Abstract
The role for mechanical stimulation in the control of cell fate has been previously proposed, suggesting that there may be a role of mechanical conditioning in directing mesenchymal stromal cells (MSCs) towards specific lineage for tissue engineering applications. Although previous studies have reported that calcium signalling is involved in regulating many cellular processes in many cell types, its role in managing cellular responses to tensile loading (mechanotransduction) of MSCs has not been fully elucidated. In order to establish this, we disrupted calcium signalling by blocking stretch-activated calcium channel (SACC) in human MSCs (hMSCs) in vitro. Passaged-2 hMSCs were exposed to cyclic tensile loading (1 Hz + 8% for 6, 24, 48, and 72 hours) in the presence of the SACC blocker, gadolinium. Analyses include image observations of immunochemistry and immunofluorescence staining from extracellular matrix (ECM) production, and measuring related tenogenic and apoptosis gene marker expression. Uniaxial tensile loading increased the expression of tenogenic markers and ECM production. However, exposure to strain in the presence of 20 μM gadolinium reduced the induction of almost all tenogenic markers and ECM staining, suggesting that SACC acts as a mechanosensor in strain-induced hMSC tenogenic differentiation process. Although cell death was observed in prolonged stretching, it did not appear to be apoptosis mediated. In conclusion, the knowledge gained in this study by elucidating the role of calcium in MSC mechanotransduction processes, and that in prolonged stretching results in non-apoptosis mediated cell death may be potential useful for regenerative medicine applications.
Highlights
To explore the possibility of the role for calcium signalling in the management of cellular responses to tensile loading, we investigated the involvement of extracellular-intracellular calcium movement by disrupting the function of stretch-activated calcium channel (SACC) of human mesenchymal stromal cells (MSCs)
Our results demonstrated that human MSCs (hMSCs) could successfully commit towards osteoblast lineage visualized by Alizarin Red S staining; adipocyte lineage observed by Oil Red O; and chondrogenic lineage in the pellet culture system demonstrated positively by Safranin O staining
The results of our study demonstrate that SACC blocker used concurrently with uniaxial stretching partially affects tenogenic differentiation and at prolonged periods, induce cell death
Summary
Harvesting bone marrow specimens from humanThe use of bone marrow stromal cells in this study was based on the approval from the Medical Ethics Committee in University Malaya Medical Centre (reference number: 369.19). Isolation and expansion of hMSCs. Human bone marrow MSCs isolation was performed using our standard laboratory protocol, previously described in other publications [19, 22]. The mononuclear cells were extracted after undergoing gradient density centrifugation at 2,200 rpm for 25 min. The cell pellet was extracted after a centrifugation at 1,600 rpm for 10 min. The mixture was transplanted in culture flask to be cultured in cell culture medium (low-glucose DMEM supplemented with 10% FBS, 1% penicillin-streptomycin and 1% Glutamax-1) (Invitrogen-Gibco, USA). Growth medium was replaced every 72 hrs to achieve optimal cell proliferation. This continued until the cell cultures became 80–85% confluent. The hMSCs surface markers analysis and tri-lineage differentiation was performed following standards established as our standard laboratory protocol, which was mentioned in our previous publications [19]
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