Skip to main content
SpringerLink
Log in

The response of foetal annulus fibrosus cells to growth factors: modulation of matrix synthesis by TGF-β1 and IGF-1

  • Original Paper
  • Published:
Histochemistry and Cell Biology Aims and scope Submit manuscript

Abstract

The annulus fibrosus of the intervertebral disc is a complex radial-ply tissue that derives initially from segmental condensations of axial mesenchyme surrounding the notochord. These mesenchymal condensations differentiate into the early annulus fibrosus during foetal development—their outer part becoming fibrous, containing collagen type I; and their inner part cartilaginous, containing type II collagen and aggrecan. With post-natal growth and ageing, there is a switch from type I to type II collagen and an increase in proteoglycan synthesis in the outer annulus. This fibrocartilaginous metaplasia appears to occur in response to compressive loading of the tissue as occurs in tendons that wrap around bony pulleys, and driven by growth factors, such as TGF-β. In this study, using high-density micromass cultures, we have assessed the response of foetal outer annulus cells to growth factor stimulation with TGF-β1 and IGF-1, growth factors known to occur within the developing disc. We qualitatively and quantitatively describe the stimulatory effects of these growth factors, both alone and in combination, on the synthesis of sulphated glycosaminoglycan, and collagen types I and II by annulus cells. We show a potential role for TGF-β1 in pushing cells towards a fibrocartilaginous phenotype, with possible complementary effects of IGF-1.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

References

  • Ahrens PB, Solursh M, Reiter RS (1977) Stage-related capacity for limb chondrogenesis in cell culture. Dev Biol 60:69–82

    Article  PubMed  CAS  Google Scholar 

  • Allan GJ, Flint DJ, Patel K (2001) Insulin-like growth factor axis during embryonic development. Reproduction 122:31–39

    Article  PubMed  CAS  Google Scholar 

  • Baffi MO, Moran MA, Serra R (2006) Tgfbr2 regulates the maintenance of boundaries in the axial skeleton. Dev Biol 296:363–374

    Article  PubMed  CAS  Google Scholar 

  • Bastiaansen-Jenniskens YM, Koevoet W, De Bart ACW, Zuurmond A-M, Bank RA, Verhaar JAN, DeGroot J, Van Osch GJVM (2008) TGFβ affects collagen cross-linking independent of chondrocyte phenotype but strongly depending on physical environment. Tissue Eng 14:1059–1066

    Article  CAS  Google Scholar 

  • Benjamin M, Ralphs JR (1998) Fibrocartilage in tendons and ligaments—an adaptation to compressive load. J Anat 193:481–494

    Article  PubMed  Google Scholar 

  • Benya PD, Shaffer JD (1982) De-differentiated chondrocytes re-express the differentiated collagen phenotype when cultured in agarose gels. Cell 30:373–384

    Article  Google Scholar 

  • Benya PD, Padilla SR, Nimni ME (1978) Independent regulation of collagen types by chondrocytes during the loss of differentiated function in culture. Cell 15:1313–1321

    Article  PubMed  CAS  Google Scholar 

  • Bernick S, Walker JM, Paule WJ (1991) Age changes to the annulus fibrosus in human intervertebral discs. Spine 16:520–524

    Article  PubMed  CAS  Google Scholar 

  • Bikle DD, Sakata T, Leary C, Elalieh H, Ginzinger D, Rosen CJ, Beamer W, Majumdar S, Halloran BP (2002) Insulin-like growth factor I is required for the anabolic actions of parathyroid hormone on mouse bone. J Bone Miner Res 17:1570–1578

    Article  PubMed  CAS  Google Scholar 

  • Bogduk N (2005) Clinical anatomy of the lumbar spine and sacrum, 4th edn. Churchill Livingstone, London

    Google Scholar 

  • Cottrill CP, Archer CW, Wolpert L (1987) Cell sorting and chondrogenic aggregate formation in micromass culture. Dev Biol 122:503–515

    Article  PubMed  CAS  Google Scholar 

  • Daniels K, Reiter R, Solursh M (1996) Micromass cultures of limb and other mesenchyme. Methods Cell Biol 51:237–247

    Article  PubMed  CAS  Google Scholar 

  • De Wreede R, Ralphs JR (2009) Deposition of collagenous matrices by tendon fibroblasts in vitro: a comparison of fibroblasts behaviour in pellet cultures and a novel three-dimensional long-term scaffoldless culture system. Tissue Eng Part A 15:2707–2715

    Article  PubMed  Google Scholar 

  • Eyre DR (1979) Biochemistry of the intervertebral disc. Int Rev Connect Tissue Res 8:227–291

    PubMed  CAS  Google Scholar 

  • Eyre DR, Muir H (1976) Types I and II collagens in human intervertebral disc. Interchanging radial distributions in annulus fibrosus. Biochem J 157:267–270

    PubMed  CAS  Google Scholar 

  • Eyre DR, Muir H (1977) Quantitative analysis of types I and II collagens in human intervertebral disc at various ages. Biochim Biophys Acta 492:29–42

    PubMed  CAS  Google Scholar 

  • Farndale RW, Buttle DJ, Barrett AJ (1986) Improved quantitation and discrimination of sulphated glycosaminoglycans by use of dimethylmethylene blue. Biochem Biophys Acta 883:173–177

    PubMed  CAS  Google Scholar 

  • Gay S, Fine J (1987) Characterisation and isolation of polyclonal and monoclonal antibodies against collagen for use in immunohistochemistry. Method Enzymol 145:148–167

    Article  CAS  Google Scholar 

  • Glowacki J, Trepman E, Folkman J (1983) Cell shape and phenotypic expression in chondrocytes. Proc Soc Exp Biol Med 172:93–98

    PubMed  CAS  Google Scholar 

  • Gruber HE, Fisher EC, Desai B, Stasky AA, Hoelscher G, Hanley EN (1997) Human intervertebral disc cells from the annulus: three-dimensional culture in agarose or alginate and responsiveness to TGF-beta 1. Exp Cell Res 235:13–21

    Article  PubMed  CAS  Google Scholar 

  • Gruber HE, Norton JH, Hanley EN (2000) Anti-apoptotic effects of IGF-1 and PDGF on human intervertebral disc cells in vitro. Spine 25:2153–2157

    Article  PubMed  CAS  Google Scholar 

  • Gruber HE, Leslie K, Ingram J, Hoelscher G, Norton HJ, Hanley EN (2004) Colony formation and matrix production by human annulus cells: modulation in three-dimensional culture. Spine 29:267–274

    Article  Google Scholar 

  • Gruber HE, Hoelscher GL, Ingram JA, Bethea S, Hanley EN (2008) IGF-1 rescues human intervertebral annulus cells from in vitro stress-induced premature senescence. Growth Factors 26:220–225

    Article  PubMed  CAS  Google Scholar 

  • Gruber HE, Chow Y, Hoelscher GL, Ingram JA, Zinchenko N, Norton HJ, Sun Y, Hanley EN (2010) Micromass culture of human annulus cells: morphology and extracellular matrix production. Spine 35:1033–1039

    PubMed  Google Scholar 

  • Hayes AJ, Benjamin M, Ralphs JR (1999) Roles of actin stress fibres in the development of the intervertebral disc: Cytoskeletal control of extracellular matrix assembly. Dev Dyn 215:179–189

    Article  PubMed  CAS  Google Scholar 

  • Hayes AJ, Benjamin M, Ralphs JR (2001) The extracellular matrix of the developing intervertebral disc. Matrix Biol 20:107–121

    Article  PubMed  CAS  Google Scholar 

  • Hayes AJ, Hughes CE, Ralphs JR, Caterson B (2011) Chondroitin sulphate sulphation motif expression in the ontogeny of the intervertebral disc. Eur Cells Mater 21:1–14

    CAS  Google Scholar 

  • Heine UI, Munoz EF, Flanders KC, Ellingsworth LR, Lam HY, Thompson NL, Roberts AB, Sporn MB (1987) Role of transforming growth factor-β in the development of the mouse embryo. J Cell Biol 105:2861–2876

    Article  PubMed  CAS  Google Scholar 

  • Hill DJ, Logan A (1992) Peptide growth factors and their interactions during chondrogenesis. Prog Growth Factor Res 4:45–68

    Article  PubMed  CAS  Google Scholar 

  • Jin H, Shen J, Wang B, Wang M, Shu B, Chen D (2011) TGF-β signaling plays an essential role in the growth and maintenance of intervertebral disc tissue. FEBS Lett 585:1209–1215

    Article  PubMed  CAS  Google Scholar 

  • Kim YJ, Sah RL, Doong JY, Grodzinsky AJ (1988) Fluorometric assay of DNA in cartilage explants using Hoechst 33258. Anal Biochem 174:168–176

    Article  PubMed  CAS  Google Scholar 

  • Koob TJ, Clark PE, Hernandez DJ, Thurmond FA, Vogel KG (1992) Compression loading in vitro regulates proteoglycan synthesis by tendon fibrocartilage. Arch Biochem Biophys 298:303–312

    Article  PubMed  CAS  Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    Article  PubMed  CAS  Google Scholar 

  • Masuda K, An HS (2004) Growth factors and the intervertebral disc. Spine J 4:330S–340S

    Article  PubMed  Google Scholar 

  • Matsunaga S, Nagano S, Onishi T, Morimoto N, Suzuki S, Komiya S (2003) Age-related changes in expression of transforming growth factor-beta and intervertebral discs. J Neurosurg 98:63–67

    PubMed  CAS  Google Scholar 

  • Millan FA, Denhez F, Kondaiah P, Akhurst RJ (1991) Embryonic expression patterns of TGFβ1, β2 and β3 suggest different developmental functions in vivo. Development 111:131–144

    PubMed  CAS  Google Scholar 

  • Miller EJ (1971) Collagen cross-linking: Identification of two cyanogen bromide peptides containing sites of intermolecular cross-link formation in cartilage collagen. Biochem Biophys Res Comm 45:444–451

    Article  PubMed  CAS  Google Scholar 

  • Miller EJ, Epstein EH, Piez KA (1971) Identification of three genetically distinct collagens by cyanogen bromide cleavage of insoluble human skin and cartilage collagen. Biochem Biophys Res Comm 42:1024–1029

    Article  PubMed  CAS  Google Scholar 

  • Murakami H, Yoon TS, Attallah-Wasif ES, Tsai K-J, Qinming F, Hutton WC, Kang JD (2006) The expression of anabolic cytokines in intervertebral discs in age-related degeneration. Spine 31:1770–1775

    Article  PubMed  Google Scholar 

  • Nagano S, Matsunaga S, Takae R, Morimoto N, Suzuki S, Yoshida H (2000) Immunolocalisation of transforming growth factor-betas and their receptors in the intervertebral disk of senescence-accelerated mouse. Int J Oncol 17:461–466

    PubMed  CAS  Google Scholar 

  • Osada R, Ohshima H, Ishihara H, Yudoh K, Matsui H, Tsuji H (1996) Autocrine/paracrine mechanism of insulin-like growth factor-1 secretion, and the effect of insulin-like growth factor-1 on proteoglycan synthesis in bovine intervertebral discs. J Orthop Res 14:690–699

    Article  PubMed  CAS  Google Scholar 

  • Peacock A (1951a) Observations on the post-natal structure of the intervertebral disc in man. J Anat 86:162–179

    Google Scholar 

  • Peacock A (1951b) Observations on the pre-natal development of the intervertebral disc in man. J Anat 85:260–274

    PubMed  CAS  Google Scholar 

  • Pelton RW, Dickinson ME, Moses HL, Hogan BLM (1990) In situ hybridization analysis of TGFβ3 RNA expression during mouse development: comparative studies with TGFβ1 and β2. Development 110:609–620

    PubMed  CAS  Google Scholar 

  • Peterkofsky B (1991) Ascorbate requirement for hydroxylation and secretion of procollagen: relationship to inhibition of collagen synthesis in scurvy. Am J Clin Nutr 1135S–1140S

  • Pinnel SR, Murad S, Darr D (1987) Induction of collagen synthesis by ascorbic acid. A possible mechanism. Arch Dermatol 123:1684–1686

    Article  PubMed  CAS  Google Scholar 

  • Pintar JE, Wood TL, Streck RD, Havton L, Hsu MS (1991) Expression of IGF-II, the IGF-II/mannose-6-phosphate receptor and IGFBP-2 during rat embryogenesis. Adv Exp Med Biol 293:325–333

    PubMed  CAS  Google Scholar 

  • Powell-Braxton L, Hollingshead P, Warburton C, Dowd M, Pitts-Meek S, Dalton D, Gillett N, Stewart TA (1993) IGF-1 is required for normal embryonic growth in mice. Genes Dev 7:2609–2617

    Article  PubMed  CAS  Google Scholar 

  • Pratsinis H, Kletsas D (2008) Growth factors in intervertebral disc homeostasis. Connect Tissue Res 49:273–276

    Article  PubMed  CAS  Google Scholar 

  • Ralphs JR, Wylie L, Hill D (1990) Distribution of insulin-like growth factor peptides in the developing chick embryo. Development 109:51–58

    PubMed  CAS  Google Scholar 

  • Risbud MV, Di Martino A, Guttapalli A, Seghatoleslami R, Denaro V, Vaccaro AR, Albert TJ, Shapiro IM (2006) Toward an optimum system for intervertebral disc organ culture: TGF-β3 enhances nucleus pulposus and annulus fibrosus survival and function through modulation of TGF-β-R expression and ERK signalling. Spine 31:884–890

    Article  PubMed  Google Scholar 

  • Robbins JR, Evanko SP, Vogel KG (1997) Mechanical loading and TGF-beta regulate proteoglycan synthesis in tendon. Arch Biochem Biophys 342:203–211

    Article  PubMed  CAS  Google Scholar 

  • Rufai A, Benjamin M, Ralphs JR (1995) The development of fibrocartilage in the rat intervertebral disc. Anat Embryol (Berl) 192:53–62

    Article  CAS  Google Scholar 

  • Schmid P, Cox D, Bilbe G, Maier R, McMaster GK (1991) Differential expression of TGFβ1, β2 and β3 genes during mouse embryogenesis. Development 111:117–130

    PubMed  CAS  Google Scholar 

  • Shakbaei M, Seifarth C, John T, Rahmanzadeh M, Mobasheri A (2006) IGF-1 extends the chondrogenic potential of human articular chondrocytes in vitro: molecular association between Sox9 and Erk1/2. Biochem Pharmacol 30:1382–1395

    Article  Google Scholar 

  • Sohn P, Cox M, Chen D, Serra R (2010) Molecular profiling of the developing mouse axial skeleton: a role for Tgfbr2 in the development of the intervertebral disc. BMC Dev Biol 10:29

    Article  PubMed  Google Scholar 

  • Stokes DG, Liu G, Dharmavaram R, Hawkins D, Piera-Velazquez S, Jiminez SA (2001) Regulation of type II collagen gene expression during human chondrocyte de-differentiation and recovery of chondrocyte-specific phenotype in culture involves Sry-type high mobility-group box (SOX) transcription factors. Biochem J 360:461–470

    Article  PubMed  CAS  Google Scholar 

  • Vogel KG, Koob TJ (1989) Structural specializations in tendons under compression. Int Rev Cytol 115:267–293

    Article  PubMed  CAS  Google Scholar 

  • Walmsley R (1953) The development and growth of the intervertebral disc. Edinb Med J 60:341–364

    PubMed  CAS  Google Scholar 

  • Wan M, Shi X, Cao X (2002) TGF-β/BMP signalling in cartilage and bone cells. Curr Opin Orthop 13:368–374

    Article  Google Scholar 

  • Wang JY, Baer AE, Kraus VB, Setton LA (2001) Intervertebral disc cells exhibit differences in gene expression in alginate and monolayer culture. Spine 26:1747–1752

    Article  PubMed  CAS  Google Scholar 

  • Watt FM (1988) Effect of seeding density on stability of the differentiated phenotype of pig articular chondrocytes. J Cell Sci 89:373–378

    PubMed  Google Scholar 

  • Yang KG, Saris DB, Geuze RE, Helm YJ, Rijen MH, Verbout AJ, Dhert WJ et al (2006) Impact of expansion and redifferentiation conditions on chondrogenic capacity of cultured chondrocytes. Tissue Eng 12:2435–2447

    Article  PubMed  CAS  Google Scholar 

  • Zhang R, Ruan D, Zhang C (2006) Effects of TGF-β1 and IGF-1 on proliferation of human nucleus pulposus cells in medium with different serum concentrations. J Orthop Surg Res 1:9

    Article  PubMed  Google Scholar 

Download references

Acknowledgments

Thanks to Dr. Andrew Waggett for assistance in many of the biochemical procedures employed in this study, to Dr. John Wardale for providing the types I and II collagen antibodies and standards used for Western blot analyses and to Mr. Guy Pitt for assistance with macro-photography.

Author information

Authors and Affiliations

  1. School of Biosciences, Cardiff University, Biomedical Sciences Building, Museum Avenue, PO Box 911, Cardiff, CF10 3AX, UK

    Anthony J. Hayes & James R. Ralphs

Authors
  1. Anthony J. Hayes
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. James R. Ralphs
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Anthony J. Hayes.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Hayes, A.J., Ralphs, J.R. The response of foetal annulus fibrosus cells to growth factors: modulation of matrix synthesis by TGF-β1 and IGF-1. Histochem Cell Biol 136, 163–175 (2011). https://doi.org/10.1007/s00418-011-0835-x

Download citation

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00418-011-0835-x

Keywords

Search

Navigation