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Journal of Orthopaedic Surgery and Research
Published in: Journal of Orthopaedic Surgery and Research 1/2014

Open Access 01-12-2014 | Research article

Effects of initial boost with TGF-beta 1 and grade of intervertebral disc degeneration on 3D culture of human annulus fibrosus cells

Authors: Aldemar Andres Hegewald, Jessie Cluzel, Jan Philipp Krüger, Michaela Endres, Christian Kaps, Claudius Thomé

Published in: Journal of Orthopaedic Surgery and Research | Issue 1/2014

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Abstract

Background

Three-dimensional (3D) culture in porous biomaterials as well as stimulation with growth factors are known to be supportive for intervertebral disc cell differentiation and tissue formation. Unless sophisticated releasing systems are used, however, effective concentrations of growth factors are maintained only for a very limited amount of time in in vivo applications. Therefore, we investigated, if an initial boost with transforming growth factor-beta 1 (TGF-beta 1) is capable to induce a lasting effect of superior cartilaginous differentiation in slightly and severely degenerated human annulus fibrosus (AF) cells.

Methods

Human AF tissue was harvested during surgical treatment of six adult patients with lumbar spinal diseases. Grading of disc degeneration was performed with magnet resonance imaging. AF cells were isolated and expanded in monolayer culture and rearranged three-dimensionally in a porous biomaterial consisting of stepwise absorbable poly-glycolic acid and poly-(lactic-co-glycolic) acid and a supportive fine net of non-absorbable polyvinylidene fluoride. An initial boost of TGF-beta 1 or TGF-beta 1 and hyaluronan was applied and compared with controls. Matrix formation was assessed at days 7 and 21 by (1) histological staining of the typical extracellular matrix molecules proteoglycan and type I and type II collagens and by (2) real-time gene expression analysis of aggrecan, decorin, biglycan, type I, II, III, and X collagens as well as of catabolic matrix metalloproteinases MMP-2 and MMP-13.

Results

An initial boost with TGF-beta 1 or TGF-beta 1 and hyaluronan did not enhance the expression of characteristic AF matrix molecules in our 3D culture system. AF cells showed high viability in the progressively degrading biomaterial. Stratification by grade of intervertebral disc degeneration showed that AF cells from both, slightly degenerated, or severely degenerated tissue are capable of significant up-regulations of characteristic matrix molecules in 3D culture. AF cells from severely degenerated tissue, however, displayed significantly lower up-regulations in some matrix molecules such as aggrecan.

Conclusions

We failed to show a supportive effect of an initial boost with TGF-beta 1 in our 3D culture system. This underlines the need for further investigations on growth factor releasing systems.
Appendix
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Literature
1.
Bailey A, Araghi A, Blumenthal S, Huffmon GV: Prospective, multicenter, randomized, controlled study of anular repair in lumbar discectomy: two-year follow-up. Spine. 2013, 38: 1161-1169. 10.1097/BRS.0b013e31828b2e2f.CrossRefPubMed
2.
Wilke HJ, Heuer F, Neidlinger-Wilke C, Claes L: Is a collagen scaffold for a tissue engineered nucleus replacement capable of restoring disc height and stability in an animal model?. Eur Spine J. 2006, 15 (Suppl 3): S433-S438. 10.1007/s00586-006-0177-x.CrossRefPubMed
3.
Le Maitre CL, Pockert A, Buttle DJ, Freemont AJ, Hoyland JA: Matrix synthesis and degradation in human intervertebral disc degeneration. Biochem Soc Trans. 2007, 35: 652-655. 10.1042/BST0350652.CrossRefPubMed
4.
Sivan SS, Tsitron E, Wachtel E, Roughley PJ, Sakkee N, van der Ham F, DeGroot J, Roberts S, Maroudas A: Aggrecan turnover in human intervertebral disc as determined by the racemization of aspartic acid. J Biol Chem. 2006, 281: 13009-13014. 10.1074/jbc.M600296200.CrossRefPubMed
5.
Sivan SS, Wachtel E, Tsitron E, Sakkee N, van der Ham F, Degroot J, Roberts S, Maroudas A: Collagen turnover in normal and degenerate human intervertebral discs as determined by the racemization of aspartic acid. J Biol Chem. 2008, 283: 8796-8801. 10.1074/jbc.M709885200.CrossRefPubMed
6.
Götz W, Barnert S, Bertagnoli R, Miosge N, Kresse H, Herken R: Immunohistochemical localization of the small proteoglycans decorin and biglycan in human intervertebral discs. Cell Tissue Res. 1997, 289: 185-190. 10.1007/s004410050864.CrossRefPubMed
7.
Cs-Szabo G, Ragasa-San Juan D, Turumella V, Masuda K, Thonar EJ, An HS: Changes in mRNA and protein levels of proteoglycans of the anulus fibrosus and nucleus pulposus during intervertebral disc degeneration. Spine. 2002, 27: 2212-2219. 10.1097/00007632-200210150-00006.CrossRefPubMed
8.
Singh K, Masuda K, Thonar EJ, An HS, Cs-Szabo G: Age-related changes in the extracellular matrix of nucleus pulposus and anulus fibrosus of human intervertebral disc. Spine. 2009, 34: 10-16. 10.1097/BRS.0b013e31818e5ddd.PubMedCentralCrossRefPubMed
9.
Gruber HE, Hoelscher GL, Ingram JA, Bethea S, Zinchenko N, Hanley EN: Variations in aggrecan localization and gene expression patterns characterize increasing stages of human intervertebral disk degeneration. Exp Mol Pathol. 2011, 91: 534-539. 10.1016/j.yexmp.2011.06.001.CrossRefPubMed
10.
Guterl CC, See EY, Blanquer SB, Pandit A, Ferguson SJ, Benneker LM, Grijpma DW, Sakai D, Eglin D, Alini M, Iatridis JC, Grad S: Challenges and strategies in the repair of ruptured annulus fibrosus. Eur Cell Mater. 2013, 25: 1-21.PubMedCentralPubMed
11.
Hegewald AA, Zouhair S, Endres M, Cabraja M, Woiciechowsky C, Thomé C, Kaps C: Towards biological anulus repair: TGF-beta3, FGF-2 and human serum support matrix formation by human anulus fibrosus cells. Tissue Cell. 2012, 45: 68-76. 10.1016/j.tice.2012.09.011.CrossRefPubMed
12.
Hegewald AA, Neumann K, Kalwitz G, Freymann U, Endres M, Schmieder K, Kaps C, Thomé C: The chemokines CXCL10 and XCL1 recruit human annulus fibrosus cells. Spine. 2012, 37: 101-107. 10.1097/BRS.0b013e318210ed55.CrossRefPubMed
13.
Vadalà G, Mozetic P, Rainer A, Centola M, Loppini M, Trombetta M, Denaro V: Bioactive electrospun scaffold for annulus fibrosus repair and regeneration. Eur Spine J. 2012, 21 (Suppl 1): S20-S26. 10.1007/s00586-012-2235-x.CrossRefPubMed
14.
Cho H, Park SH, Park K, Shim JW, Huang J, Smith R, Elder S, Min BH, Hasty KA: Construction of a tissue-engineered annulus fibrosus. Artif Organs. 2013, 37: E131-E138. 10.1111/aor.12066.CrossRefPubMed
15.
Cabraja M, Endres M, Hegewald AA, Vetterlein S, Thomé C, Woiciechowsky C, Kaps C: A 3D environment for anulus fibrosus regeneration. J Neurosurg Spine. 2012, 17: 177-183. 10.3171/2012.4.SPINE111095.CrossRefPubMed
16.
Hegewald AA, Medved FM, Feng DF, Tsagogiorgas CT, Schindler GS, Arshi AA, Deichmann TD, Kaps CK, Schmieder KS, Thomé CT: Biological sealing of annulus fibrosus defects with a polymer-based implant in an ovine model. Eur Spine J. 2010, 19 (Suppl 3): 254-
17.
Griffith JF, Wang YX, Antonio GE, Choi KC, Yu A, Ahuja AT, Leung PC: Modified Pfirrmann grading system for lumbar intervertebral disc degeneration. Spine. 2007, 32: E708-E712. 10.1097/BRS.0b013e31815a59a0.CrossRefPubMed
18.
Chomczynski P: A reagent for the single-step simultaneous isolation of RNA, DNA and proteins from cell and tissue samples. Biotechniques. 1993, 15: 532-534. 536–7PubMed
19.
Winer J, Jung CK, Shackel I, Williams PM: Development and validation of real-time quantitative reverse transcriptase-polymerase chain reaction for monitoring gene expression in cardiac myocytes in vitro. Anal Biochem. 1999, 270: 41-49. 10.1006/abio.1999.4085.CrossRefPubMed
20.
O'Brien MC, Bolton WE: Comparison of cell viability probes compatible with fixation and permeabilization for combined surface and intracellular staining in flow cytometry. Cytometry. 1995, 19: 243-255. 10.1002/cyto.990190308.CrossRefPubMed
21.
Neidlinger-Wilke C, Würtz K, Liedert A, Schmidt C, Börm W, Ignatius A, Wilke HJ, Claes L: A three-dimensional collagen matrix as a suitable culture system for the comparison of cyclic strain and hydrostatic pressure effects on intervertebral disc cells. J Neurosurg Spine. 2005, 2: 457-465. 10.3171/spi.2005.2.4.0457.CrossRefPubMed
22.
Reza AT, Nicoll SB: Hydrostatic pressure differentially regulates outer and inner annulus fibrosus cell matrix production in 3D scaffolds. Ann Biomed Eng. 2008, 36: 204-213. 10.1007/s10439-007-9407-6.CrossRefPubMed
23.
Melrose J, Smith SM, Fuller ES, Young AA, Roughley PJ, Dart A, Little CB: Biglycan and fibromodulin fragmentation correlates with temporal and spatial annular remodelling in experimentally injured ovine intervertebral discs. Eur Spine J. 2007, 16: 2193-2205. 10.1007/s00586-007-0497-5.PubMedCentralCrossRefPubMed
24.
Schmid TM, Bonen DK, Luchene L, Linsenmayer TF: Late events in chondrocyte differentiation: hypertrophy, type X collagen synthesis and matrix calcification. In Vivo. 1991, 5: 533-540.PubMed
25.
Weiler C, Nerlich AG, Zipperer J, Bachmeier BE, Boos N: SSE award competition in basic science: expression of major matrix metalloproteinases is associated with intervertebral disc degradation and resorption. Eur Spine J. 2002, 2002 (11): 308-320. 10.1007/s00586-002-0472-0.CrossRef
26.
Le Maitre CL, Freemont AJ, Hoyland JA: Localization of degradative enzymes and their inhibitors in the degenerate human intervertebral disc. J Pathol. 2004, 204: 47-54. 10.1002/path.1608.CrossRefPubMed
27.
Gruber HE, Chow Y, Hoelscher GL, Ingram JA, Zinchenko N, Norton HJ, Sun Y, Hanley EN: Micromass culture of human anulus cells: morphology and extracellular matrix production. Spine. 2010, 35: 1033-1038.PubMed
28.
Hegewald AA, Ringe J, Bartel J, Krüger I, Notter M, Barnewitz D, Kaps C, Sittinger M: Hyaluronic acid and autologous synovial fluid induce chondrogenic differentiation of equine mesenchymal stem cells: a preliminary study. Tissue Cell. 2004, 36: 431-438. 10.1016/j.tice.2004.07.003.CrossRefPubMed
29.
Bhang SH, Jeon JY, La WG, Seong JY, Hwang JW, Ryu SE, Kim BS: Enhanced chondrogenic marker expression of human mesenchymal stem cells by interaction with both TGF-beta3 and hyaluronic acid. Biotechnol Appl Biochem. 2011, 58: 271-276. 10.1002/bab.39.CrossRefPubMed
30.
Wallach CJ, Kim JS, Sobajima S, Lattermann C, Oxner WM, McFadden K, Robbins PD, Gilbertson LG, Kang JD: Safety assessment of intradiscal gene transfer: a pilot study. Spine J. 2006, 6: 107-112. 10.1016/j.spinee.2005.05.002.CrossRefPubMed
31.
Sohier J, Moroni L, van Blitterswijk C, de Groot K, Bezemer JM: Critical factors in the design of growth factor releasing scaffolds for cartilage tissue engineering. Expert Opin Drug Deliv. 2008, 5: 543-566. 10.1517/17425247.5.5.543.CrossRefPubMed
Metadata
Title
Effects of initial boost with TGF-beta 1 and grade of intervertebral disc degeneration on 3D culture of human annulus fibrosus cells
Authors
Aldemar Andres Hegewald
Jessie Cluzel
Jan Philipp Krüger
Michaela Endres
Christian Kaps
Claudius Thomé
Publication date
01-12-2014
Publisher
BioMed Central
Published in
Journal of Orthopaedic Surgery and Research / Issue 1/2014
Electronic ISSN: 1749-799X
DOI
https://doi.org/10.1186/s13018-014-0073-8

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