title: Hypoxic culture of bone marrow-derived mesenchymal stromal stem cells differentially enhances in vitro chondrogenesis within cell-seeded collagen and hyaluronic acid porous scaffolds.
abstract: BACKGROUND
  The quality of cartilaginous tissue derived from bone marrow mesenchymal stromal stem cell (BMSC) transplantation has been correlated with clinical outcome. Therefore, culture conditions capable of modulating tissue phenotype, such as oxygen tension and scaffold composition, are under investigation. The objective of this study was to assess the effect of hypoxia on in vitro BMSC chondrogenesis within clinically approved porous scaffolds composed of collagen and hyaluronic acid (HA). It was hypothesized that hypoxic isolation/expansion and differentiation would improve BMSC chondrogenesis in each construct.
  
  
  METHODS
  Ovine BMSCs were isolated and expanded to passage 2 under hypoxia (3% oxygen) or normoxia (21% oxygen). Cell proliferation and colony-forming characteristics were assessed. BMSCs were seeded at 10 million cells per cubic centimeter on cylindrical scaffolds composed of either collagen I sponge or esterified HA non-woven mesh. Chondrogenic differentiation was performed in a defined medium under hypoxia or normoxia for 14 days. Cultured constructs were assessed for gene expression, proteoglycan staining, glycosaminoglycan (GAG) quantity, and diameter change.
  
  
  RESULTS
  Isolation/expansion under hypoxia resulted in faster BMSC population doublings per day (P <0.05), whereas cell and colony counts were not significantly different (P = 0.60 and 0.30, respectively). Collagen and HA scaffolds seeded with BMSCs that were isolated, expanded, and differentiated under hypoxia exhibited superior aggrecan and collagen II mRNA expressions (P <0.05), GAG quantity (P <0.05), and proteoglycan staining in comparison with normoxia. GAG/DNA was augmented with hypoxic isolation/expansion in all constructs (P <0.01). Comparison by scaffold composition indicated increased mRNA expressions of hyaline cartilage-associated collagen II, aggrecan, and SOX9 in collagen scaffolds, although expression of collagen X, which is related to hypertrophic cartilage, was also elevated (P <0.05). Proteoglycan deposition was not significantly improved in collagen scaffolds unless culture involved normoxic isolation/expansion followed by hypoxic differentiation. During chondrogenesis, collagen-based constructs progressively contracted to 60.1% ± 8.9% of the initial diameter after 14 days, whereas HA-based construct size was maintained (109.7% ± 4.2%).
  
  
  CONCLUSIONS
  Hypoxic isolation/expansion and differentiation enhance in vitro BMSC chondrogenesis within porous scaffolds. Although both collagen I and HA scaffolds support the creation of hyaline-like cartilaginous tissue, variations in gene expression, extracellular matrix formation, and construct size occur during chondrogenesis.
authors.affiliation: Laboratory of Stem Cell Biology and Orthopaedic Tissue Engineering, Li Ka Shing Centre for Health Research Innovation, Divisions of Orthopaedic Surgery and Surgical Research, Department of Surgery, University of Alberta, Edmonton, AB, T6G 2E1, Canada. tbornes@ualberta.ca.
authors.forename: Troy D
authors.initials: TD
authors.lastname: Bornes
authors.affiliation: Laboratory of Stem Cell Biology and Orthopaedic Tissue Engineering, Li Ka Shing Centre for Health Research Innovation, Divisions of Orthopaedic Surgery and Surgical Research, Department of Surgery, University of Alberta, Edmonton, AB, T6G 2E1, Canada. njomha@ualberta.ca.
authors.forename: Nadr M
authors.initials: NM
authors.lastname: Jomha
authors.affiliation: Laboratory of Stem Cell Biology and Orthopaedic Tissue Engineering, Li Ka Shing Centre for Health Research Innovation, Divisions of Orthopaedic Surgery and Surgical Research, Department of Surgery, University of Alberta, Edmonton, AB, T6G 2E1, Canada. muletsie@ualberta.ca.
authors.forename: Aillette
authors.initials: A
authors.lastname: Mulet-Sierra
authors.affiliation: Laboratory of Stem Cell Biology and Orthopaedic Tissue Engineering, Li Ka Shing Centre for Health Research Innovation, Divisions of Orthopaedic Surgery and Surgical Research, Department of Surgery, University of Alberta, Edmonton, AB, T6G 2E1, Canada. adesida@ualberta.ca.
authors.forename: Adetola B
authors.initials: AB
authors.lastname: Adesida
mesh_terms: D053674:Aggrecans / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology*; D002454:Cell Differentiation; D015687:Cell Hypoxia; D049109:Cell Proliferation; D002478:Cells, Cultured; D020219:Chondrogenesis; D003094:Collagen / Q000737:chemistry; D024043:Collagen Type II / Q000235:genetics / Q000378:metabolism; D004247:DNA / Q000032:analysis; D005260:Female; D006025:Glycosaminoglycans / Q000032:analysis; D006820:Hyaluronic Acid / Q000737:chemistry; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000378:metabolism*; D016062:Porosity; D055758:SOX9 Transcription Factor / Q000235:genetics / Q000378:metabolism; D012756:Sheep; D054457:Tissue Scaffolds / Q000737:chemistry*
chemical_list: D053674:Aggrecans; D024043:Collagen Type II; D006025:Glycosaminoglycans; D055758:SOX9 Transcription Factor; D006820:Hyaluronic Acid; D003094:Collagen; D004247:DNA