Hypoxic Conditions during Expansion Culture Prime Human Mesenchymal Stromal Precursor Cells for Chondrogenic Differentiation in Three-Dimensional Cultures

Abstract

Cell-based approaches using mesenchymal stromal precursor cells (MSCs) for the regeneration of intervertebral discs are attracting increased interest, even though the intervertebral disc is a very demanding environment. Implanted cells eventually face acidic pH, hypoxia, and a lack of nutrients. While the regenerative potential of MSCs for skeletal tissues has been well described, it is still questionable whether human MSCs can be prepared for prolonged survival and proper functioning and whether they can differentiate under the adverse conditions encountered in the disc. Here we examined the influence of hypoxia during expansion and differentiation on the chondrogenesis of MSCs. Chondrogenic differentiation was performed in in situ solidifying gelatin hydrogels, which represent a suitable matrix for delivering and anchoring cells within the disc tissue. To consider limitations in nutrition in the intervertebral disc, differentiation was performed at low cell concentrations in the gelatin hydrogels. Standard high-density micromass cultures served as reference controls. To determine the quality of chondrogenesis we analyzed typical marker molecules such as collagen types I, II, X, Sox-9, MIA, and aggrecan mRNA using RT-qPCR and determined protein deposition by histological stainings and biochemical methods. We could demonstrate that in gelatin-based hydrogels chondrogenic differentiation of human MSCs is possible at low cell concentrations. The quality of chondrogenic differentiation could be improved by hypoxia. Best results were obtained when the entire in vitro process, including MSC expansion and subsequent differentiation, was done under hypoxic conditions. MSCs that were expanded under reduced oxygen tension were primed for a chondrogenic differentiation.

Strategies to engineer tendon/ligament-to-bone interface: Biomaterials, cells and growth factors

“…High oxygen concentration promotes osteogenic differentiation and induces bone formation and mineralization [156,157]. Low oxygen concentration favors chondrogenic differentiation, upregulates collagen type II and type X expression, and downregulates collagen I expression [150,158,159]. Thus, tight coordination of oxygen concentration and spatial and temporal presentation of angiogenic and/or antiangiogenic factors will be essential to generate a tendon/ligament-to-bone construct with appropriate vascularization.…”

Evaluating the Impact of Oxygen Concentration and Plating Density on Human Wharton's Jelly-Derived Mesenchymal Stromal Cells

“…There was no effect of growth in serum-free medium when it was compared to our standard growth medium which contains 2% serum, growth factors and cytokines [14]. We hypothesized that reduced oxygen concentration during culture would increase WJC expansion rates and CFE since previous work had shown that reduced oxygen concentration during culture has potent effects on expansion of bone marrow-derived MSCs [29][30][31][32][33][34][35] and WJCs [36,37]. We speculated that plating density would impact WJC expansion based upon previous work using bone marrow-derived MSCs which had shown that MSC have more rapid expansion after plating a low density [38].…”

Mass Transfer Analysis of Growth and Substance Metabolism of NSCs Cultured in Collagen-Based Scaffold In Vitro

“…Research by O'Connor [44] has shown that NSCs can proliferate and differentiate in 3D collagen gels; Ma [12] has vaccined NSCs in collagen matrix for proliferation and Successful 3D cell bioprocessing depends on the supply of nutrients and cytokines [45]. At the same time, a stable physiological environment is also necessary, i.e., temperature, pH, and oxygen [45][46][47]. The concentration of nutrients and metabolites should be strictly monitored and controlled during cultivation since it affects cell growth, viability, and differentiation.…”