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Cell and Tissue Banking

, Volume 16, Issue 2, pp 195–207 | Cite as

Alternative protocols to induce chondrogenic differentiation: transforming growth factor-β superfamily

  • Claudia Cicione
  • Emma Muiños-López
  • Tamara Hermida-Gómez
  • Isaac Fuentes-Boquete
  • Silvia Díaz-PradoEmail author
  • Francisco J. BlancoEmail author
Original Paper

Abstract

Mesenchymal stem cells (MSCs) are an accepted candidate for cell-based therapy of multiple diseases. The interest in MSCs and their possible application in cell therapy have resulted in a better understanding of the basic biology of these cells. Recently, like aggregation and transforming growth factor beta (TGFβ) delivery, hypoxia has been indicated as crucial for complete chondrogenesis. The aim of this study was to test different culture conditions for directing stem cell differentiation into the chondrogenic lineage in vitro by testing different TGFβ superfamily members into the culture media under normoxic conditions. All chondrogenic culture conditions used allowed the differentiation of bone marrow-MSCs (BM-MSCs) into chondrogenic lineage. Chondrogenic induction capacity depended on the growth factor added to the culture media. In particular, the chondrogenic culture condition that better induced chondrogenesis was the medium that included the combination of three growth factors: bone morphogenetic protein-2 (BMP-2), BMP-7 and TGFβ-3. In this culture media, differentiated cells showed the highest levels expression of two markers of chondrogenesis, SOX9 and COL2A1, compared to the control points (p < 0.05, two-tailed t test). In our experimental conditions, the combination of BMP-2, BMP-7 and TGFβ-3 was the most effective in promoting chondrogenesis of BM-MSCs. These results underline the importance of determining in each experimental design the best protocol for in vitro directing stem cell differentiation into the chondrogenic lineage.

Keywords

Cell therapy Autologous chondrocyte implantation Focal lesions Hyaline articular cartilage 

Abbreviations

AA

Ascorbic acid

AGG

Aggrecan

ALP

Alkaline phosphatase

APM1

Adiponectin

BM-MSCs

Bone marrow-mesenchymal stem cells

BMP-2

Bone morphogenetic protein-2

BMP-7

Bone morphogenetic protein-7

cDNA

Complementary deoxyribonucleic acid

COL10A1

Collagen type XA1

COL1A1

Collagen type IA1

COL2A1

Collagen type IIA1

DMEM

Dulbecco’s modified Eagle’s medium

FABP4

Fatty acid-binding protein 4

FBS

Fetal bovine serum

HE

Hematoxylin–eosin

IGF-1

Insulin-like growth factor-1

KO

Knockout serum

LPL

Lipoprotein lipase

MMP13

Matrix metalloproteinase 13

MSCs

Mesenchymal stem cells

MT

Masson’s trichrome

MTG

Monotioglycerol

OP

Osteoprotegerin

OP-1

Osteogenic protein 1

P/S

Penicillin and streptomycin

PCR

Polymerase chain reaction

qPCR

Real-Time PCR

REL

Relative expression levels

rHuBMP-2

Recombinant human bone morphogenetic protein 2

RNA

Ribonucleic acid

SaO

Safranin O

SOX9

Sex determining region Y-box 9

TB

Toluidine blue

TBP

TATA box binding protein

TGFβ

Transforming growth factor beta

TGFβ-3

Transforming growth factor beta-3

Notes

Acknowledgments

This study was supported by grants: Servizo Galego de Saúde, Xunta de Galicia (PS07/84), Cátedra Bioiberica de la Universidade da Coruña and Instituto de Salud Carlos III CIBER BBN; Ministerio Ciencia e Innovacion PLE2009-0144; Fondo Investigacion Sanitaria-PI 08/2028 with participation of funds from FEDER (European Community), Tamara Hermida-Gómez is the beneficiary of a contract from Fondo de Investigación Sanitaria (2008), Spain. We would like to thank P.Filgueira and M.J.Sánchez for technical assistance.

Conflict of interest

The authors declare that no competing financial interest exists.

Supplementary material

10561_2014_9472_MOESM1_ESM.doc (34 kb)
Supplementary material 1 (DOC 34 kb)
10561_2014_9472_MOESM2_ESM.doc (29 kb)
Supplementary material 2 (DOC 29 kb)

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Copyright information

© Springer Science+Business Media Dordrecht 2014

Authors and Affiliations

  • Claudia Cicione
    • 1
    • 2
    • 3
  • Emma Muiños-López
    • 1
    • 2
    • 3
  • Tamara Hermida-Gómez
    • 1
    • 2
    • 3
  • Isaac Fuentes-Boquete
    • 2
    • 4
  • Silvia Díaz-Prado
    • 2
    • 4
    • 6
    Email author
  • Francisco J. Blanco
    • 1
    • 2
    • 3
    • 5
    • 6
    Email author
  1. 1.División de ReumatologíaINIBIC-Hospital Universitario A CoruñaA CoruñaSpain
  2. 2.Servicio de Reumatología, Grupo de Bioingeniería Tisular y Terapia Celular (CBTTC-CHUAC), CIBER-BBN/ISCIII, Instituto de Investigación Biomédica de A Coruña (INIBIC), Complexo Hospitalario Universitario de A Coruña (CHUAC), SERGASUniversidade da Coruña (UDC)A CoruñaSpain
  3. 3.Catedra BioibericaUniversidad de A Coruña, Hospital Universitario A CoruñaA CoruñaSpain
  4. 4.Grupo de Terapia Celular y Medicina Regenerativa, Departamento de Medicina, Facultad de Ciencias de la Salud, Instituto de Investigación Biomédica de A Coruña (INIBIC), Complejo Hospitalario Universitario de A Coruña (CHUAC), SERGASUniversidade da CoruñaA CoruñaSpain
  5. 5.Grupo de Reumatología, Departamento de Medicina, Facultad de Medicina, Instituto de Investigación Biomédica de A Coruña (INIBIC), Complejo Hospitalario Universitario de A Coruña (CHUAC), SERGASUniversidad de Santiago de CompostelaSantiago de Compostela, A CoruñaSpain
  6. 6.Osteoarticular and Aging Research LaboratoryHospital Universitario A CoruñaA CoruñaSpain

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