Abstract
Summary
Within hours after exposure to hypoxic circumstances hMSCs start producing AGFs. Initially hypoxia does not affect hMSC proliferation and metabolic activity, but after 7 days both are decreased, compared to hMSCs cultured under ambient oxygen conditions.
Introduction
At the moment of implantation of a large cell seeded scaffold, usually a vascular network is lacking within the scaffold. Therefore, the cells seeded on the scaffold are exposed to hypoxic circumstances. Human mesenchymal stem cells (hMSCs) exposed to hypoxic circumstances, start to produce angiogenic factors (AGF)1 and to proliferate faster than at ambient oxygen levels2. Under severe, continued hypoxia, hMSC metabolism slows down and ultimately stops3. We hypothesise that there is a threshold oxygen level above which hMSCs at hypoxia will both produce AGF and still proliferate, and below which cells slow down their metabolism. If hMSCs are provided with oxygen levels just above this threshold, effective tissue regeneration, which requires cell proliferation and vascular ingrowth, may be accomplished.
Methods
Human mesenchymal stem cells (hMSCs) were isolated using Ficoll density gradient centrifugation from reaming debris that was collected during total hip replacement. Experiments were performed in a hypoxic chamber at 5% CO2 and 1%, 2%, 3%, 4%, 6%, 8% and 10% O2, temperature was 37°C and humidity was 100%. The cells were transfected with a pCDNA\HRE-luciferase 2 plasmid to find the oxygen range at which hMSCs start producing AGF. Subsequently, AGF production was quantified using qPCR. Cell proliferation and cell metabolism rate under hypoxic circumstances was assayed using the CyQuant DNA quantification method and a (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, respectively.
Results
In hMSCs cultured at 1% O2, the hypoxia responsive element was activated. A selection of AGFs was expressed at 1%, 2% and 3% O2. AGF production started four hours after exposure to hypoxic circumstances commenced. Initially, at 1 day of exposure to hypoxia, cell proliferation and cell metabolic activity were not influenced by hypoxia. After 7 days, hMSC proliferation and metabolic activity were lower in cells grown under hypoxic circumstances than in cells grown under ambient oxygen circumstances.
Discussion/Conclusion
The threshold level above which hMSCs at hypoxia will both produce AGF and still proliferate seems lower than 1% O2. Within hours after exposure to hypoxic circumstances hMSCs start producing AGFs. Initially, hMSC proliferation and metabolic activity were not affected by hypoxia, but after 7 days both proliferation and metabolic activity were decreased compared to hMSCs cultured under ambient oxygen circumstances.
