Dysregulation of STAT3 signaling is associated with endplate-oriented herniations of the intervertebral disc in Adgrg6 mutant mice

English version

Autoři: Zhaoyang Liu ^aff001; Garrett W. D. Easson ^aff003; Jingjing Zhao ^aff004; Nadja Makki ^aff004; Nadav Ahituv ^aff004; Matthew J. Hilton ^aff006; Simon Y. Tang ^aff003; Ryan S. Gray ^aff001
Působiště autorů: Department of Nutrional Sciences, University of Texas at Austin, Austin, Texas, United States of America ^aff001; Department of Pediatrics, Dell Pediatric Research Institute, University of Texas at Austin Dell Medical School, Austin, Texas, United States of America ^aff002; Department of Orthopedics, Washington University School of Medicine, Saint Louis, Missouri, United States of America ^aff003; Department of Bioengineering and Therapeutic Sciences and Institute for Human Genetics, University of California San Francisco, San Francisco, California, United States of America ^aff004; Department of Anatomy and Cell Biology, University of Florida, College of Medicine, Gainesville, Florida, United States of America ^aff005; Department of Orthopedic Surgery and Cell Biology, Duke University School of Medicine, Durham, North Carolina, United States of America ^aff006
Vyšlo v časopise: Dysregulation of STAT3 signaling is associated with endplate-oriented herniations of the intervertebral disc in Adgrg6 mutant mice. PLoS Genet 15(10): e32767. doi:10.1371/journal.pgen.1008096
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.pgen.1008096

Souhrn

Degenerative changes of the intervertebral disc (IVD) are a leading cause of disability affecting humans worldwide and has been attributed primarily to trauma and the accumulation of pathology during aging. While genetic defects have also been associated with disc degeneration, the precise mechanisms driving the initiation and progression of disease have remained elusive due to a paucity of genetic animal models. Here, we discuss a novel conditional mouse genetic model of endplate-oriented disc herniations in adult mice. Using conditional mouse genetics, we show increased mechanical stiffness and reveal dysregulation of typical gene expression profiles of the IVD in adhesion G-protein coupled receptor G6 (Adgrg6) mutant mice prior to the onset of endplate-oriented disc herniations in adult mice. We observed increased STAT3 activation prior to IVD defects and go on to demonstrate that treatment of Adgrg6 conditional mutant mice with a small molecule inhibitor of STAT3 activation ameliorates endplate-oriented herniations. These findings establish ADGRG6 and STAT3 as novel regulators of IVD endplate and growth plate integrity in the mouse, and implicate ADGRG6/STAT3 signaling as promising therapeutic targets for endplate-oriented disc degeneration.

Klíčová slova:

Gene expression – Mouse models – Collagens – Fibrosis – Cartilage – Stiffness – STAT signaling

Zdroje

1. Brinjikji W, Diehn FE, Jarvik JG, Carr CM, Kallmes DF, Murad MH, et al. MRI Findings of Disc Degeneration are More Prevalent in Adults with Low Back Pain than in Asymptomatic Controls: A Systematic Review and Meta-Analysis. AJNR Am J Neuroradiol. 2015;36(12):2394–9. Epub 2015/09/12. doi: 10.3174/ajnr.A4498 26359154.

2. Sun D, Liu P, Cheng J, Ma Z, Liu J, Qin TJBMD. Correlation between intervertebral disc degeneration, paraspinal muscle atrophy, and lumbar facet joints degeneration in patients with lumbar disc herniation. 2017;18(1):167. doi: 10.1186/s12891-017-1522-4 28427393

3. Dudli S, Ferguson SJ, Haschtmann D. Severity and pattern of post-traumatic intervertebral disc degeneration depend on the type of injury. Spine J. 2014;14(7):1256–64. Epub 2014/03/04. doi: 10.1016/j.spinee.2013.07.488 24583791.

4. Zhu F, Bao H, Yan P, Liu S, Bao M, Zhu Z, et al. Do the disc degeneration and osteophyte contribute to the curve rigidity of degenerative scoliosis? 2017;18(1):128. doi: 10.1186/s12891-017-1471-y 28356146

5. Cortes DH, Elliott DM. The Intervertebral Disc: Overview of Disc Mechanics. In: Shapiro IM, Risbud MV, editors. The Intervertebral Disc: Molecular and Structural Studies of the Disc in Health and Disease. Vienna: Springer Vienna; 2014. p. 17–31.

6. Wong J, Sampson SL, Bell-Briones H, Ouyang A, Lazar AA, Lotz JC, et al. Nutrient supply and nucleus pulposus cell function: effects of the transport properties of the cartilage endplate and potential implications for intradiscal biologic therapy. Osteoarthritis Cartilage. 2019;27(6):956–64. Epub 2019/02/06. doi: 10.1016/j.joca.2019.01.013 30721733; PubMed Central PMCID: PMC6536352.

7. Sambrook PN, MacGregor AJ, Spector TD. Genetic influences on cervical and lumbar disc degeneration: a magnetic resonance imaging study in twins. Arthritis Rheum. 1999;42(2):366–72. Epub 1999/02/20. doi: 10.1002/1529-0131(199902)42:2<366::AID-ANR20>3.0.CO;2-6 10025932.

8. Munir S, Rade M, Maatta JH, Freidin MB, Williams FMK. Intervertebral Disc Biology: Genetic Basis of Disc Degeneration. Curr Mol Biol Rep. 2018;4(4):143–50. Epub 2018/11/23. doi: 10.1007/s40610-018-0101-2 30464887; PubMed Central PMCID: PMC6223888.

9. Patra C, van Amerongen MJ, Ghosh S, Ricciardi F, Sajjad A, Novoyatleva T, et al. Organ-specific function of adhesion G protein-coupled receptor GPR126 is domain-dependent. Proc Natl Acad Sci U S A. 2013;110(42):16898–903. doi: 10.1073/pnas.1304837110 24082093; PubMed Central PMCID: PMC3801000.

10. Geng FS, Abbas L, Baxendale S, Holdsworth CJ, Swanson AG, Slanchev K, et al. Semicircular canal morphogenesis in the zebrafish inner ear requires the function of gpr126 (lauscher), an adhesion class G protein-coupled receptor gene. Development. 2013;140(21):4362–74. doi: 10.1242/dev.098061 24067352; PubMed Central PMCID: PMC4007713.

11. Karner CM, Long F, Solnica-Krezel L, Monk KR, Gray RS. Gpr126/Adgrg6 deletion in cartilage models idiopathic scoliosis and pectus excavatum in mice. Hum Mol Genet. 2015;24(15):4365–73. doi: 10.1093/hmg/ddv170 25954032; PubMed Central PMCID: PMC4492399.

12. Zhu F, Bao H, Yan P, Liu S, Bao M, Zhu Z, et al. Do the disc degeneration and osteophyte contribute to the curve rigidity of degenerative scoliosis? BMC Musculoskeletal Disorders. 2017;18(1):128. doi: 10.1186/s12891-017-1471-y 28356146

13. Dy P, Wang W, Bhattaram P, Wang Q, Wang L, Ballock RT, et al. Sox9 directs hypertrophic maturation and blocks osteoblast differentiation of growth plate chondrocytes. Dev Cell. 2012;22(3):597–609. doi: 10.1016/j.devcel.2011.12.024 22421045; PubMed Central PMCID: PMC3306603.

14. Adams M, Simms RJ, Abdelhamed Z, Dawe HR, Szymanska K, Logan CV, et al. A meckelin-filamin A interaction mediates ciliogenesis. Hum Mol Genet. 2012;21(6):1272–86. Epub 2011/11/29. doi: 10.1093/hmg/ddr557 22121117; PubMed Central PMCID: PMC3284117.

15. Lin KH, Tang SY. The Quantitative Structural and Compositional Analyses of Degenerating Intervertebral Discs Using Magnetic Resonance Imaging and Contrast-Enhanced Micro-Computed Tomography. Ann Biomed Eng. 2017;45(11):2626–34. Epub 2017/07/27. doi: 10.1007/s10439-017-1891-8 28744842; PubMed Central PMCID: PMC5665707.

16. Yee A, Chan D. Genetic Basis of Intervertebral Disc Degeneration. In: Shapiro IM, Risbud MV, editors. The Intervertebral Disc: Molecular and Structural Studies of the Disc in Health and Disease. Vienna: Springer Vienna; 2014. p. 157–76.

17. Kadow T, Sowa G, Vo N, Kang JD. Molecular basis of intervertebral disc degeneration and herniations: what are the important translational questions? Clin Orthop Relat Res. 2015;473(6):1903–12. Epub 2014/07/16. doi: 10.1007/s11999-014-3774-8 25024024; PubMed Central PMCID: PMC4418989.

18. Tonge DP, Pearson MJ, Jones SW. The hallmarks of osteoarthritis and the potential to develop personalised disease-modifying pharmacological therapeutics. Osteoarthritis Cartilage. 2014;22(5):609–21. Epub 2014/03/19. doi: 10.1016/j.joca.2014.03.004 24632293.

19. Kalb S, Martirosyan NL, Kalani MY, Broc GG, Theodore N. Genetics of the degenerated intervertebral disc. World Neurosurg. 2012;77(3–4):491–501. Epub 2011/11/29. doi: 10.1016/j.wneu.2011.07.014 22120330.

20. Nguyen QT, Jacobsen TD, Chahine NO. Effects of Inflammation on Multiscale Biomechanical Properties of Cartilaginous Cells and Tissues. ACS Biomater Sci Eng. 2017;3(11):2644–56. Epub 2017/11/21. doi: 10.1021/acsbiomaterials.6b00671 29152560; PubMed Central PMCID: PMC5686563.

21. Liu JW, Abraham AC, Tang SY. The high-throughput phenotyping of the viscoelastic behavior of whole mouse intervertebral discs using a novel method of dynamic mechanical testing. J Biomech. 2015;48(10):2189–94. Epub 2015/05/26. doi: 10.1016/j.jbiomech.2015.04.040 26004435; PubMed Central PMCID: PMC4492880.

22. Huang da W, Sherman BT, Lempicki RA. Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists. Nucleic Acids Res. 2009;37(1):1–13. Epub 2008/11/27. doi: 10.1093/nar/gkn923 19033363; PubMed Central PMCID: PMC2615629.

23. Kizawa H, Kou I, Iida A, Sudo A, Miyamoto Y, Fukuda A, et al. An aspartic acid repeat polymorphism in asporin inhibits chondrogenesis and increases susceptibility to osteoarthritis. Nat Genet. 2005;37(2):138–44. Epub 2005/01/11. doi: 10.1038/ng1496 15640800.

24. Balakrishnan L, Nirujogi RS, Ahmad S, Bhattacharjee M, Manda SS, Renuse S, et al. Proteomic analysis of human osteoarthritis synovial fluid. Clin Proteomics. 2014;11(1):6. Epub 2014/02/19. doi: 10.1186/1559-0275-11-6 24533825; PubMed Central PMCID: PMC3942106.

25. Zhang Q, Lin S, Liu Y, Yuan B, Harris SE, Feng JQ. Dmp1 Null Mice Develop a Unique Osteoarthritis-like Phenotype. Int J Biol Sci. 2016;12(10):1203–12. Epub 2016/10/22. doi: 10.7150/ijbs.15833 27766035; PubMed Central PMCID: PMC5069442.

26. Snelling SJ, Davidson RK, Swingler TE, Le LT, Barter MJ, Culley KL, et al. Dickkopf-3 is upregulated in osteoarthritis and has a chondroprotective role. Osteoarthritis Cartilage. 2016;24(5):883–91. Epub 2015/12/22. doi: 10.1016/j.joca.2015.11.021 26687825; PubMed Central PMCID: PMC4863878.

27. Mahr S, Burmester GR, Hilke D, Gobel U, Grutzkau A, Haupl T, et al. Cis- and trans-acting gene regulation is associated with osteoarthritis. Am J Hum Genet. 2006;78(5):793–803. Epub 2006/04/28. doi: 10.1086/503849 16642435; PubMed Central PMCID: PMC1474041.

28. Antoniou J, Steffen T, Nelson F, Winterbottom N, Hollander AP, Poole RA, et al. 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. 1996;98(4):996–1003. Epub 1996/08/15. doi: 10.1172/JCI118884 8770872; PubMed Central PMCID: PMC507515.

29. Yee A, Lam MP, Tam V, Chan WC, Chu IK, Cheah KS, et al. Fibrotic-like changes in degenerate human intervertebral discs revealed by quantitative proteomic analysis. Osteoarthritis Cartilage. 2016;24(3):503–13. Epub 2015/10/16. doi: 10.1016/j.joca.2015.09.020 26463451.

30. Chen CH, Chiang CJ, Wu LC, Yang CH, Kuo YJ, Tsuang YH, et al. Time course investigation of intervertebral disc degeneration in a rat-tail puncture model. Life Sci. 2016;156:15–20. Epub 2016/05/20. doi: 10.1016/j.lfs.2016.05.020 27197027.

31. Zhang Y, Xiong C, Kudelko M, Li Y, Wang C, Wong YL, et al. Early onset of disc degeneration in SM/J mice is associated with changes in ion transport systems and fibrotic events. Matrix Biol. 2018;70:123–39. Epub 2018/04/13. doi: 10.1016/j.matbio.2018.03.024 29649547.

32. Fearing BV, Hernandez PA, Setton LA, Chahine NO. Mechanotransduction and cell biomechanics of the intervertebral disc. JOR Spine. 2018;1(3). Epub 2018/12/21. doi: 10.1002/jsp2.1026 30569032; PubMed Central PMCID: PMC6296470.

33. Erickson GR, Alexopoulos LG, Guilak F. Hyper-osmotic stress induces volume change and calcium transients in chondrocytes by transmembrane, phospholipid, and G-protein pathways. J Biomech. 2001;34(12):1527–35. Epub 2001/11/22. doi: 10.1016/s0021-9290(01)00156-7 11716854.

34. Matta C, Zakany R, Mobasheri A. Voltage-dependent calcium channels in chondrocytes: roles in health and disease. Curr Rheumatol Rep. 2015;17(7):43. Epub 2015/05/20. doi: 10.1007/s11926-015-0521-4 25980668.

35. Carow B, Rottenberg ME. SOCS3, a Major Regulator of Infection and Inflammation. Front Immunol. 2014;5:58. Epub 2014/03/07. doi: 10.3389/fimmu.2014.00058 24600449; PubMed Central PMCID: PMC3928676.

36. O'Reilly S, Ciechomska M, Cant R, Hugle T, van Laar JM. Interleukin-6, its role in fibrosing conditions. Cytokine Growth Factor Rev. 2012;23(3):99–107. Epub 2012/05/09. doi: 10.1016/j.cytogfr.2012.04.003 22561547.

37. Deng X, Zhao F, Kang B, Zhang X. Elevated interleukin-6 expression levels are associated with intervertebral disc degeneration. Exp Ther Med. 2016;11(4):1425–32. doi: 10.3892/etm.2016.3079 27073460; PubMed Central PMCID: PMC4812581.

38. Suzuki S, Fujita N, Fujii T, Watanabe K, Yagi M, Tsuji T, et al. Potential Involvement of the IL-6/JAK/STAT3 Pathway in the Pathogenesis of Intervertebral Disc Degeneration. Spine (Phila Pa 1976). 2017;42(14):E817–E24. Epub 2016/11/24. doi: 10.1097/BRS.0000000000001982 27879577.

39. Oishi Y, Manabe I. Macrophages in inflammation, repair and regeneration. Int Immunol. 2018;30(11):511–28. Epub 2018/08/31. doi: 10.1093/intimm/dxy054 30165385.

40. Yao Y, Wang Y. ATDC5: an excellent in vitro model cell line for skeletal development. J Cell Biochem. 2013;114(6):1223–9. Epub 2012/11/30. doi: 10.1002/jcb.24467 23192741.

41. Bi W, Huang W, Whitworth DJ, Deng JM, Zhang Z, Behringer RR, et al. Haploinsufficiency of Sox9 results in defective cartilage primordia and premature skeletal mineralization. Proc Natl Acad Sci U S A. 2001;98(12):6698–703. Epub 2001/05/24. doi: 10.1073/pnas.111092198 11371614; PubMed Central PMCID: PMC34415.

42. Arnold MA, Kim Y, Czubryt MP, Phan D, McAnally J, Qi X, et al. MEF2C transcription factor controls chondrocyte hypertrophy and bone development. Dev Cell. 2007;12(3):377–89. Epub 2007/03/06. doi: 10.1016/j.devcel.2007.02.004 17336904.

43. Garbers C, Aparicio-Siegmund S, Rose-John S. The IL-6/gp130/STAT3 signaling axis: recent advances towards specific inhibition. Curr Opin Immunol. 2015;34:75–82. Epub 2015/03/10. doi: 10.1016/j.coi.2015.02.008 25749511.

44. Kapoor M, Martel-Pelletier J, Lajeunesse D, Pelletier JP, Fahmi H. Role of proinflammatory cytokines in the pathophysiology of osteoarthritis. Nat Rev Rheumatol. 2011;7(1):33–42. Epub 2010/12/02. doi: 10.1038/nrrheum.2010.196 21119608.

45. Schust J, Sperl B, Hollis A, Mayer TU, Berg T. Stattic: a small-molecule inhibitor of STAT3 activation and dimerization. Chem Biol. 2006;13(11):1235–42. Epub 2006/11/23. doi: 10.1016/j.chembiol.2006.09.018 17114005.

46. Wuertz K, Haglund L. Inflammatory mediators in intervertebral disk degeneration and discogenic pain. Global Spine J. 2013;3(3):175–84. Epub 2014/01/18. doi: 10.1055/s-0033-1347299 24436868; PubMed Central PMCID: PMC3854585.

47. Eskola PJ, Kjaer P, Sorensen JS, Okuloff A, Wedderkopp N, Daavittila I, et al. Gender difference in genetic association between IL1A variant and early lumbar disc degeneration: a three-year follow-up. Int J Mol Epidemiol Genet. 2012;3(3):195–204. Epub 2012/10/11. 23050050; PubMed Central PMCID: PMC3459213.

48. Livshits G, Zhai G, Hart DJ, Kato BS, Wang H, Williams FM, et al. Interleukin-6 is a significant predictor of radiographic knee osteoarthritis: The Chingford Study. Arthritis Rheum. 2009;60(7):2037–45. Epub 2009/07/01. doi: 10.1002/art.24598 19565477; PubMed Central PMCID: PMC2841820.

49. Akiyama H, Chaboissier MC, Martin JF, Schedl A, de Crombrugghe B. The transcription factor Sox9 has essential roles in successive steps of the chondrocyte differentiation pathway and is required for expression of Sox5 and Sox6. Genes Dev. 2002;16(21):2813–28. doi: 10.1101/gad.1017802 12414734; PubMed Central PMCID: PMC187468.

50. Henry SP, Liang S, Akdemir KC, de Crombrugghe B. The postnatal role of Sox9 in cartilage. J Bone Miner Res. 2012;27(12):2511–25. doi: 10.1002/jbmr.1696 22777888; PubMed Central PMCID: PMC3502666.

51. El-Brolosy MA, Stainier DYR. Genetic compensation: A phenomenon in search of mechanisms. PLoS Genet. 2017;13(7):e1006780. Epub 2017/07/14. doi: 10.1371/journal.pgen.1006780 28704371; PubMed Central PMCID: PMC5509088.

52. Adams MA, McMillan DW, Green TP, Dolan P. Sustained loading generates stress concentrations in lumbar intervertebral discs. Spine (Phila Pa 1976). 1996;21(4):434–8. Epub 1996/02/15. doi: 10.1097/00007632-199602150-00006 8658246.

53. Wilke HJ, Kienle A, Maile S, Rasche V, Berger-Roscher N. A new dynamic six degrees of freedom disc-loading simulator allows to provoke disc damage and herniation. Eur Spine J. 2016;25(5):1363–72. Epub 2016/02/04. doi: 10.1007/s00586-016-4416-5 26838335.

54. Vergroesen PP, Kingma I, Emanuel KS, Hoogendoorn RJ, Welting TJ, van Royen BJ, et al. Mechanics and biology in intervertebral disc degeneration: a vicious circle. Osteoarthritis Cartilage. 2015;23(7):1057–70. Epub 2015/04/02. doi: 10.1016/j.joca.2015.03.028 25827971.

55. Berger-Roscher N, Casaroli G, Rasche V, Villa T, Galbusera F, Wilke HJ. Influence of Complex Loading Conditions on Intervertebral Disc Failure. Spine (Phila Pa 1976). 2017;42(2):E78–E85. Epub 2016/05/18. doi: 10.1097/BRS.0000000000001699 27187053.

56. Veres SP, Robertson PA, Broom ND. The influence of torsion on disc herniation when combined with flexion. Eur Spine J. 2010;19(9):1468–78. Epub 2010/05/04. doi: 10.1007/s00586-010-1383-0 20437184; PubMed Central PMCID: PMC2989279.

57. Yang B, O'Connell GD. Effect of collagen fibre orientation on intervertebral disc torsion mechanics. Biomech Model Mechanobiol. 2017;16(6):2005–15. Epub 2017/07/25. doi: 10.1007/s10237-017-0934-2 28733922.

58. Urban JP, Smith S, Fairbank JC. Nutrition of the intervertebral disc. Spine (Phila Pa 1976). 2004;29(23):2700–9. Epub 2004/11/27. doi: 10.1097/01.brs.0000146499.97948.52 15564919.

59. Smith LJ, Nerurkar NL, Choi KS, Harfe BD, Elliott DM. Degeneration and regeneration of the intervertebral disc: lessons from development. Dis Model Mech. 2011;4(1):31–41. doi: 10.1242/dmm.006403 21123625; PubMed Central PMCID: PMC3008962.

60. Zhao CQ, Wang LM, Jiang LS, Dai LY. The cell biology of intervertebral disc aging and degeneration. Ageing Res Rev. 2007;6(3):247–61. Epub 2007/09/18. doi: 10.1016/j.arr.2007.08.001 17870673.

61. Osuka K, Usuda N, Aoyama M, Yamahata H, Takeuchi M, Yasuda M, et al. Expression of the JAK/STAT3/SOCS3 signaling pathway in herniated lumbar discs. Neurosci Lett. 2014;569:55–8. Epub 2014/04/02. doi: 10.1016/j.neulet.2014.03.045 24686183.

62. Stannus O, Jones G, Cicuttini F, Parameswaran V, Quinn S, Burgess J, et al. Circulating levels of IL-6 and TNF-alpha are associated with knee radiographic osteoarthritis and knee cartilage loss in older adults. Osteoarthritis Cartilage. 2010;18(11):1441–7. Epub 2010/09/08. doi: 10.1016/j.joca.2010.08.016 20816981.

63. Liu Z, Chen J, Mirando AJ, Wang C, Zuscik MJ, O'Keefe RJ, et al. A dual role for NOTCH signaling in joint cartilage maintenance and osteoarthritis. Sci Signal. 2015;8(386):ra71. doi: 10.1126/scisignal.aaa3792 26198357; PubMed Central PMCID: PMC4607068.

64. Latourte A, Cherifi C, Maillet J, Ea HK, Bouaziz W, Funck-Brentano T, et al. Systemic inhibition of IL-6/Stat3 signalling protects against experimental osteoarthritis. Ann Rheum Dis. 2017;76(4):748–55. doi: 10.1136/annrheumdis-2016-209757 27789465.

65. Mogha A, Benesh AE, Patra C, Engel FB, Schoneberg T, Liebscher I, et al. Gpr126 functions in Schwann cells to control differentiation and myelination via G-protein activation. J Neurosci. 2013;33(46):17976–85. doi: 10.1523/JNEUROSCI.1809-13.2013 24227709; PubMed Central PMCID: PMC3828454.

66. Soriano P. Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat Genet. 1999;21(1):70–1. doi: 10.1038/5007 9916792.

67. Long F, Zhang XM, Karp S, Yang Y, McMahon AP. Genetic manipulation of hedgehog signaling in the endochondral skeleton reveals a direct role in the regulation of chondrocyte proliferation. Development. 2001;128(24):5099–108. 11748145.

68. Martin M. Cutadapt removes adapter sequences from high-throughput sequencing reads. 2011. 2011;17(1):3%J EMBnet.journal. Epub 2011-08-02. doi: 10.14806/ej.17.1.200

69. Kim D, Langmead B, Salzberg SL. HISAT: a fast spliced aligner with low memory requirements. Nature Methods. 2015;12:357. doi: 10.1038/nmeth.3317 https://www.nature.com/articles/nmeth.3317#supplementary-information. 25751142

70. Frazee AC, Pertea G, Jaffe AE, Langmead B, Salzberg SL, Leek JT. Ballgown bridges the gap between transcriptome assembly and expression analysis. Nat Biotechnol. 2015;33(3):243–6. doi: 10.1038/nbt.3172 25748911; PubMed Central PMCID: PMC4792117.

71. Pertea M, Pertea GM, Antonescu CM, Chang T-C, Mendell JT, Salzberg SL. StringTie enables improved reconstruction of a transcriptome from RNA-seq reads. Nature Biotechnology. 2015;33:290. doi: 10.1038/nbt.3122, https://www.nature.com/articles/nbt.3122#supplementary-information. 25690850

72. Lin KH, Wu Q, Leib DJ, Tang SY. A novel technique for the contrast-enhanced microCT imaging of murine intervertebral discs. J Mech Behav Biomed Mater. 2016;63:66–74. Epub 2016/06/25. doi: 10.1016/j.jmbbm.2016.06.003 27341292; PubMed Central PMCID: PMC4983496.

73. Sabiston P, Adams ME, Ho YA. Automation of 1,9-dimethylmethylene blue dye-binding assay for sulfated glycosaminoglycans with application to cartilage microcultures. Anal Biochem. 1985;149(2):543–8. Epub 1985/09/01. doi: 10.1016/0003-2697(85)90611-6 4073509.

74. Woessner JF Jr. The determination of hydroxyproline in tissue and protein samples containing small proportions of this imino acid. Arch Biochem Biophys. 1961;93:440–7. Epub 1961/05/01. doi: 10.1016/0003-9861(61)90291-0 13786180.