted significantly more proliferation and chondrogenic differentiation, whereas matrices with a high modulus (G 0 ¼ 2 kPa) promoted osteogenic differentiation. Imaging performed via confocal and scanning electron microscopes validated cell survival and highlighted stiffness-dependent cell-matrix interactions. These results underscore the effect of the matrix modulus on the fate of NP-SCs. This research may facilitate eluci- dation of the complex cross-talk between NP-SCs and their surrounding matrix in healthy as well as pathological conditions. © 2015 Elsevier Ltd. All rights reserved. 1. Introduction Intervertebral disc (IVD) degeneration and consequent low-back pain present a major medical challenge with no optimal solution in sight. In Western society, this pathological condition is prevalent among people younger than 45 years of age and percentage of the work force affected varied from 2% to 8% with days of absence from work per patient [1]. The IVD consists of three major anatomic zones: the nucleus pulposus (NP), the annulus fibrosus (AF), and cartilage endplates. These three anatomic zones are distinct but uniquely attached, contributing to the mechanical function of the IVD [2,3]. The NP is a gel-like substance that contributes to the load-bearing capacity of the IVD and sustains the flexion/extension and lateral bending are spine movements required for many daily activities. This gelatinous substance plays an important role in the IVD's mechanical function by redistributing spinal compressive loads [4]. During IVD degen- eration, the disc's biophysical and biochemical properties are altered. The pathogenesis of IVD degeneration is complex and principally relies on resident NP cells to revitalize tissue [3,4]. The NP is originally derived from the notochord [5e7]; the NP cells in the immature nucleus are smaller and contain more * Corresponding author. Board of Governors Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA. Fax: þ1 (310) 248 8066. E-mail address: zulmag@ekmd.huji.ac.il (Z. Gazit). Contents lists available at ScienceDirect Biomaterials journal homepage: www.elsevier.com/locate/biomaterials http://dx.doi.org/10.1016/j.biomaterials.2015.01.021 0142-9612/© 2015 Elsevier Ltd. All rights reserved. Biomaterials 49 (2015) 68e76