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

Journal of Orthopaedic Surgery and Research

Claudius Thomé³  Christian Kaps¹  Michaela Endres¹  Jan Philipp Krüger²  Jessie Cluzel²  Aldemar Andres Hegewald⁴ 

[1] Tissue Engineering Laboratory, Department of Rheumatology, Charité University Medicine Berlin, Charitéplatz 1, Berlin 10117, Germany;TransTissue Technologies GmbH, Charitéplatz 1, Berlin 10117, Germany;Department of Neurosurgery, Innsbruck Medical University, Anichstr. 35, Innsbruck 6020, Austria;Department of Neurosurgery, University Medical Center Mannheim, Heidelberg University, Theodor-Kutzer-Ufer 1-3, Mannheim 68167, Germany

关键词: Growth factors;    Biomaterial;    Annulus fibrosus;    Intervertebral disc;    Spine;    Regenerative medicine;    Tissue engineering;   

Others  :  1152196
DOI  :  10.1186/s13018-014-0073-8

 received in 2014-03-23, accepted in 2014-07-25,  发布年份 2014

【 摘 要 】

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.

【 授权许可】

   
2014 Hegewald et al.; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [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.
• [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.
• [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.
• [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.
• [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.
• [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.
• [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.
• [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.
• [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]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.
• [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.
• [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.
• [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.
• [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.
• [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.
• [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.
• [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. 536–7
• [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.
• [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.
• [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.
• [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.
• [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.
• [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.
• [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.
• [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.
• [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.
• [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.
• [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.
• [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.
• [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.