3D assessment of intervertebral disc degeneration in zebrafish identifies changes in bone density that prime disc disease

Abstract

Back pain is a common condition with a high social impact and represents a global health burden. Intervertebral disc disease (IVDD) is one of the major causes of back pain; no therapeutics are currently available to reverse this disease. The impact of bone mineral density (BMD) on IVDD has been controversial, with some studies suggesting osteoporosis as causative for IVDD and others suggesting it as protective for IVDD. Functional studies to evaluate the influence of genetic components of BMD in IVDD could highlight opportunities for drug development and repurposing. By taking a holistic 3D approach, we established an aging zebrafish model for spontaneous IVDD. Increased BMD in aging, detected by automated computational analysis, is caused by bone deformities at the endplates. However, aged zebrafish spines showed changes in bone morphology, microstructure, mineral heterogeneity, and increased fragility that resembled osteoporosis. Elements of the discs recapitulated IVDD symptoms found in humans: the intervertebral ligament (equivalent to the annulus fibrosus) showed disorganized collagen fibers and herniation, while the disc center (nucleus pulposus equivalent) showed dehydration and cellular abnormalities. We manipulated BMD in young zebrafish by mutating sp7 and cathepsin K, leading to low and high BMD, respectively. Remarkably, we detected IVDD in both groups, demonstrating that low BMD does not protect against IVDD, and we found a strong correlation between high BMD and IVDD. Deep learning was applied to high-resolution synchrotron µCT image data to analyze osteocyte 3D lacunar distribution and morphology, revealing a role of sp7 in controlling the osteocyte lacunar 3D profile. Our findings suggest potential avenues through which bone quality can be targeted to identify beneficial therapeutics for IVDD.

References

[1] GBD 2016 Disease and Injury Incidence and Prevalence Collaborators. Global, regional, and national incidence, prevalence, and years lived with disability for 328 diseases and injuries for 195 countries, 1990-2016: a systematic analysis for the Global Burden of Disease Study 2016. Lancet, 2017, 390: 1211-1259

[2] Zheng CJ, Chen J. Disc degeneration implies low back pain. Theor. Biol. Med. Model., 2015, 12

[3] Hoy D et al. A systematic review of the global prevalence of low back pain. Arthritis Rheum., 2012, 64: 2028-2037

[4] Hayes AJ, Benjamin M, Ralphs JR. Extracellular matrix in development of the intervertebral disc. Matrix Biol., 2001, 20: 107-121

[5] Feng Y, Egan B, Wang J. Genetic factors in intervertebral disc degeneration. Genes Dis., 2016, 3: 178-185

[6] Ashinsky BG et al. Intervertebral disc degeneration is associated with aberrant endplate remodeling and reduced small molecule transport. J. Bone Miner. Res., 2020, 35: 1572-1581

[7] Munir S, Rade M, Määttä JH, Freidin MB, Williams FMK. Intervertebral disc biology: genetic basis of disc degeneration. Curr. Mol. Biol. Rep., 2018, 4: 143-150

[8] Williams FM et al. Novel genetic variants associated with lumbar disc degeneration in northern Europeans: a meta-analysis of 4600 subjects. Ann. Rheum. Dis., 2013, 72: 1141-1148

[9] Ikuno A et al. Genome-wide analysis of DNA methylation profile identifies differentially methylated loci associated with human intervertebral disc degeneration. PLoS One, 2019, 14: e0222188

[10] Freidin MB et al. Insight into the genetic architecture of back pain and its risk factors from a study of 509 000 individuals. Pain, 2019, 160: 1361-1373

[11] Bjornsdottir G et al. Sequence variant at 8q24.21 associates with sciatica caused by lumbar disc herniation. Nat. Commun., 2017, 8

[12] Yong-Hing K, Kirkaldy-Willis WH. The pathophysiology of degenerative disease of the lumbar spine. Orthop. Clin. North Am., 1983, 14: 491-504

[13] Mattei TA. Osteoporosis delays intervertebral disc degeneration by increasing intradiscal diffusive transport of nutrients through both mechanical and vascular pathophysiological pathways. Med. Hypotheses, 2013, 80: 582-586

[14] Miyakoshi N et al. Inverse relation between osteoporosis and spondylosis in postmenopausal women as evaluated by bone mineral density and semiquantitative scoring of spinal degeneration. Spine (Philos. Pa 1976), 2003, 28: 492-495

[15] Nanjo Y, Morio Y, Nagashima H, Hagino H, Teshima R. Correlation between bone mineral density and intervertebral disk degeneration in pre- and postmenopausal women. J. Bone Min. Metab., 2003, 21: 22-27

[16] Cooper, C., Campion, G. & Melton, L. J. 3rd. Hip fractures in the elderly: a world-wide projection. Osteoporos. Int. 2, 285–289 (1992).

[17] Verstraeten, A. et al. Osteoarthrosis retards the development of osteoporosis. Observation of the coexistence of osteoarthrosis and osteoporosis. Clin. Orthop. Relat. Res. (264)169–177 (1991).

[18] Zhou Z et al. Alendronate prevents intervertebral disc degeneration adjacent to a lumbar fusion in ovariectomized rats. Spine (Philos. Pa 1976), 2015, 40: E1073-E1083

[19] Xiao ZF et al. Osteoporosis of the vertebra and osteochondral remodeling of the endplate causes intervertebral disc degeneration in ovariectomized mice. Arthritis Res. Ther., 2018, 20: 207

[20] Luo Y et al. Alendronate retards the progression of lumbar intervertebral disc degeneration in ovariectomized rats. Bone, 2013, 55: 439-448

[21] Tian FM et al. Calcitonin suppresses intervertebral disk degeneration and preserves lumbar vertebral bone mineral density and bone strength in ovariectomized rats. Osteoporos. Int., 2015, 26: 2853-2861

[22] Liu CC et al. Protective effect of calcitonin on lumbar fusion-induced adjacent-segment disc degeneration in ovariectomized rat. BMC Musculoskelet. Disord., 2015, 16

[23] Livshits G et al. Lumbar disc degeneration and genetic factors are the main risk factors for low back pain in women:the UK Twin Spine Study. Ann Rheum Dis., 2011, 70: 1740-1745 Epub 6 Jun 2011.

[24] Kaiser J et al. Correspondence between bone mineral density and intervertebral disc degeneration across age and sex. Arch. Osteoporos., 2018, 13

[25] Samelson, E. J. et al. Older adults with greater severity of lumbar disc height narrowing and facet joint osteoarthritis have higher lumbar volumetric BMD, independently of body weight: Framingham QCT Study. J. Bone. Miner Res. 32 (2018).

[26] Bergen DJM, Kague E, Hammond CL. Zebrafish as an emerging model for osteoporosis: a primary testing platform for screening new osteo-active compounds. Front. Endocrinol. (Lausanne), 2019, 10: 6

[27] Kwon RY, Watson CJ, Karasik D. Using zebrafish to study skeletal genomics. Bone, 2019, 126: 37-50

[28] Fisher S, Jagadeeswaran P, Halpern ME. Radiographic analysis of zebrafish skeletal defects. Dev. Biol., 2003, 264: 64-76

[29] Gistelinck C et al. Zebrafish type I collagen mutants faithfully recapitulate human type I collagenopathies. Proc. Natl Acad. Sci. USA, 2018, 115: E8037-E8046

[30] Tonelli, F. et al. Crtap and p3h1 knock out zebrafish support defective collagen chaperoning as the cause of their osteogenesis imperfecta phenotype. Matrix Biol. 90, 40–60 (2020).

[31] Kague E et al. Osterix/Sp7 limits cranial bone initiation sites and is required for formation of sutures. Dev. Biol., 2016, 413: 160-172

[32] Symmons S. Notochordal and elastic components of the axial skeleton of fishes and their functions in locomotion. J. Zool., 1979, 189: 157-206

[33] Witten PE et al. Bone without minerals and its secondary mineralization in Atlantic salmon (Salmo salar): the recovery from phosphorus deficiency. J. Exp. Biol., 2019, 222: jeb188763

[34] Schmitz RJ. Ultrastructure and function of cellular components of the intercentral joint in the percoid vertebral column. J. Morphol., 1995, 226: 1-24

[35] Witten PE, Obach A, Huysseuned A, Baeverfjorda G. Vertebrae fusion in Atlantic salmon (Salmo salar): development, aggravation and pathways of containment. Aquaculture, 2006, 258: 164-172

[36] Hayes AJ et al. Spinal deformity in aged zebrafish is accompanied by degenerative changes to their vertebrae that resemble osteoarthritis. PLoS One, 2013, 8: e75787

[37] Monma Y et al. Aging-associated microstructural deterioration of vertebra in zebrafish. Bone Rep., 2019, 11: 100215

[38] Kushchayev SV et al. ABCs of the degenerative spine. Insights Imaging, 2018, 9: 253-274

[39] Suniaga S et al. Increased mechanical loading through controlled swimming exercise induces bone formation and mineralization in adult zebrafish. Sci. Rep., 2018, 8

[40] Boskey AL et al. Examining the relationships between bone tissue composition, compositional heterogeneity, and fragility fracture: a matched case-controlled FTIRI study. J. Bone Min. Res, 2016, 31: 1070-1081

[41] Yu A et al. Spatial differences in the distribution of bone between femoral neck and trochanteric fractures. J. Bone Min. Res, 2017, 32: 1672-1680

[42] Newham E et al. Finite element and deformation analyses predict pattern of bone failure in loaded zebrafish spines. J. R. Soc. Interface, 2019, 16: 20190430

[43] Tenne M, McGuigan F, Besjakov J, Gerdhem P, Akesson K. Degenerative changes at the lumbar spine-implications for bone mineral density measurement in elderly women. Osteoporos. Int, 2013, 24: 1419-1428

[44] Nakashima K et al. The novel zinc finger-containing transcription factor osterix is required for osteoblast differentiation and bone formation. Cell, 2002, 108: 17-29

[45] Sinha KM, Zhou X. Genetic and molecular control of osterix in skeletal formation. J. Cell Biochem, 2013, 114: 975-984

[46] Timpson NJ et al. Common variants in the region around Osterix are associated with bone mineral density and growth in childhood. Hum. Mol. Genet., 2009, 18: 1510-1517

[47] Medina-Gomez C et al. Life-course genome-wide association study meta-analysis of total body BMD and assessment of age-specific effects. Am. J. Hum. Genet., 2018, 102: 88-102

[48] Lapunzina P et al. Identification of a frameshift mutation in Osterix in a patient with recessive osteogenesis imperfecta. Am. J. Hum. Genet, 2010, 87: 110-114

[49] Fiscaletti M et al. Novel variant in Sp7/Osx associated with recessive osteogenesis imperfecta with bone fragility and hearing impairment. Bone, 2018, 110: 66-75

[50] Hayat A et al. Biallelic variants in four genes underlying recessive osteogenesis imperfecta. Eur. J. Med Genet., 2020, 63: 103954

[51] Kague E et al. Zebrafish sp7 mutants show tooth cycling independent of attachment, eruption and poor differentiation of teeth. Dev. Biol., 2018, 435: 176-184

[52] Currey JD. The relationship between the stiffness and the mineral content of bone. J. Biomech., 1969, 2: 477-480

[53] Mammone JF, Hudson SM. Micromechanics of bone strength and fracture. J. Biomech., 1993, 26: 439-446

[54] Cotti S et al. More bone with less minerals? The effects of dietary phosphorus on the post-cranial skeleton in zebrafish. Int. J. Mol. Sci., 2020, 21: 5429

[55] Milovanovic, P. & Busse, B. Phenomenon of osteocyte lacunar mineralization: indicator of former osteocyte death and a novel marker of impaired bone quality? Endocr. Connect 9, R70–R80 (2020).

[56] Rolvien T et al. Long-term immobilization in elderly females causes a specific pattern of cortical bone and osteocyte deterioration different from postmenopausal osteoporosis. J. Bone Min. Res., 2020, 35: 1343-1351

[57] Hemmatian H, Bakker AD, Klein-Nulend J, van Lenthe GHAging. Osteocytes, and mechanotransduction. Curr. Osteoporos. Rep., 2017, 15: 401-411

[58] Rochefort GY. The osteocyte as a therapeutic target in the treatment of osteoporosis. Ther. Adv. Musculoskelet. Dis., 2014, 6: 79-91

[59] Ofer L, Zaslansky P, Shahar R. A comparison of the structure, composition and mechanical properties of anosteocytic vertebrae of medaka (O. latipes) and osteocytic vertebrae of zebrafish (D. rerio). J. Fish Biol., 2021, 98: 995-1006

[60] Bredfeldt JS et al. Computational segmentation of collagen fibers from second-harmonic generation images of breast cancer. J. Biomed. Opt., 2014, 19: 16007

[61] Gelb BD, Shi GP, Chapman HA, Desnick RJ. Pycnodysostosis, a lysosomal disease caused by cathepsin K deficiency. Science, 1996, 273: 1236-1238

[62] Watson CJ et al. Phenomics-based quantification of CRISPR-induced mosaicism in zebrafish. Cell Syst., 2020, 10: 275-286.e5

[63] Daly C, Ghosh P, Jenkin G, Oehme D, Goldschlager T. A review of animal models of intervertebral disc degeneration: pathophysiology, regeneration, and translation to the clinic. Biomed. Res. Int., 2016, 2016: 5952165

[64] Wang F et al. Formation, function, and exhaustion of notochordal cytoplasmic vacuoles within intervertebral disc: current understanding and speculation. Oncotarget, 2017, 8: 57800-57812

[65] Risbud MV, Shapiro IM. Notochordal cells in the adult intervertebral disc: new perspective on an old question. Crit. Rev. Eukaryot. Gene Expr., 2011, 21: 29-41

[66] Chanchairujira K et al. Intervertebral disk calcification of the spine in an elderly population: radiographic prevalence, location, and distribution and correlation with spinal degeneration. Radiology, 2004, 230: 499-503

[67] Novais EJ et al. Comparison of inbred mouse strains shows diverse phenotypic outcomes of intervertebral disc aging. Aging Cell, 2020, 19: e13148

[68] Wickrematilake GW. Calcium pyrophosphate dihydrate deposition disease in young patients: two case reports. Arch. Rheumatol., 2017, 32: 80-83

[69] Huxeley TH. Observations on the development of some parts of the skeleton of fishes. Q. J. Microsc. Sci. (continued J. Cell Sci.), 1859, 7: 33-46

[70] Lleras Forero L et al. Segmentation of the zebrafish axial skeleton relies on notochord sheath cells and not on the segmentation clock. Elife, 2018, 7: e33843

[71] Pogoda HM et al. Direct activation of chordoblasts by retinoic acid is required for segmented centra mineralization during zebrafish spine development. Development, 2018, 145: dev159418

[72] Kaiser J et al. Heterogeneity and spatial distribution of intravertebral trabecular bone mineral density in the lumbar spine is associated with prevalent vertebral fracture. J. Bone Min. Res., 2020, 35: 641-648

[73] Wang YXJ. Senile osteoporosis is associated with disc degeneration. Quant. Imaging Med. Surg., 2018, 8: 551-556

[74] Khajuria DK, Karasik D. Novel model of restricted mobility induced osteopenia in zebrafish. J. Fish Biol., 2020, 98: 1031-1038

[75] Harada A, Okuizumi H, Miyagi N, Genda E. Correlation between bone mineral density and intervertebral disc degeneration. Spine (Philos. Pa 1976), 1998, 23: 857-861 discussion 862

[76] Dequeker J, Aerssens J, Luyten FP. Osteoarthritis and osteoporosis: clinical and research evidence of inverse relationship. Aging Clin. Exp. Res., 2003, 15: 426-439

[77] Weintroub S et al. Osteoarthritis of the hip and fracture of the proximal end of the femur. Acta Orthop. Scand., 1982, 53: 261-264

[78] Marcelli C et al. The relationship between osteoarthritis of the hands, bone mineral density, and osteoporotic fractures in elderly women. Osteoporos. Int, 1995, 5: 382-388

[79] Lee S et al. Association between low-back pain and lumbar spine bone density: a population-based cross-sectional study. J. Neurosurg. Spine, 2013, 19: 307-313

[80] Lindstrom E et al. Nonclinical and clinical pharmacological characterization of the potent and selective cathepsin K inhibitor MIV-711. J. Transl. Med., 2018, 16

[81] Reid IR. Short-term and long-term effects of osteoporosis therapies. Nat. Rev. Endocrinol., 2015, 11: 418-428

[82] Westerfield, M. The Zebrafish Book. A Guide for the Laboratory Use of Zebrafish (Danio rerio) (Univ. of Oregon Press, 2000).

[83] Wienholds E et al. Efficient target-selected mutagenesis in zebrafish. Genome Res., 2003, 13: 2700-2707

[84] DeLaurier A et al. Zebrafish sp7:EGFP: a transgenic for studying otic vesicle formation, skeletogenesis, and bone regeneration. Genesis, 2010, 48: 505-511

[85] Brunt LH, Begg K, Kague E, Cross S, Hammond CL. Wnt signalling controls the response to mechanical loading during zebrafish joint development. Development, 2017, 144: 2798-2809

[86] Carrington B, Varshney GK, Burgess SM, Sood R. CRISPR-STAT: an easy and reliable PCR-based method to evaluate target-specific sgRNA activity. Nucleic Acids Res, 2015, 43: e157

[87] Shah AN, Davey CF, Whitebirch AC, Miller AC, Moens CB. Rapid reverse genetic screening using CRISPR in zebrafish. Nat. Methods, 2015, 12: 535-540

[88] De Clercq A et al. The external phenotype-skeleton link in post-hatch farmed Chinook salmon (Oncorhynchus tshawytscha). J. Fish. Dis., 2018, 41: 511-527

[89] Bensimon-Brito A et al. Revisiting in vivo staining with alizarin red S-a valuable approach to analyse zebrafish skeletal mineralization during development and regeneration. BMC Dev. Biol., 2016, 16: 2

[90] Kellgren JH, Lawrence JS. Radiological assessment of osteo-arthrosis. Ann. Rheum. Dis., 1957, 16: 494-502

[91] Bouxsein ML et al. Guidelines for assessment of bone microstructure in rodents using micro-computed tomography. J. Bone Min. Res., 2010, 25: 1468-1486

[92] Kague E et al. Scleraxis genes are required for normal musculoskeletal development and for rib growth and mineralization in zebrafish. FASEB J., 2019, 33: 9116-9130

[93] Schindelin J et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods, 2012, 9: 676-682

[94] Ronneberger OFP, Brox T. U-Net: convolutional networks for biomedical image segmentation. Lecture Notes in Computer Science, 2015, 9351: 1-8

[95] Rueden CT et al. ImageJ2: ImageJ for the next generation of scientific image data. BMC Bioinformatics, 2017, 18

[96] Arganda-Carreras, I. et al. Trainable_Segmentation: Release v3.1.2. Zenodo https://doi.org/10.5281/zenodo.59290 (2016).

[97] Cross, S. MIA v0.14.13. Zenodo https://doi.org/10.5281/zenodo.3976622 (2020).

[98] Legland D, Arganda-Carreras I, Andrey P. MorphoLibJ: integrated library and plugins for mathematical morphology with ImageJ. Bioinformatics, 2016, 32: 3532-3534

[99] Chollet, F. Keras. GitHub Repository (2015).

[100] Abadi, A. et al. Tensorflow: Large-scale machine learning on heterogeneous systems. Preprint at https://arxiv.org/abs/1603.04467 (2015).

[101] Doube M et al. BoneJ: free and extensible bone image analysis in ImageJ. Bone, 2010, 47: 1076-1079

[102] Rohlf, F. J. TpsDig2. Digitise landmarks and outlines version 2.17. Department of Ecology and Evolution, State University of New York (2013).

[103] Haines AJ, Crampton JS. Improvements to the method of Fourier shape analysis as applied in morphometric studies. Paleontology, 2000, 43: 765-783

[104] Hammer O, Harper DAT, Ryan PD. Paleontological statistics software package for education and data analysis. Paleontologia Electron., 2001, 4: 9

[105] Chen X, Nadiarynkh O, Plotnikov S, Campagnola PJ. Second harmonic generation microscopy for quantitative analysis of collagen fibrillar structure. Nat. Protoc., 2012, 7: 654-669

[106] Schneider CA, Rasband WS, Eliceiri KW. NIH image to ImageJ: 25 years of image analysis. Nat. Methods, 2012, 9: 671-675

Funding

Uk Space agency ST/T000678/1 Versus Arthritis 21937, 21211

EC | EC Seventh Framework Programm | FP7 Ideas: European Research Council (FP7-IDEAS-ERC - Specific Programme: "Ideas" Implementing the Seventh Framework Programme of the European Community for Research, Technological Development and Demonstration Activities (2007 to 2013))(617266 “NANOPRS”)