In the end stage of intervertebral disc degeneration, cartilage, bone, endothelial cells, and neurons appear in association with the worsening condition. The origin of the abnormal cells is not clear. This study investigated the properties of progenitor cells in the annulus fibrosus (AF) using one in vitro and two in vivo models. Cultivation of rabbit AF cells with chondrogenic media significantly increased expressions of collagen and aggrecan. Upon exposure to osteogenic conditions, the cultures showed increased mineralization and expression of osteopontin, runx2, and bmp2 genes. Two models were used in the in vivo subcutaneous implantation experiments: 1) rabbit AF tissue in a demineralized bone matrix (DBM) cylinder (DBM/AF), and, 2) rat intact and needle punctured lumbar discs. Bone formation in the AF tissue was detected and hypertrophic chondrocytes and osteoblasts were present 1 month after implantation of the DBM/AF to nude mice. In addition to collagen I and II, immunostaining shows collagen X and osteocalcin expression in DBM/AF specimens 4 months after implantation. Similar changes were detected in the injured discs. Almost the entire needle punctured disc had ossified at 6 months. The results suggest that AF cells have characteristics of progenitor cells and, under appropriate stimuli, are capable of differentiating into chondrocytes and osteoblasts in vitro as well as in vivo. Importantly, these cells may be a target for biological treatment of disc degeneration.
References
[1]
Diamond S, Borenstein D (2006) Chronic low back pain in a working-age adult. Best Pract Res Clin Rheumatol 20: 707–720 10.1016/j.berh.2006.04.002.
[2]
Battie MC, Videman T (2006) Lumbar disc degeneration: Epidemiology and genetics. J Bone Joint Surg Am 88 Suppl 2: 3–9 10.2106/JBJS.E.01313.
[3]
Wang JC, Mummaneni PV, Haid RW (2005) Current treatment strategies for the painful lumbar motion segment: Posterolateral fusion versus interbody fusion. Spine (Phila Pa 1976) 30: S33–43. doi: 10.1097/01.brs.0000174559.13749.83
[4]
Awad JN, Moskovich R (2006) Lumbar disc herniations: Surgical versus nonsurgical treatment. Clin Orthop Relat Res 443: 183–197 10.1097/01.blo.0000198724.54891.3a.
[5]
Adams MA, Stefanakis M, Dolan P (2010) Healing of a painful intervertebral disc should not be confused with reversing disc degeneration: Implications for physical therapies for discogenic back pain. Clin Biomech (Bristol, Avon) 25: 961–971 10.1016/j.clinbiomech.2010.07.016.
[6]
Antoniou J, Steffen T, Nelson F, Winterbottom N, Hollander AP, et al. (1996) The human lumbar intervertebral disc: Evidence for changes in the biosynthesis and denaturation of the extracellular matrix with growth, maturation, ageing, and degeneration. J Clin Invest 98: 996–1003 10.1172/JCI118884.
[7]
Lotz JC, Staples A, Walsh A, Hsieh AH (2004) Mechanobiology in intervertebral disc degeneration and regeneration. Conf Proc IEEE Eng Med Biol Soc 7: 5459.
[8]
Battie MC, Videman T, Levalahti E, Gill K, Kaprio J (2008) Genetic and environmental effects on disc degeneration by phenotype and spinal level: A multivariate twin study. Spine (Phila Pa 1976) 33: 2801–2808 10.1097/BRS.0b013e31818043b7.
[9]
Anderson DG, Tannoury C (2005) Molecular pathogenic factors in symptomatic disc degeneration. Spine J 5: 260S–266S 10.1016/j.spinee.2005.02.010.
[10]
Coppes MH, Marani E, Thomeer RT, Oudega M, Groen GJ (1990) Innervation of annulus fibrosis in low back pain. Lancet 336: 189–190. doi: 10.1016/0140-6736(90)91723-n
[11]
Inoue G, Ohtori S, Aoki Y, Ozawa T, Doya H, et al. (2006) Exposure of the nucleus pulposus to the outside of the anulus fibrosus induces nerve injury and regeneration of the afferent fibers innervating the lumbar intervertebral discs in rats. Spine (Phila Pa 1976) 31: 1433–1438. doi: 10.1097/01.brs.0000219946.25103.db
[12]
Freemont AJ, Peacock TE, Goupille P, Hoyland JA, O'Brien J, et al. (1997) Nerve ingrowth into diseased intervertebral disc in chronic back pain. Lancet 350: 178–181. doi: 10.1016/s0140-6736(97)02135-1
[13]
Peng B, Chen J, Kuang Z, Li D, Pang X, et al. (2009) Expression and role of connective tissue growth factor in painful disc fibrosis and degeneration. Spine (Phila Pa 1976) 34: E178–82 10.1097/BRS.0b013e3181908ab3; 10.1097/BRS.0b013e3181908ab3.
[14]
Roberts S, Evans H, Trivedi J, Menage J (2006) Histology and pathology of the human intervertebral disc. J Bone Joint Surg Am 88 Suppl 210–14 10.2106/JBJS.F.00019.
[15]
Feng G, Yang X, Shang H, Marks IW, Shen FH, et al. (2010) Multipotential differentiation of human anulus fibrosus cells: An in vitro study. J Bone Joint Surg Am 92: 675–685. doi: 10.2106/jbjs.h.01672
[16]
Rutges J, Creemers LB, Dhert W, Milz S, Sakai D, et al. (2010) Variations in gene and protein expression in human nucleus pulposus in comparison with annulus fibrosus and cartilage cells: Potential associations with aging and degeneration. Osteoarthritis Cartilage 18: 416–423. doi: 10.1016/j.joca.2009.09.009
[17]
Risbud MV, Guttapalli A, Tsai TT, Lee JY, Danielson KG, et al. (2007) Evidence for skeletal progenitor cells in the degenerate human intervertebral disc. Spine (Phila Pa 1976) 32: 2537–2544. doi: 10.1097/brs.0b013e318158dea6
[18]
Henriksson H, Thornemo M, Karlsson C, Hagg O, Junevik K, et al. (2009) Identification of cell proliferation zones, progenitor cells and a potential stem cell niche in the intervertebral disc region: A study in four species. Spine (Phila Pa 1976) 34: 2278–2287 10.1097/BRS.0b013e3181a95ad2; 10.1097/BRS.0b013e3181a95ad2.
[19]
Blanco JF, Graciani IF, Sanchez-Guijo FM, Muntion S, Hernandez-Campo P, et al. (2010) Isolation and characterization of mesenchymal stromal cells from human degenerated nucleus pulposus: Comparison with bone marrow mesenchymal stromal cells from the same subjects. Spine (Phila Pa 1976) 35: 2259–2265 10.1097/BRS.0b013e3181cb8828; 10.1097/BRS.0b013e3181cb8828.
[20]
Mizrahi O, Sheyn D, Tawackoli W, Ben-David S, Su S, et al. (2013) Nucleus pulposus degeneration alters properties of resident progenitor cells. Spine J 13: 803–814 10.1016/j.spinee.2013.02.065; 10.1016/j.spinee.2013.02.065.
[21]
Sakai D, Nakamura Y, Nakai T, Mishima T, Kato S, et al. (2012) Exhaustion of nucleus pulposus progenitor cells with ageing and degeneration of the intervertebral disc. Nat Commun 3: 1264 10.1038/ncomms2226; 10.1038/ncomms2226.
[22]
Zeng Q, Li X, Beck G, Balian G, Shen FH (2007) Growth and differentiation factor-5 (GDF-5) stimulates osteogenic differentiation and increases vascular endothelial growth factor (VEGF) levels in fat-derived stromal cells in vitro. Bone 40: 374–381 10.1016/j.bone.2006.09.022.
[23]
Cui M, Wan Y, Anderson DG, Shen FH, Leo BM, et al. (2008) Mouse growth and differentiation factor-5 protein and DNA therapy potentiates intervertebral disc cell aggregation and chondrogenic gene expression. Spine J 8: 287–295. doi: 10.1016/j.spinee.2007.05.012
[24]
Gong M, Zhang D, Wan Y, Edmondson B, Cui Q, et al. (2010) Engineered synovial joint condyle using demineralized bone matrix. 30: 531–539. doi: 10.1016/j.msec.2010.01.015
[25]
Li X, Leo BM, Beck G, Balian G, Anderson GD (2004) Collagen and proteoglycan abnormalities in the GDF-5-deficient mice and molecular changes when treating disk cells with recombinant growth factor. Spine (Phila Pa 1976) 29: 2229–2234. doi: 10.1097/01.brs.0000142427.82605.fb
[26]
Li X, Jin L, Balian G, Laurencin CT, Greg Anderson D (2006) Demineralized bone matrix gelatin as scaffold for osteochondral tissue engineering. Biomaterials 27: 2426–2433 10.1016/j.biomaterials.2005.11.040.
[27]
Jin L, Wan Y, Shimer AL, Shen FH, Li XJ (2012) Intervertebral disk-like biphasic scaffold-demineralized bone matrix cylinder and poly(polycaprolactone triol malate)-for interbody spine fusion. J Tissue Eng 3: 2041731412454420 10.1177/2041731412454420; 10.1177/2041731412454420.
[28]
Zhang D, Jin L, Reames DL, Shen FH, Shimer AL, et al. (2013) Intervertebral disc degeneration and ectopic bone formation in apolipoprotein E knockout mice. J Orthop Res 31: 210–217 10.1002/jor.22216; 10.1002/jor.22216.
[29]
Urist MR, Strates BS (1970) Bone formation in implants of partially and wholly demineralized bone matrix. including observations on acetone-fixed intra and extracellular proteins. Clin Orthop Relat Res 71: 271–278. doi: 10.1097/00003086-197007000-00031
Videman T, Gibbons LE, Battie MC (2008) Age- and pathology-specific measures of disc degeneration. Spine (Phila Pa 1976) 33: 2781–2788 10.1097/BRS.0b013e31817e1d11.
[32]
Kelempisioti A, Eskola PJ, Okuloff A, Karjalainen U, Takatalo J, et al. (2011) Genetic susceptibility of intervertebral disc degeneration among young finnish adults. BMC Med Genet 12: 153 10.1186/1471-2350-12-153.
[33]
Hadjipavlou AG, Tzermiadianos MN, Bogduk N, Zindrick MR (2008) The pathophysiology of disc degeneration: A critical review. J Bone Joint Surg Br 90: 1261–1270 10.1302/0301-620X.90B10.20910.
[34]
Widschwendter M, Fiegl H, Egle D, Mueller-Holzner E, Spizzo G, et al. (2007) Epigenetic stem cell signature in cancer. Nat Genet 39: 157–158 10.1038/ng1941.
[35]
Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, et al. (1999) Multilineage potential of adult human mesenchymal stem cells. Science 284: 143–147. doi: 10.1126/science.284.5411.143
[36]
Jiang Y, Jahagirdar BN, Reinhardt RL, Schwartz RE, Keene CD, et al. (2002) Pluripotency of mesenchymal stem cells derived from adult marrow. Nature 418: 41–49 10.1038/nature00870.
[37]
Freemont TJ, LeMaitre C, Watkins A, Hoyland JA (2001) Degeneration of intervertebral discs: Current understanding of cellular and molecular events, and implications for novel therapies. Expert Rev Mol Med 2001: 1–10 doi:10.1017/S1462399401002885.
[38]
Anderson DG, Li X, Balian G (2005) A fibronectin fragment alters the metabolism by rabbit intervertebral disc cells in vitro. Spine (Phila Pa 1976) 30: 1242–1246. doi: 10.1097/01.brs.0000164097.47091.4c
[39]
Freemont AJ, Watkins A, Le Maitre C, Baird P, Jeziorska M, et al. (2002) Nerve growth factor expression and innervation of the painful intervertebral disc. J Pathol 197: 286–292. doi: 10.1002/path.1108
[40]
Abe Y, Akeda K, An HS, Aoki Y, Pichika R, et al. (2007) Proinflammatory cytokines stimulate the expression of nerve growth factor by human intervertebral disc cells. Spine (Phila Pa 1976) 32: 635–642 10.1097/01.brs.0000257556.90850.53.
[41]
Malinsky J (1959) The ontogenetic development of nerve terminations in the intervertebral discs of man. (histology of intervertebral discs, 11th communication). Acta Anat (Basel) 38: 96–113. doi: 10.1159/000141490
[42]
Johnson WE, Caterson B, Eisenstein SM, Roberts S (2005) Human intervertebral disc aggrecan inhibits endothelial cell adhesion and cell migration in vitro. Spine (Phila Pa 1976) 30: 1139–1147. doi: 10.1097/01.brs.0000162624.95262.73
[43]
Chelberg MK, Banks GM, Geiger DF, Oegema TR Jr (1995) Identification of heterogeneous cell populations in normal human intervertebral disc. J Anat 186 (Pt 1): 43–53.
[44]
Hegewald AA, Neumann K, Kalwitz G, Freymann U, Endres M, et al. (2011) The chemokines CXCL10 and XCL1 recruit human annulus fibrosus cells. Spine (Phila Pa 1976). doi: 10.1097/brs.0b013e318210ed55
[45]
Nosikova Y, Santerre JP, Grynpas MD, Kandel RA (2013) Annulus fibrosus cells can induce mineralization: An in vitro study. Spine J 13: 443–453 10.1016/j.spinee.2012.11.050; 10.1016/j.spinee.2012.11.050.
[46]
Mirza SK, Deyo RA (2007) Systematic review of randomized trials comparing lumbar fusion surgery to nonoperative care for treatment of chronic back pain. Spine (Phila Pa 1976) 32: 816–823 10.1097/01.brs.0000259225.37454.38.
[47]
Jopling C, Boue S, Izpisua Belmonte JC (2011) Dedifferentiation, transdifferentiation and reprogramming: Three routes to regeneration. Nat Rev Mol Cell Biol 12: 79–89 10.1038/nrm3043; 10.1038/nrm3043.
[48]
Tata PR, Mou H, Pardo-Saganta A, Zhao R, Prabhu M, et al. (2013) Dedifferentiation of committed epithelial cells into stem cells in vivo. Nature 503: 218–223 10.1038/nature12777; 10.1038/nature12777.
[49]
Poss KD (2010) Advances in understanding tissue regenerative capacity and mechanisms in animals. Nat Rev Genet 11: 710–722 10.1038/nrg2879; 10.1038/nrg2879.
[50]
Nye HL, Cameron JA, Chernoff EA, Stocum DL (2003) Regeneration of the urodele limb: A review. Dev Dyn 226: 280–294 10.1002/dvdy.10236.
[51]
Chen ZL, Yu WM, Strickland S (2007) Peripheral regeneration. Annu Rev Neurosci 30: 209–233 10.1146/annurev.neuro.30.051606.094337.
[52]
Elaut G, Henkens T, Papeleu P, Snykers S, Vinken M, et al. (2006) Molecular mechanisms underlying the dedifferentiation process of isolated hepatocytes and their cultures. Curr Drug Metab 7: 629–660. doi: 10.2174/138920006778017759
[53]
Diaz-Romero J, Nesic D, Grogan SP, Heini P, Mainil-Varlet P (2008) Immunophenotypic changes of human articular chondrocytes during monolayer culture reflect bona fide dedifferentiation rather than amplification of progenitor cells. J Cell Physiol 214: 75–83 10.1002/jcp.21161.
[54]
Kluba T, Niemeyer T, Gaissmaier C, Grunder T (2005) Human anulus fibrosis and nucleus pulposus cells of the intervertebral disc: Effect of degeneration and culture system on cell phenotype. Spine (Phila Pa 1976) 30: 2743–2748. doi: 10.1097/01.brs.0000192204.89160.6d
[55]
Gruber HE, Fisher EC Jr, Desai B, Stasky AA, Hoelscher G, et al. (1997) Human intervertebral disc cells from the annulus: Three-dimensional culture in agarose or alginate and responsiveness to TGF-beta1. Exp Cell Res 235: 13–21. doi: 10.1006/excr.1997.3647
[56]
Warnke PH, Springer IN, Wiltfang J, Acil Y, Eufinger H, et al. (2004) Growth and transplantation of a custom vascularised bone graft in a man. Lancet 364: 766–770 10.1016/S0140-6736(04)16935-3.
[57]
Yu PB, Deng DY, Lai CS, Hong CC, Cuny GD, et al. (2008) BMP type I receptor inhibition reduces heterotopic [corrected] ossification. Nat Med 14: 1363–1369 10.1038/nm.1888; 10.1038/nm.1888.
[58]
Seyedjafari E, Soleimani M, Ghaemi N, Sarbolouki MN (2011) Enhanced osteogenic differentiation of cord blood-derived unrestricted somatic stem cells on electrospun nanofibers. J Mater Sci Mater Med 22: 165–174 10.1007/s10856-010-4174-6; 10.1007/s10856-010-4174-6.
[59]
Kempen DH, Lu L, Hefferan TE, Creemers LB, Heijink A, et al. (2010) Enhanced bone morphogenetic protein-2-induced ectopic and orthotopic bone formation by intermittent parathyroid hormone (1-34) administration. Tissue Eng Part A 16: 3769–3777 10.1089/ten.TEA.2010.0173; 10.1089/ten.TEA.2010.0173.
[60]
Gurevitch O, Khitrin S, Valitov A, Slavin S (2007) Osteoporosis of hematologic etiology. Exp Hematol 35: 128–136 10.1016/j.exphem.2006.09.010.
[61]
Scott MA, Levi B, Askarinam A, Nguyen A, Rackohn T, et al. (2012) Brief review of models of ectopic bone formation. Stem Cells Dev 21: 655–667 10.1089/scd.2011.0517; 10.1089/scd.2011.0517.
