Skip to main content

Advertisement

SpringerLink
Log in

Hypertonic conditions enhance cartilage formation in scaffold-free primary chondrocyte cultures

  • Regular Article
  • Published:
Cell and Tissue Research Aims and scope Submit manuscript

Abstract

The potential of hypertonic conditions at in vivo levels to promote cartilage extracellular matrix accumulation in scaffold-free primary chondrocyte cultures was investigated. Six million bovine primary chondrocytes were cultured in transwell inserts in low glucose (LG), high glucose (HG), or hypertonic high glucose (HHG) DMEM supplemented with fetal bovine serum, antibiotics, and ascorbate under 5 % or 20 % O2 tension with and without transforming growth factor (TGF)-β3 for 6 weeks. Samples were collected for histological staining of proteoglycans (PGs) and type II collagen, analysis by quantitative reverse transcription plus the polymerase chain reaction (RT-PCR) of mRNA expression of aggrecan and procollagen α1 (II) and of Sox9 and procollagen α2 (I), and quantitation of PGs and PG separation in agarose gels. Cartilage tissues produced at 20 % O2 tension were larger than those formed at 5 % O2 tension. Compared with LG, the tissues grew to larger sizes in HG or HHG medium. Histological staining showed the strongest PG and type II collagen staining in cartilage generated in HG or HHG medium at 20 % O2 tension. Quantitative RT-PCR results indicated significantly higher expression of procollagen α1 (II) mRNA in cartilage generated in HHG medium at 20 % O2 tension compared with that in the other samples. TGF-β3 supplements in the culture medium provided no advantage for cartilage formation. Thus, HHG medium used at 20 % O2 tension is the most beneficial combination of the tested culture conditions for scaffold-free cartilage production in vitro and should improve cell culture for research into cartilage repair or tissue engineering.

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

  • Barry F, Boynton RE, Liu B, Murphy JM (2001) Chondrogenic differentiation of mesenchymal stem cells from bone marrow: differentiation-dependent gene expression of matrix components. Exp Cell Res 268:189–200

    Article  PubMed  CAS  Google Scholar 

  • Boeuf S, Richter W (2010) Chondrogenesis of mesenchymal stem cells: role of tissue source and inducing factors. Stem Cell Res Ther 1:31

    Article  PubMed  PubMed Central  Google Scholar 

  • Bosnakovski D, Mizuno M, Kim G, Takagi S, Okumura M, Fujinaga T (2006) Chondrogenic differentiation of bovine bone marrow mesenchymal stem cells (MSCs) in different hydrogels: influence of collagen type II extracellular matrix on MSC chondrogenesis. Biotechnol Bioeng 93:1152–1163

    Article  PubMed  CAS  Google Scholar 

  • Byers BA, Mauck RL, Chiang IE, Tuan RS (2008) Transient exposure to transforming growth factor beta 3 under serum-free conditions enhances the biomechanical and biochemical maturation of tissue-engineered cartilage. Tissue Eng A 14:1821–1834

    Article  CAS  Google Scholar 

  • Capito RM, Spector M (2003) Scaffold-based articular cartilage repair. IEEE Eng Med Biol Mag 22:42–50

    Article  PubMed  Google Scholar 

  • Cigan AD, Nims RJ, Albro MB, Esau JD, Dreyer MP, Vunjak-Novakovic G, Hung CT, Ateshian GA (2013) Insulin, ascorbate, and glucose have a much greater influence than transferrin and selenous acid on the in vitro growth of engineered cartilage in chondrogenic media. Tissue Eng A 19:1941–1948

    Article  CAS  Google Scholar 

  • Clouet J, Vinatier C, Merceron C, Pot-vaucel M, Maugars Y, Weiss P, Grimandi G, Guicheux J (2009) From osteoarthritis treatments to future regenerative therapies for cartilage. Drug Discov Today 14:913–925

    Article  PubMed  CAS  Google Scholar 

  • de Haart M, Marijnissen WJ, van Osch GJ, Verhaar JA (1999) Optimization of chondrocyte expansion in culture. Effect of TGF beta-2, bFGF and L-ascorbic acid on bovine articular chondrocytes. Acta Orthop Scand 70:55–61

    Article  PubMed  Google Scholar 

  • Dillon CF, Rasch EK, Gu Q, Hirsch R (2006) Prevalence of knee osteoarthritis in the United States: arthritis data from the Third National Health and Nutrition Examination Survey 1991–94. J Rheumatol 33:2271–2279

    PubMed  Google Scholar 

  • Donaldson L, Vuocolo T, Gray C, Strandberg Y, Reverter A, McWilliam S, Wang Y, Byrne K, Tellam R (2005) Construction and validation of a bovine innate immune microarray. BMC Genomics 6:135

    Article  PubMed  PubMed Central  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  • Felson DT, Lawrence RC, Dieppe PA, Hirsch R, Helmick CG, Jordan JM, Kington RS, Lane NE, Nevitt MC, Zhang Y, Sowers M, McAlindon T, Spector TD, Poole AR, Yanovski SZ, Ateshian G, Sharma L, Buckwalter JA, Brandt KD, Fries JF (2000) Osteoarthritis: new insights. Part 1: the disease and its risk factors. Ann Intern Med 133:635–646

    Article  PubMed  CAS  Google Scholar 

  • Frenkel SR, Di Cesare PE (2004) Scaffolds for articular cartilage repair. Ann Biomed Eng 32:26–34

    Article  PubMed  Google Scholar 

  • Galois L, Hutasse S, Cortial D, Rousseau CF, Grossin L, Ronziere MC, Herbage D, Freyria AM (2006) Bovine chondrocyte behaviour in three-dimensional type I collagen gel in terms of gel contraction, proliferation and gene expression. Biomaterials 27:79–90

    Article  PubMed  CAS  Google Scholar 

  • Grande DA, Halberstadt C, Naughton G, Schwartz R, Manji R (1997) Evaluation of matrix scaffolds for tissue engineering of articular cartilage grafts. J Biomed Mater Res 34:211–220

    Article  PubMed  CAS  Google Scholar 

  • Hayes AJ, Hall A, Brown L, Tubo R, Caterson B (2007) Macromolecular organization and in vitro growth characteristics of scaffold-free neocartilage grafts. J Histochem Cytochem 55:853–866

    Article  PubMed  CAS  Google Scholar 

  • Heywood HK, Bader DL, Lee DA (2006a) Glucose concentration and medium volume influence cell viability and glycosaminoglycan synthesis in chondrocyte-seeded alginate constructs. Tissue Eng 12:3487–3496

    Article  PubMed  CAS  Google Scholar 

  • Heywood HK, Bader DL, Lee DA (2006b) Rate of oxygen consumption by isolated articular chondrocytes is sensitive to medium glucose concentration. J Cell Physiol 206:402–410

    Article  PubMed  CAS  Google Scholar 

  • Holmdahl R, Rubin K, Klareskog L, Larsson E, Wigzell H (1986) Characterization of the antibody response in mice with type II collagen-induced arthritis, using monoclonal anti-type II collagen antibodies. Arthritis Rheum 29:400–410

    Article  PubMed  CAS  Google Scholar 

  • Hopewell B, Urban JP (2003) Adaptation of articular chondrocytes to changes in osmolality. Biorheology 40:73–77

    PubMed  CAS  Google Scholar 

  • Hunziker EB (2002) Articular cartilage repair: basic science and clinical progress. A review of the current status and prospects. Osteoarthritis Cartilage 10:432–463

    Article  PubMed  CAS  Google Scholar 

  • Hutmacher DW, Goh JC, Teoh SH (2001) An introduction to biodegradable materials for tissue engineering applications. Ann Acad Med Singapore 30:183–191

    PubMed  CAS  Google Scholar 

  • Jurgens WJ, Lu Z, Zandieh-Doulabi B, Kuik DJ, Ritt MJ, Helder MN (2012) Hyperosmolarity and hypoxia induce chondrogenesis of adipose-derived stem cells in a collagen type 2 hydrogel. J Tissue Eng Regen Med 6:570–578

    Article  PubMed  CAS  Google Scholar 

  • Kaitainen S, Mähönen AJ, Lappalainen R, Kröger H, Lammi MJ, Qu C (2013) TiO2 coating promotes human mesenchymal stem cell proliferation without the loss of their capacity for chondrogenic differentiation. Biofabrication 5:025009

    Article  PubMed  Google Scholar 

  • Lee RB, Urban JP (1997) Evidence for a negative Pasteur effect in articular cartilage. Biochem J 321:95–102

    PubMed  CAS  PubMed Central  Google Scholar 

  • Lee RB, Urban JP (2002) Functional replacement of oxygen by other oxidants in articular cartilage. Arthritis Rheum 46:3190–3200

    Article  PubMed  CAS  Google Scholar 

  • Lima EG, Bian L, Ng KW, Mauck RL, Byers BA, Tuan RS, Ateshian GA, Hung CT (2007) The beneficial effect of delayed compressive loading on tissue-engineered cartilage constructs cultured with TGF-beta3. Osteoarthritis Cartilage 15:1025–1033

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Majno G, Joris I (1995) Apoptosis, oncosis, and necrosis. An overview of cell death. Am J Pathol 146:3–15

    PubMed  CAS  PubMed Central  Google Scholar 

  • Mayer-Wagner S, Schiergens TS, Sievers B, Docheva D, Schieker M, Betz OB, Jansson V, Muller PE (2010) Membrane-based cultures generate scaffold-free neocartilage in vitro: influence of growth factors. Tissue Eng Part A 16:513–521

    Article  PubMed  CAS  Google Scholar 

  • Miyazaki T, Miyauchi S, Matsuzaka S, Yamagishi C, Kobayashi K (2010) Formation of proteoglycan and collagen-rich scaffold-free stiff cartilaginous tissue using two-step culture methods with combinations of growth factors. Tissue Eng Part A 16:1575–1584

    Article  PubMed  CAS  Google Scholar 

  • Mobasheri A, Vannucci SJ, Bondy CA, Carter SD, Innes JF, Arteaga MF, Trujillo E, Ferraz I, Shakibaei M, Martin-Vasallo P (2002) Glucose transport and metabolism in chondrocytes: a key to understanding chondrogenesis, skeletal development and cartilage degradation in osteoarthritis. Histol Histopathol 17:1239–1267

    PubMed  CAS  Google Scholar 

  • Negoro K, Kobayashi S, Takeno K, Uchida K, Baba H (2008) Effect of osmolarity on glycosaminoglycan production and cell metabolism of articular chondrocyte under three-dimensional culture system. Clin Exp Rheumatol 26:534–541

    PubMed  CAS  Google Scholar 

  • Ng KW, O’Conor CJ, Kugler LE, Cook JL, Ateshian GA, Hung CT (2011) Transient supplementation of anabolic growth factors rapidly stimulates matrix synthesis in engineered cartilage. Ann Biomed Eng 39:2491–2500

    Article  PubMed  PubMed Central  Google Scholar 

  • Oswald ES, Chao PH, Bulinski JC, Ateshian GA, Hung CT (2008) Dependence of zonal chondrocyte water transport properties on osmotic environment. Cell Mol Bioeng 1:339–348

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Oswald ES, Ahmed HS, Kramer SP, Bulinski JC, Ateshian GA, Hung CT (2011) Effects of hypertonic (NaCl) two-dimensional and three-dimensional culture conditions on the properties of cartilage tissue engineered from an expanded mature bovine chondrocyte source. Tissue Eng Part C Methods 17:1041–1049

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Qu CJ, Karjalainen HM, Helminen HJ, Lammi MJ (2006) The lack of effect of glucosamine sulphate on aggrecan mRNA expression and (35) S-sulphate incorporation in bovine primary chondrocytes. Biochim Biophys Acta 1762:453–459

    Article  PubMed  CAS  Google Scholar 

  • Qu CJ, Jauhiainen M, Auriola S, Helminen HJ, Lammi MJ (2007) Effects of glucosamine sulfate on intracellular UDP-hexosamine and UDP-glucuronic acid levels in bovine primary chondrocytes. Osteoarthritis Cartilage 15:773–779

    Article  PubMed  Google Scholar 

  • Qu CJ, Pöytäkangas T, Jauhiainen M, Auriola S, Lammi MJ (2009) Glucosamine sulphate does not increase extracellular matrix production at low oxygen tension. Cell Tissue Res 337:103–111

    Article  PubMed  CAS  Google Scholar 

  • Qu C, Lindeberg H, Ylärinne JH, Lammi MJ (2012) Five percent oxygen tension is not beneficial for neocartilage formation in scaffold-free cell cultures. Cell Tissue Res 348:109–117

    Article  PubMed  CAS  Google Scholar 

  • Richmon JD, Sage AB, Shelton E, Schumacher BL, Sah RL, Watson D (2005) Effect of growth factors on cell proliferation, matrix deposition, and morphology of human nasal septal chondrocytes cultured in monolayer. Laryngoscope 115:1553–1560

    Article  PubMed  CAS  Google Scholar 

  • Sachlos E, Czernuszka JT (2003) Making tissue engineering scaffolds work. Review: the application of solid freeform fabrication technology to the production of tissue engineering scaffolds. Eur Cell Mater 5:29–39

    PubMed  CAS  Google Scholar 

  • Schneider N, Mouithys-Mickalad A, Lejeune JP, Duyckaerts C, Sluse F, Deby-Dupont G, Serteyn D (2007) Oxygen consumption of equine articular chondrocytes: influence of applied oxygen tension and glucose concentration during culture. Cell Biol Int 31:878–886

    Article  PubMed  CAS  Google Scholar 

  • Sittinger M, Reitzel D, Dauner M, Hierlemann H, Hammer C, Kastenbauer E, Planck H, Burmester GR, Bujia J (1996) Resorbable polyesters in cartilage engineering: affinity and biocompatibility of polymer fiber structures to chondrocytes. J Biomed Mater Res 33:57–63

    Article  PubMed  CAS  Google Scholar 

  • Tchetina E, Mwale F, Poole AR (2003) Distinct phases of coordinated early and late gene expression in growth plate chondrocytes in relationship to cell proliferation, matrix assembly, remodeling, and cell differentiation. J Bone Miner Res 18:844–851

    Article  PubMed  CAS  Google Scholar 

  • Urban JP, Hall AC, Gehl KA (1993) Regulation of matrix synthesis rates by the ionic and osmotic environment of articular chondrocytes. J Cell Physiol 154:262–270

    Article  PubMed  CAS  Google Scholar 

  • Vijayan S, Bentley G, Briggs T, Skinner J, Carrington R, Pollock R, Flanagan A (2010) Cartilage repair: a review of Stanmore experience in the treatment of osteochondral defects in the knee with various surgical techniques. Indian J Orthop 44:238–245

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Xu X, Urban JP, Tirlapur UK, Cui Z (2010) Osmolarity effects on bovine articular chondrocytes during three-dimensional culture in alginate beads. Osteoarthritis Cartilage 18:433–439

    Article  PubMed  CAS  Google Scholar 

  • Zhou S, Cui Z, Urban JP (2008) Nutrient gradients in engineered cartilage: metabolic kinetics measurement and mass transfer modeling. Biotechnol Bioeng 101:408–421

    Article  PubMed  CAS  Google Scholar 

  • Zimber MP, Tong B, Dunkelman N, Pavelec R, Grande D, New L, Purchio AF (1995) TGF-beta promotes the growth of bovine chondrocytes in monolayer culture and the formation of cartilage tissue on three-dimensional scaffolds. Tissue Eng 1:289–300

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

We are grateful to Eija Rahunen, Heli Lindeberg, and Juha Prittinen for their excellent assistance in the laboratory.

Author information

Authors and Affiliations

  1. Department of Applied Physics, University of Eastern Finland, Yliopistonranta 1 F, 70211, Kuopio, Finland

    Janne H. Ylärinne, Chengjuan Qu & Mikko J. Lammi

  2. Biocenter Kuopio, University of Eastern Finland, Kuopio, Finland

    Mikko J. Lammi

Authors
  1. Janne H. Ylärinne
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Chengjuan Qu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Mikko J. Lammi
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Chengjuan Qu.

Additional information

This research was supported by strategic funding from the University of Eastern Finland, Kuopio University Foundation, and North Savo Regional Fund of the Finnish Cultural Foundation.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Ylärinne, J.H., Qu, C. & Lammi, M.J. Hypertonic conditions enhance cartilage formation in scaffold-free primary chondrocyte cultures. Cell Tissue Res 358, 541–550 (2014). https://doi.org/10.1007/s00441-014-1970-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00441-014-1970-1

Keywords

Search

Navigation