Tissue and Cell 41 (2009) 414–420 Contents lists available at ScienceDirect Tissue and Cell journal homepage: www.elsevier.com/locate/tice Human intervertebral disc-derived cells are recruited by human serum and form nucleus pulposus-like tissue upon stimulation with TGF-3 or hyaluronan in vitro K. Haberstroh a , A. Enz b , M.L. Zenclussen c , A.A. Hegewald d , K. Neumann b , A. Abbushi c , C. Thomé d , M. Sittinger a , M. Endres a,b , C. Kaps a,b,∗ a Charité-University Medical Center Berlin, Dept. of Rheumatology, Berlin, Germany b TransTissue Technologies GmbH, Berlin, Germany c Department of Neurosurgery, Charité Medical University Berlin, Berlin, Germany d Department of Neurosurgery, Medical Faculty Mannheim, University of Heidelberg, Germany article info Article history: Received 18 February 2009 Received in revised form 12 May 2009 Accepted 25 May 2009 Available online 23 July 2009 Keywords: Nucleus pulposus cells Intervertebral disc Hyaluronan Cell recruitment Regeneration abstract The aims of this work were to test whether human intervertebral disc-derived nucleus pulposus cells (hNP-cells) are attracted by human serum and to analyze if matrix generation from hNP-cells is promoted under the influence of transforming growth factor-3 (TGF-3) or hyaluronan (HA) in vitro. Using the multi-well chemotaxis assay to determine cell migration under the influence of different concentrations of human serum, it was demonstrated that dedifferentiated hNP-cells are able to migrate towards a serum fraction gradient in a concentration-dependent manner. Re-differentiation capacity of hNP-cells in 3D micro-masses under the influence of TGF-3 or hyaluronan was also tested. Gene expression analysis of types I, II, III and IX collagen, as well as aggrecan, COMP and LINK of hNP-cells in 3D-micro-mass cell- culture revealed a strong increase of these markers in TGF-3 treated cells. Furthermore, histochemical and immuno-histochemical staining after 28d showed proteoglycan and type II collagen-rich matrix for both, the TGF-3 and the hyaluronan treated cells. These findings show that TGF-3 or hyaluronan are able to induce the differentiation and that human serum stimulates the migration of hNP-cells in vitro. Therefore, hyaluronan and serum are suited for cell-free biomaterials as cell migration and differentiation inducing factors intended for biological treatment strategies of the intervertebral disc. © 2009 Elsevier Ltd. All rights reserved. 1. Introduction Intervertebral disc herniations are often complicated by re- herniations due to structural defects of the annulus fibrosus and in the long term by advancing degeneration of the intervertebral disc (IVD) resulting in disabling low back pain (Barth et al., 2008). A large proportion of the population is affected by painful IVD degeneration and its treatment entails immense costs. Whereas commonly used surgical decompression, spinal arthrodesis and total disc replacement are symptomatic therapies, biological treat- ment options might offer preventive and curative treatment options (Hegewald et al., 2008). Intervertebral discs consist of two chondro- cytic tissues: the annulus fibrosus (AF) and the nucleus pulposus (NP). The AF is rich in specifically aligned type I collagen, ideal for resisting tensile forces. NP, however, consists mainly of type II collagen, containing large amounts of hyaluronan and proteo- ∗ Corresponding author at: TransTissue Technologies GmbH, Tucholskystrasse 2, D-10117 Berlin, Germany. Tel.: +49 30 450 513 239; fax: +49 30 450 513 957. E-mail address: christian.kaps@transtissue.com (C. Kaps). glycans, mostly aggrecan. In addition, the extracellular matrix of developing or mature intervertebral discs shows, amongst other matrix molecules, types III and IX collagens as well as cartilage oligomeric matrix protein (COMP) and cartilage link protein (Robert et al., 1994; Hayes et al., 2001; Wu and Eyre, 2003; Ishii et al., 2006). During degeneration of the disc, the expression of types I and III collagens increases while aggrecan is repressed (Le Maitre et al., 2007). In particular, the hydration properties of the large molecules hyaluronan and proteoglycan account for the ability of the nucleus pulposus to resist compression forces and maintain spinal stability. Potential strategies to avoid complications after IVD herniations are the restoration of the integrity of the AF and the prevention or rever- sion of advancing NP degeneration. Different biological treatment modalities have been suggested and include the administration of growth factors, the application of autologous or allogenic cells (disc or progenitor cells), gene therapy and the introduction of bioma- terials or a combination thereof (Hegewald et al., 2008; Kandel et al., 2008). However, cell-based approaches are discussed controver- sially due to nutrition deprivation and very different estimations of cell death in the human IVD (Hutton et al., 2004; Paesold et al., 2007; Johnson and Roberts, 2007). 0040-8166/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.tice.2009.05.006