Hypoxic Expansion of Human Mesenchymal Stem Cells Enhances Three-Dimensional Maturation of Tissue-Engineered Intervertebral Discs

Abstract

Culture of three-dimensional (3D) constructs in hypoxic conditions (1-5% O) has been shown to increase production of extracellular matrix components in primary intervertebral disc (IVD) cells and drive chondrogenesis of human mesenchymal stem cells (hMSCs). Growing evidence suggests that two-dimensional (2D) expansion under hypoxic conditions may have an even greater influence on chondrogenesis in MSCs. This study aims to determine the effects of hypoxia during 2D expansion and subsequent 3D culture on the in vitro maturation of tissue-engineered IVDs (TE-IVDs) made with hMSCs, using a previously developed TE-IVD system. hMSCs were expanded in either hypoxic (5% O) or normoxic (21% O) conditions before construction of TE-IVDs. Discs were cultured in 3D in either hypoxic or normoxic conditions to create four experimental groups. Discs made from MSCs expanded in hypoxia were up to 141% stiffer than those made with normoxia-expanded MSCs. Similar patterns were seen in all mechanical properties. Increases in glycosaminoglycan content and collagen content in the nucleus pulposus (NP) were associated with 3D hypoxic culture. A boundary region between the manufactured fibrosus and NP regions developed by 2 weeks and mimicked the organization of the native disc. Hypoxic conditions in both 2D expansion and subsequent 3D culture improved the maturation of TE-IVDs made with hMSCs.

Promise, progress, and problems in whole disc tissue engineering

“…For the fabrication of engineered discs with human MSCs, monolayer expansion in hypoxia (5% O 2 ) prior to seeding into the 3D construct improved compressive mechanical properties compared to normoxic expansion (21% O 2 ). This may have been due to improved integration between the AF and NP components of the engineered discs, as there was little effect of hypoxic culture on construct biochemistry in the individual compartments . Interestingly, the mechanical properties and biochemical content of disc cell‐seeded constructs and human MSC‐seeded constructs in the aforementioned studies fell within a similar range.…”

“…However, divergent approaches exist regarding the most appropriate manner in which to culminate in cell‐seeded engineered discs that are mature and mechanically functional in the in vivo environment. Two main strategies have been pursued—maturation of the construct in vitro via pre‐culture prior to in vivo implantation, and implantation of an immature construct without preculture that subsequently undergoes maturation in the in vivo space …”

“…Other recent studies have investigated the effects of alternative exogenous stimuli, including hypoxia and dynamic compressive loading, on engineered disc maturation in vitro . In these studies, engineered discs were cultured in a serum containing media without TGF‐β, and were composed of either MSC or disc cell‐seeded alginate hydrogels for the NP region, combined with collagen gels for the AF region that self‐assembled in culture to become circumferentially aligned.…”

“…As another approach, acclimation of engineered discs to the harsh low glucose and oxygen environment of the native disc may also improve in vivo performance, and could potentially be achieved via preculture methods to reduce construct metabolic activity prior to implantation. This could include mild hypothermia, serum starvation, hypoxic culture or other strategies that are applied for a period of time prior to implantation . The design of engineered discs could also be modified to include material inclusions that provide the sustained release of small molecules (ie, IL‐1Ra, dexamethasone, glucose, TGF‐β) from within the construct to mitigate the inflammatory and nutrient‐poor environment of the degenerative spine and assist in maintaining engineered disc phenotype …”

“…Additionally, MSCs are more sensitive than disc cells to nutrient deprivation, and therefore the challenges with scale up of engineered discs to larger sizes will likely be exacerbated in MSC seeded constructs . Because of this, strategies to “precondition” MSCs to the harsh, environment of the disc may be necessary, such as preculture in hypoxia or low glucose media …”