Table 4.
Summarizes the studies that apply hypoxia for osteogenic and adipogenic differentiation of mesenchymal stem cells
| Reference | Material | Conditions and device used | Cell Source | Medium/Growth Factors Used | Reported Result |
|---|---|---|---|---|---|
| Burian et al.[ 24 ] | 2D cell culture flask and 3D tricalcium phosphate (TCP) scaffolds with PHB | Hypoxic culture (2% O2) in humidified incubator (MCO‐5M, Sanyo) for 21 days |
Porcine bone marrow‐derived MSC (pbMSC) and porcine adipose‐derived MSC (paMSC) Cell density 5 × 103 cells mL−1 |
Osteogenic medium (DMEM with 10% FBS, 100 × 10−9 m dexamethasone, 50 × 10−6 m ascorbic acid 2‐phosphate and 10 × 10−3 m β‐glycerophosphate disodium) | Hypoxia attenuated osteogenic differentiation of pbMSCs compared to normoxia, and slightly increased osteogenic differentiation in paMSC |
| Volkmer et al.[ 45 ] | 6‐well plate | Hypoxic culture (2% O2) for 21 days in multigas incubator; |
hMSC Cell density 3000 cells cm−2 |
Osteogenic medium (High glucose DMEM supplemented with 10% FBS, 100 × 10−9 m dexamethasone, 10 × 10−3 m b‐glycerophosphate, 50 × 10−3 m l‐ascorbic acid 2‐phosphate and antibiotics). |
Hypoxia increased proliferation of hMSCs but inhibited osteogenesis. Hypoxic preconditioning prior osteogenesis restored osteogenic potential. |
| Boyette et al.[ 5 ] | 6‐well or 24‐well plates (osteogenic differentiation); | Hypoxic culture (5% O2) for 21 days in closed incubators; |
hbMSC Cell density 1 × 104 cells cm−2 |
Osteogenic medium (DMEM supplemented with 10% (FBS), 50 µg mL−1 L‐ascorbate‐2‐phosphate, 0.1 × 10−6 m dexamethasone, 10 × 10−3 m β‐glycerophosphate, and 10 × 10−9 m 1α,25‐(OH)2 vitamin D3) | Hypoxia during differentiation upregulated osteogenesis associated genes, alkaline phosphatase activity and total mineral deposition in hbMSCs. |
| Sheehy et al.[ 49 ] | 6‐well plates (osteogenic differentiation) | Hypoxic culture (5% O2) for 14 days |
pbMSC Cell density 3 × 103 cells cm−2 |
Osteogenic Medium (DMEM GlutaMAX supplemented with 10% FBS, 20 µg mL−1 β‐glycerophosphate,100 × 10−9 m dexamethasone and l‐ascorbic acid‐2‐phosphate and antibiotics) | Osteogenic potential and calcium accumulation higher when pbMSCs were both expanded and differentiated under hypoxia compared to normoxia. |
| Zhang et al.[ 44 ] | 6‑well plates | Hypoxic culture (2% O2) for 14 days in three‑gas modular hypoxic incubator (IG750, Jouan) |
Rat bone marrow‐derived MSC (rbMSC) Cell density 2 × 104 cells cm−2 |
Osteogenic medium (DMEM supplemented with 10% FBS, 0.1 × 10−3 m dexamethasone, 10 × 10−3 m β‐glycerophosphate and 50 × 10−3 m ascorbic acid) | Hypoxia reduces osteogenesis, ALP activity and mRNA expression of osteocalcin, ALP and collagen I in rbMSCs |
| Liu et al.[ 50 ] | Tissue culture plates | Preconditioning with hypoxia (5% O2) for 6 h |
rbMSC Cell density 5 × 103 cells cm−2 |
Osteogenic medium (DMEM supplemented with 10% FBS, dexamethasone, β‐glycerol phosphate and ascorbate); Adipogenic medium (DMEM supplemented with 1‐methyl‐3‐isobutylxanthine, dexamethasone, insulin, and indomethacin) | No difference in osteogenesis or adipogenesis of rbMSCs under hypoxia. Hypoxia enhanced survival of rbMSCs. |
| Lennon et al.[ 17 ] | Tissue culture plates | Hypoxic culture (5% O2) for 21 days in closed incubator chambers |
rbMSC Cell density 5 × 107 cells 100 mm−1 |
Osteogenic medium (Low‐glucose DMEM supplemented with 10% FBS,100 × 10−9 m dexamethasone, 80 × 10−3 m ascorbic acid 2‐phosphate, 10 × 10−3 m β‐glycerophosphate) | Hypoxia increased osteogenesis, with increased ALP activity and calcium content. |
| Basciano et al.[ 23c ] | 60 cm2 petri dishes | Hypoxic culture (5% O2) for 4 passages in incubator (Sanyo). |
hbMSC Cell density 100 cells cm−2 |
Osteogenic medium (α‐MEM supplemented with 10% FBS, 2 × 10−3 m glutamine, 60 × 10−6 m ascorbic acid, 10 × 10−3 m β‐glycerol phosphate and 0.1 × 10−6 m dexamethasone) | Post expansion in hypoxia, cells showed higher potential for osteogenic differentiation with increased ALP and RUNX2 expression. |
| dos Santos et al.[ 19b ] | 12‐well plates | Hypoxic culture (2% O2) in C‐Chamber connected to Proox Model 21 controller (BioSpherix) |
hbMSC Cell density 1000 cells cm−2 |
Osteogenic medium (Low glucose DMEM supplemented with 10% FBS, 100 × 10−9 m dexamethasone, 10 × 10−3 m β‐glycerophophate and 0.05 × 10−3 m 2‐phospho‐L‐ascorbic acid), Adipogenic medium (DMEM supplemented with 10% FBS, 170 × 10−9 m insulin, 0.5 × 10−3 m 3‐isobutyl‐1‐methyl‐xanthine, 0.2 × 10−3 m indomethacin, and 1 × 10−3 m dexamethasone) | No difference in osteogenic and adipogenic differentiation observed between cells expanded in hypoxia and normoxia. |
| Fehrer et al.[ 12 ] | 6‐well plates | Hypoxic culture (3% O2) in Thermo Electron Corporation 3110 incubators |
hbMSC Cell density 50 cells cm−2 for differentiation |
Osteogenic medium (MEM supplemented with 20% FCS, 20 × 10−3 m β‐glycerol phosphate, 1 × 10−9 m dexamethasone, 0.5 × 10−6 m ascorbate‐2‐phosphate and antibiotics), Adipogenic medium (MEM supplemented with 20% FCS, 1 × 10−6 m dexamethasone, 50 × 10−6 m indomethacine, 0.5 × 10−6 m 3‐iso‐butyl‐1‐methylxanthine, 0.5 × 10−6 m hydrocortisone and antibiotics) |
Reduced adipogenic differentiation and no osteogenic differentiation under both expansion and differentiation under hypoxia. Post expansion in hypoxia and differentiation in normoxia, cells showed higher osteogenic and adipogenic differentiation. |
| Malladi et al.[ 47a ] | 12‐well plates |
Hypoxic culture (2% O2) for 15 days |
Mouse adipose derived MSC (maMSC) Cell density 10 000 cells/well |
Osteogenic medium (DMEM supplemented with 10% FBS, 100 µg mL−1 ascorbic acid, 10 × 10−3 m β‐glycerophosphate, antibiotics and with 1 × 10−6 m retinoic acid or 50 × 10−9 m vitamin D) | Hypoxia reduced osteogenesis, with decreased alkaline phosphatase activity and mineralization being observed. |
| Holzwarth et al.[ 26 ] | 96‐well plates | Hypoxic culture (5%, 3% or 1% O2) for 14 days in Heracell gas addition incubators (Heraeus Instruments GmbH) |
hbMSC Cell density 6250 cells cm−2 |
Basal medium (Low glucose DMEM supplemented with 5% human fresh frozen plasma, 107 mL−1 platelets, 80 IU mL−1 heparin sulphate, 1 × 10−3 m glutamine and antibiotics), Adipogenic medium (Basal medium supplemented with 1 × 10−6 m dexamethasone, 60 × 10−6 m indomethacin, 0.5 × 10−3 m isobuthylmethylxanthine and 10 × 10−6 m insulin), Osteogenic medium (Basal medium supplemented with 10 × 10−9 m dexamethasone, 0.1 × 10−3 m L‐ascorbic acid‐2‐phosphate, 10 × 10−3 m β‐glycerol phosphate and 100 ng mL BMP‐2) | hbMSCs showed impaired adipogenic and osteogenic differentiation under 1% O2. Cells cultured at 3% O2 showed osteogenesis comparable to normoxia. |
| Fink et al.[ 52 ] | 6‐well plates |
Hypoxic culture (1% O2) for 3 days in In Vivo 400 hypoxic workstation (Maltec) for adipogenic differentiation |
Immortalized hbMSC Cell density 2 × 105 cells/well |
Basal medium (EMEM with 10% FBS and antibiotics) for expansion; Adipogenic medium (DMEM supplemented with 10% FCS, 1 × 10−6 m dexamethasone, 0.45 × 10−3 m isobutyl methylxanthine, 170 × 10−9 m insulin, 0.2 × 10−3 m indomethacin, 1 × 10−6 m rosiglitazone and antibiotics) | hbMSCs showed morphological changes with cytoplasmic lipid inclusions under hypoxia, but adipocyte‐specific genes were not induced. |
| Ren et al.[ 18 ] | T25 culture flasks | Hypoxic culture (8% O2) for 7–8 days in modular airtight humidified chamber |
mbMSC Cell density 1 × 105 cells cm−2 |
Adipogenic medium (60% low glucose DMEM, 40% MCDB‐201 with 2% FBS and 2 × 10–9 m dexamethansone) | Cells showed 5‐ to 6‐fold increase in lipid droplets under hypoxia compared to normoxia. Hypoxia accelerated mbMSC proliferation and adipogenic differentiation. |
| Grayson et al.[ 23d ] | Synthetic poly(ethylene terephthalate) (PET) fibrous matrices with 100 to 200 µm pore size | Hypoxic culture (2% O2) for 30 days in sealed chamber |
hbMSCs Cell density 3 × 106 cells per PET disk |
Osteogenic medium (Basal medium supplemented with 100 × 10−9 m dexamethasone, 10 × 10−3 m sodium‐β‐glycerophosphate, and 0.05 × 10−3 m ascorbic acid‐2 phosphate), Adipogenic induction medium (High glucose DMEM with 10% FBS, 0.2 × 10−3 m indomethacin, 0.5 × 10−3 m isobutyl‐1‐methyl xanthine, 1 × 10−3 m dexamethasone, and 5 mg mL−1 insulin) for 2 days, Adipogenic maintenance medium (High glucose DMEM supplemented with 10% FBS and 10 mg mL−1 insulin) |
Hypoxic hbMSCs expressed higher levels of osteogenic and adipogenic differentiation markers |
| Salim et al.[ 23e ] | ‐ | Hypoxic (2% O2) or anoxic (<0.02% O2) culture for 24 h in hypoxia workstations (Bactron Anaerobic/Environmental Chamber) | hbMSC | Basal medium (Poietics MSCGM Mesenchymal Stem Cell Medium) for expansion, Osteogenic medium (Basal medium with 1 m dexamethasone, 5 × 10−3 m β‐glycerophosphate, and 100 g mL−1 ascorbic acid) | Post expansion under hypoxia, hypoxic hbMSCs showed osteogenesis comparable to normoxia. Anoxic culture inhibited osteogenesis, visualized by downregulation of Runx2 and extracellular calcium deposition. |
| Yang et al.[ 46a ] | 12‐well plates | Hypoxic culture (1% O2) for 3 days |
hbMSC Cell density 1 × 104 cells cm−2 |
Osteogenic medium (α‐MEM supplemented with 16.6% FBS, 50 mg mL−1 ascorbate‐2 phosphate, 10–8 m dexamethasone and 10 × 10−3 m β‐glycerophosphate) | Hypoxia inhibited osteogenesis of hbMSCs, visualized by downregulation of Runx2 and reduced staining by Alizarin Red as compared to normoxia. |
| Tamama et al.[ 23f ] | 24‐well plates for osteogenic and 6‐well plate for adipogeni differentiation | Hypoxic culture (1% O2) in hypoxia chamber (Stemcell Technologies) |
Primary hMSCs Cell density 5 × 104 cells/24‐well for osteogenic and 1 × 106 cells/6‐well differentiation |
Osteogenic medium (α‐MEM supplemented with 10% FBS, 100 × 10−9 m dexamethasone, 10 × 10−3 m sodium‐β‐glycerophosphate, and 0.05 × 10−3 m ascorbic acid‐2 phosphate), Adipogenic induction medium (High glucose DMEM with 10% FBS, 0.2 × 10−3 m indomethacin, 0.5 × 10−3 m isobutyl‐1‐methyl xanthine, 1 × 10−3 m dexamethasone, and 5 mg mL−1 insulin) for 2 days, Adipogenic maintenance medium (High glucose DMEM supplemented with 10% FBS and 10 mg mL−1 insulin) | HbMSCs showed decreased osteogenic and adipogenic differentiation under hypoxia. Hypoxia promoted hbMSC self‐renewal and maintained undifferentiated phenotype. |
| Huang et al.[ 47b ] | 6‐well plate | Hypoxic culture (2% O2) for 21 days in hypoxia incubator chambers (Thermo Fisher Scientific) |
rbMSC Cell density 10 000 cells cm−2 |
Basal medium (High glucose DMEM with 10% FBS and antibiotics) | Hypoxia inhibited spontaneous calcification of rbMSCs alongwith decreased ALP expression and calcium content. Osteogenic differentiation markers were downregulated in hypoxia compared to normoxia. |
| Hu et al.[ 29 ] | 35 mm petri dish | Hypoxic culture (5% O2 and 10% O2) for 14 days. | mbMSC | Osteogenic medium (DMEM with 10% FBS, 1% glutamine, 0.1 × 10−6 m dexamethasone, 10 × 10−3 m β‐glycerophosphate disodium salt hydrate, and 50 × 10−6 m L‐ascorbic acid 2‐phosphate sesquimagnesium salt hydrate), Adipogenic induction medium (DMEM with 10% FBS, 1% glutamine, 1 × 10−6 m dexamethasone, 0.125 × 10−3 m indomethacin, 0.5 × 10−3 m 3‐isobutyl‐1‐methyl‐xanthine, and 5 µg mL−1 insulin) for 3 days and adipogenic maintenance medium (DMEM with 10% FBS, 1% glutamine, and 1 × 10−6 m dexamethasone) for 1 day. |
Hypoxia (5% O2) enhanced adipogenic differentiation, while mbMSCs in both hypoxia and normoxia showed similar osteogenic differentiation. No significant difference between normoxia and hypoxia when differentiation was carried out at 10% O2 |