The regenerative capacity of the notochordal cell: tissue constructs generated in vitro under hypoxic conditions

Abstract

<jats:sec> <jats:title>Object</jats:title> <jats:p>The intervertebral disc (IVD) is a highly avascular structure that is occupied by highly specialized cells (nucleus pulposus [NP] cells) that have adapted to survive within an O<jats:sub>2</jats:sub> concentration of 2–5%. The object of this study was to investigate the effects of long-term hypoxic and normoxic tissue cultures of nonchondrodystrophic canine notochordal cells—cells that appear to protect the disc NP from degenerative change.</jats:p></jats:sec> <jats:sec> <jats:title>Methods</jats:title> <jats:p>The authors obtained notochordal cells from nonchondrodystrophic canines according to their established methods and placed them into monolayer and 3D culture using sodium alginate globules under either hypoxic (3.5% O<jats:sub>2</jats:sub>) or normoxic (21% O<jats:sub>2</jats:sub>) conditions. Histological, immunohistochemical, scanning electron microscopy, and histomorphometric methods were used to evaluate the cells within the globules after 5 months in culture.</jats:p></jats:sec> <jats:sec> <jats:title>Results</jats:title> <jats:p>Notochordal cells under in vitro hypoxic tissue culture conditions produced a highly complex, organized, 3D cellular construct that was strikingly similar to that observed in vivo. In contrast, traditional normoxic tissue culture conditions resulted in notochordal cells that failed to produce an organized matrix. Hypoxia resulted in a matrix rich in aggrecan and collagen II, whereas normoxic cultured cells did not produce any observable aggrecan or collagen II after 5 months of culture.</jats:p></jats:sec> <jats:sec> <jats:title>Conclusions</jats:title> <jats:p>Hypoxia induces notochordal cells to organize a complex 3D cellular/extracellular matrix without an external scaffold other than suspension within sodium alginate. These cells produce an extracellular matrix and large construct that shares exactly the same characteristics as the in vivo condition—robust aggrecan, and type II collagen production. Normoxic tissue culture conditions, however, lead to a failure of these cells to thrive and a lack of extracellular matrix production and significantly smaller cells. The authors suggest that future studies of NP cells and, in particular, notochordal cells should utilize hypoxic tissue culture conditions to derive meaningful, biologically relevant conclusions concerning possible biological/molecular interventions.</jats:p></jats:sec>

Coupling of small leucine-rich proteoglycans to hypoxic survival of a progenitor cell-like subpopulation in Rhesus Macaque intervertebral disc

“…We hypothesized that DPCs are able to maintain vitality under oxygen tension and therefore tested their proliferative activities under hypoxia. We found that DPCs, as well as BMSCs, showed significantly higher proliferation rate at an oxygen tension of 3.5%, which mimics the oxygen level within the IVD [30] (Fig. 4A).…”

The evolutionary importance of cell ratio between notochordal and nucleus pulposus cells: an experimental 3-D co-culture study

“…Here, we cultured the cells under normoxic conditions. It is very likely that the current results would be even more pronounced under hypoxic conditions [10]. This should be tested in a further experiment.…”

“…They can produce a better aligned ECM under hypoxic conditions [10]. Here, we cultured the cells under normoxic conditions.…”

Canonical Wnt signaling in the notochordal cell is upregulated in early intervertebral disk degeneration

“…29 Gene expression of brachyury and cytokeratin 8 was significantly decreased at all time points in culture compared with NC-rich NP tissue, indicating a loss of notochordal phenotype and potential de-differentiation of NCs in monolayer culture under normoxic conditions. 32 NCs have been shown to thrive in three-dimensional culture under hypoxic conditions, 32 and therefore we cannot exclude that the culture conditions applied may have been a possible confounding factor. Primary NCs (both clusters and single cells) showed active Wnt/b-catenin signaling with nuclear and cytoplasmic b-catenin expression.…”