Hypoxia affects mesenchymal stromal cell osteogenic differentiation and angiogenic factor expression
Introduction
Mesenchymal stromal cells (MSCs) loaded onto biocompatible scaffolds have been proposed for restoring function of lost or injured connective tissue, including bone [6], [33], [38]. Physiological oxygen tensions in bone are about 12.5% O2[22] but fall to 1% O2 in fracture hematoma [4], [22]. In tissue engineering applications, implanted MSCs undergo temporary oxygen deprivation, which may be considered as similar to fracture hematoma (i.e. 1% O2) due to the disruption of the host vascular system (as the result of injury and/or surgery) and the lack of pre-existing vascular networks within these scaffolds.
These drastic conditions of transplantation can lead to the death or functional impairment of MSCs, which can affect their ultimate bone forming potential. The exact effects of hypoxia on osteoprogenitor or osteoblast-like cells have not been clearly established, however, as several studies demonstrated a negative impact on cell growth [37], [46] and differentiation [37], [42], [48], whereas others have shown that hypoxia has positive effects on cell proliferation [48] and osteoblastic differentiation [51]. These discrepancies may be due to the differences between the cell types (primary [42], [48], [51] and cell lines [37], [42], [46]), species (rat [48], [51], human [37], [42] and mouse [42], [46]) and hypoxic conditions (from 0.02% to 5% O2) used. Since the success of bone reconstruction methods based on the use of engineered constructs depends on the maintenance of viable and functional MSCs, it is of particular interest to elucidate the effects of temporary hypoxia on primary human MSC survival and osteogenic potential.
MSCs secrete a wide variety of angiogenic factors (including vascular endothelial growth factor (VEGF) [27], transforming growth factor β1 (TGFβ1) [21], [43], and basic fibroblast growth factor (bFGF) [21], [27]) and may therefore modulate angiogenic processes and participate in the vascular invasion of engineered constructs. Since effective neo-vascularization is crucial for shortening the hypoxic episodes to which transplanted MSCs are exposed, it seemed to be worth investigating the stimulatory effects of hypoxia on angiogenic factor expression by MSCs.
The aim of the present study therefore was to investigate the effects of temporary hypoxia on primary human MSC (hMSC) proliferation, osteogenic potential and angiogenic factor expression. In this study, O2 tensions ≤ 4% are termed hypoxic conditions (as these conditions represent the hypoxia to which hMSCs transplanted in vivo are subjected) and 21% O2 tensions are termed control conditions (as these conditions represent standard cell culture conditions). Cell viability was assessed after exposing hMSCs to hypoxic conditions during various periods of time. Osteogenic differentiation was assessed after temporary (48 h) exposure of hMSCs to either control or hypoxic conditions followed by different periods of osteogenic cell culture. Expression of several angiogenic factors by hMSCs involved in new blood vessel formation (VEGF, bFGF, IL8 and TGFβ) and maturation (platelet derived growth factor BB (PDGF-BB)) was assessed after temporary (48 h) exposure of hMSCs to either control or hypoxic conditions.
Section snippets
Hypoxia
Hypoxia was obtained using a sealed jar (Oxoid Ltd, Basingstoke, United Kingdom) containing an oxygen chelator (AnaeroGen, Oxoid Ltd) [18]. Twice a day, the pO2 was measured diving an oxygen electrode directly into cell culture medium (pH 7.2) and using an Oxylab pO2™ (Oxford Optronix; Oxford, United Kingdom). The hypoxic system was left closed throughout the period of experimentation.
Cell culture
Human mesenchymal stromal cells (hMSCs) were isolated from tibia bone marrow specimens obtained as discarded
Multipotency of hMSCs
In order to determine the multipotency of the human mesenchymal stromal cells (hMSCs) used in this study, hMSCs were cultured in either osteogenic, chondrogenic, or adipogenic differentiation medium.
Culture of hMSCs in osteogenic medium for 10 and 20 days increased the levels of alkaline phosphatase (ALP) activity (Fig. 1A). Osteogenic differentiation of hMSCs was confirmed by the expression of the osteogenic differentiation markers osterix and osteocalcin (Fig. 1A).
Culture of hMSCs in
Discussion
The first step in the present study consisted in evaluating the effects of reduced oxygen tensions on hMSC survival. Our results showed that 120 h exposure to hypoxia resulted in increased cell death rates, when 48 or 72 h exposure did not, but those cell death rates may have been underestimated as the method used in the present study did not consider floating dead cells. The mechanisms underlying hMSC death upon oxygen deprivation are unclear at present. A previous study conducted on rat MSCs,
Acknowledgments
We thank Dr. Michele Guerre-Millo for providing the sealed jar for hypoxic cell culture conditions, Dr. Sylviane Dennler and Dr. Alain Mauviel for their expert assistance with the RT–PCR assays, and Dr. Sophie Le Ricousse-Roussanne for providing endothelial cells. We would also like to express special thanks to Professor Christophe Glorion and Dr. Jean-Sebastien Sylvestre for their help.
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