Influence of mechanical fluid shear stress on the osteogenic differentiation protocols for Equine adipose tissue-derived mesenchymal stem cells

Mohamed I Elashry; Shumet T Gegnaw; Michele C Klymiuk; Sabine Wenisch; Stefan Arnhold

doi:10.1016/j.acthis.2019.02.002

Influence of mechanical fluid shear stress on the osteogenic differentiation protocols for Equine adipose tissue-derived mesenchymal stem cells

Acta Histochem. 2019 Apr;121(3):344-353. doi: 10.1016/j.acthis.2019.02.002. Epub 2019 Feb 23.

Authors

Mohamed I Elashry¹, Shumet T Gegnaw², Michele C Klymiuk³, Sabine Wenisch⁴, Stefan Arnhold³

Affiliations

¹ Institute of Veterinary Anatomy-, Histology and -Embryology, University of Giessen, 35392, Giessen, Germany; Anatomy and Embryology Department, Faculty of Veterinary Medicine, University of Mansoura, 35516, Egypt. Electronic address: mohammed.elashry@vetmed.uni-giessen.de.
² Institute of Veterinary Anatomy-, Histology and -Embryology, University of Giessen, 35392, Giessen, Germany; Institute des Neurosciences Cellulaires et Integratives (INCI), University of Strasbourg, 67084, Strasbourg, France.
³ Institute of Veterinary Anatomy-, Histology and -Embryology, University of Giessen, 35392, Giessen, Germany.
⁴ Clinic of Small Animals, c/o Institute of Veterinary Anatomy, Histology and Embryology, Justus Liebig University of Giessen, 35392, Germany.

PMID: 30808518
DOI: 10.1016/j.acthis.2019.02.002

Abstract

Cell-based therapies have become a promising approach to promote tissue regeneration and the treatment of musculoskeletal disorders. Bone regeneration maintains bone homeostasis, mechanical stability and physical performance. Mechanical stimulation showed to induce stem cell differentiation into the osteogenic fate. However, the effect of various osteogenic protocols on the osteogenic commitment of equine adipose-derived stem cells is not fully elucidated. Here we examined the influence of fluid-based shear stress (FSS) via mechanical rocking to assess whether mechanical stimulation promotes osteogenic differentiation of equine adipose-derived stem cells (ASCs). ASCs were induced into osteogenic fate using osteogenic differentiation medium (ODM) protocol or additional supplementation of 5 mM CaCl₂ and 7.5 mM CaCl₂ protocol compared to cells cultivated in basal medium (BM) up to 21 day. The ASCs proliferation pattern was evaluated using the sulforhodamine B (SRB) protein assay. Osteogenic differentiation examined via semi-quantification of alizarin red staining (ARS) and alkaline phosphatase activity (ALP) as well as, via quantification of osteocalcin (OC), alkaline phosphatase (ALP), osteopontin (OP), and collagen type-1 (COL1) gene expression using RT-qPCR. We show that mechanical FSS increased the proliferation pattern of ASCs compared to the static conditions. Mechanical FSS together with 5 mM CaCl₂ and 7.5 mM CaCl₂ promoted osteogenic nodule formation and increased ARS intensity compared to the standard osteogenic protocols. We observed that combined mechanical FSS with ODM protocol increase ALP activity compared to static culture conditions. We report that ALP and OC osteogenic markers expression were upregulated under mechanical FSS culture condition particularly with the ODM protocol. Taken together, it can be assumed that mechanical stress using FSS promotes the efficiency of the osteogenic differentiation protocols of ASCs through independent mechanisms.

Keywords: Equine Adipose-derived stem cells; Fluid shear stress; Osteogenic differentiation.

MeSH terms

Adipose Tissue / cytology*
Alkaline Phosphatase / metabolism
Animals
Bone and Bones / metabolism
Cell Differentiation / physiology*
Cell Proliferation / physiology*
Cells, Cultured
Collagen Type I / metabolism
Horses
Mesenchymal Stem Cells / cytology*
Osteocalcin / genetics
Osteogenesis / physiology*

Substances

Collagen Type I
Osteocalcin
Alkaline Phosphatase