SPINE Volume 34, Number 2, pp 141–148 ©2009, Lippincott Williams & Wilkins Transplantation of Human Mesenchymal Stems Cells Into Intervertebral Discs in a Xenogeneic Porcine Model Helena B. Henriksson, Msc,* Teresia Svanvik, MD,† Marianne Jonsson, Lic.Med.Sci,* Margret Hagman, Bsc,* Michael Horn, PhD,‡ Anders Lindahl, MD, PhD,* and Helena Brisby, MD, PhD† Study Design. Experimental and descriptive study of a xenotransplantation model in minipigs. Objective. To study survival and function of human mesenchymal stem cells (hMSCs) after transplantation into injured porcine spinal discs, as a model for cell therapy. Summary of Background Data. Biologic treatment op- tions of the intervertebral disc are suggested for patients with chronic low back pain caused by disc degeneration. Methods. Three lumbar discs in each of 9 minipigs were injured by aspiration of the nucleus pulposus (NP), 2 weeks later hMSCs were injected in F12 media suspen- sion (cell/med) or with a hydrogel carrier (Puramatrix) (cell/gel). The animals were sacrificed after 1, 3, or 6 months. Disc appearance was visualized by magnetic res- onance imaging. Immunohistochemistry methods were used to detect hMSCs by antihuman nuclear antibody staining, and further performed for Collagen II, Aggrecan, and Collagen I. SOX 9, Aggrecan, Versican, Collagen IA, and Collagen IIA and Collagen IIB human mRNA expres- sion was analyzed by real-time PCR. Results. At magnetic resonance imaging all injured discs demonstrated degenerative signs. Cell/gel discs showed fewer changes compared with cell/med discs and only injured discs at later time points. hMSCs were de- tected in 9 of 10 of the cell/gel discs and in 8 of 9 of the cell/med discs. Immunostaining for Aggrecan and Colla- gen type II expression were observed in NP after 3 and 6 months in gel/cell discs and colocalized with the antihu- man nuclear antibody. mRNA expression of Collagen IIA, Collagen IIB, Versican, Collagen 1A, Aggrecan, and SOX9 were detected in both cell/med and cell/gel discs at the time points 3 and 6 months by real-time PCR. Conclusion. hMSCs survive in the porcine disc for at least 6 months and express typical chondrocyte markers suggesting differentiation toward disc-like cells. As in au- tologous animal models the combination with a three- dimensional-hydrogel carrier seems to facilitate differen- tiation and survival of MSCs in the disc. Xenotransplan- tation seems to be valuable in evaluating the possibility for human cell therapy treatment for intervertebral discs. Key words: xenotransplantation, human mesenchy- mal stem cells, intervertebral disc, disc degeneration. Spine 2009;34:141–148 In the Western world, chronic low back pain is a com- mon medical condition highly costly for the society both in the context of productivity loss and treatment expens- es. 1 Different structures in the spine have been suggested to be initiators and participate in the maintenance of pain. 2,3 However, the degeneration of intervertebral discs has been suggested to play a key role. 4 The intervertebral disc (IVD) is an avascular and fi- brocartilaginous structure that separates the vertebrae in the spine. It functions as a load absorber and participates in the movement of the spine. The IVD consists of the annulus fibrosus (AF) consisting of dense highly orga- nized collagen I rich fibers arranged in rings around the core of the IVD, the nucleus pulposus (NP). In the NP, chondrocyte-like cells are embedded in a gelatinous matrix consisting of proteoglycans, mainly collagen type II and water. The average total cell density of the disc is 5.8  10 3 cells/mm 3 . 5 The degenerated disc is characterized by increased fis- sure formation, loss of disc height, increased cell cluster- ing, and cell death as well as a decrease in fluid binding ability. On the molecular level, a decrease is seen in gly- cosaminoglycan production and an increase in matrix degrading enzymes and cytokines. 6 IL1b is one of the cytokines that have been demonstrated to be increased while a simultaneous decrease of IL1 receptor antago- nists has been seen. 6–8 Matrix degrading enzymes that are up regulated in the degenerated disc include for e.g. the metalloproteases 1, 3, 9, 13 and ADAM 4, and 5. 9 –12 The current treatment options for degenerated disc dis- ease are symptomatic treatment (analgesics, physiotherapy, and cognitive treatment) or surgical treatment (spinal fu- sion or total disc replacement using an artificial disc re- placement). None of these treatment methods attack the underlying problem. Biologic treatment options of the disc, including cell therapy, have been suggested as complemen- tary or optional treatment methods for disc degeneration. 13 Different immature cell types have been suggested for cell therapy treatment. Utilization and practical application of pluripotent embryonic stem cells in cell replacement ther- From the *The Institute of Biomedicine, and Departments of †Or- thopaedics and ‡Radiation Physics, Institute for Clinical Sciences, The Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden. Acknowledgment date: June 30, 2008. Revision date: August 14, 2008. Acceptance date: August 15, 2008. The manuscript submitted does not contain information about medical device(s)/drug(s). Foundation funds were received in support of this work. No benefits in any form have been or will be received from a commercial party related directly or indirectly to the subject of this manuscript. Supported by grants from Marianne och Marcus Wallenberg’s Foun- dation, ALF Va ¨stra Go ¨ taland, Gothenburg Medical association, The Swedish Society of Medicine, The Neubergh Foundation. MR Imaging was performed at the Center for Mouse Physiology and Bio-Imaging at the University of Gothenburg. Address correspondence and reprint requests to Helena Brisby, MD, PhD, The Institute of Clinical Sciences/Orthopaedics, The Sahlgrenska Academy, Sahlgrenska University Hospital, 413 45 Gothenburg, Swe- den; E-mail: helena.brisby@vgregion.se 141