Cartilage Tissue Engineering Using Dermis Isolated Adult Stem Cells: The Use of Hypoxia during Expansion versus Chondrogenic Differentiation

Abstract

Dermis isolated adult stem (DIAS) cells, a subpopulation of dermis cells capable of chondrogenic differentiation in the presence of cartilage extracellular matrix, are a promising source of autologous cells for tissue engineering. Hypoxia, through known mechanisms, has profound effects on in vitro chondrogenesis of mesenchymal stem cells and could be used to improve the expansion and differentiation processes for DIAS cells. The objective of this study was to build upon the mechanistic knowledge of hypoxia and translate it to tissue engineering applications to enhance chondrogenic differentiation of DIAS cells through exposure to hypoxic conditions (5% O2) during expansion and/or differentiation. DIAS cells were isolated and expanded in hypoxic (5% O2) or normoxic (20% O2) conditions, then differentiated for 2 weeks in micromass culture on chondroitin sulfate-coated surfaces in both environments. Monolayer cells were examined for proliferation rate and colony forming efficiency. Micromasses were assessed for cellular, biochemical, and histological properties. Differentiation in hypoxic conditions following normoxic expansion increased per cell production of collagen type II 2.3 fold and glycosaminoglycans 1.2 fold relative to continuous normoxic culture (p<0.0001). Groups expanded in hypoxia produced 51% more collagen and 23% more GAGs than those expanded in normoxia (p<0.0001). Hypoxia also limited cell proliferation in monolayer and in 3D culture. Collectively, these data show hypoxic differentiation following normoxic expansion significantly enhances chondrogenic differentiation of DIAS cells, improving the potential utility of these cells for cartilage engineering.

Environmental preconditioning rejuvenates adult stem cells' proliferation and chondrogenic potential

“…Hypoxia likely increased cell proliferation in a cell source-dependent manner. Kalpakci et al found that hypoxic treatment (5% oxygen) resulted in a significant decrease in number of dermis isolated adult stem cells at days 7, 9, and 11 of culture and a lower colony forming unit – fibroblast (CFU-F) compared with normoxic culture [40]. …”

Harnessing Biomechanics to Develop Cartilage Regeneration Strategies

“…This study also demonstrated the multilineage differentiation potential of DIAS cells by staining cultures for lipids, calcium deposits, and sulfated GAG and collagen type II after culture in adipogenic, osteogenic, and chondrogenic media, respectively [96]. In a separate study, chondrogenic differentiation of DIAS cells under hypoxia resulted in a 2.3-fold increase in collagen type II production per cell and a 1.2-fold increase in GAG content relative to normoxic culture [99].…”

Critical Review on the Physical and Mechanical Factors Involved in Tissue Engineering of Cartilage

“…Another study using dermis MSCs confirmed that continuous hypoxic conditions (5% O 2 ) decreases the collagen type II to total collagen ratio; however, differentiation in hypoxic conditions following normoxic expansion increases production of collagen type II and GAGs. In that same study, expansion in hypoxia increased total matrix production [101]. Yet another study suggests that hypoxia alone has no effect on chondrogenesis or matrix synthesis, but that the effect is dependent on the hydrogel-scaffolding properties [96].…”