The regenerative capacity of the notochordal cell: tissue constructs generated in vitro under hypoxic conditions.
OBJECTIVE
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(2) 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.


METHODS
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(2)) or normoxic (21% O(2)) conditions. Histological, immunohistochemical, scanning electron microscopy, and histomorphometric methods were used to evaluate the cells within the globules after 5 months in culture.


RESULTS
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.


CONCLUSIONS
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.
Erwin
W Mark
WM
Division of Orthopaedic Surgery, Department of Surgery, University of Toronto and Toronto Western Hospital, Toronto, Ontario, Canada. mark.erwin@utoronto.ca
Las Heras
Facundo
F
Islam
Diana
D
Fehlings
Michael G
MG
Inman
Robert D
RD
D053674:Aggrecans / Q000378:metabolism; D000464:Alginates; D000818:Animals; D002449:Cell Aggregation; D018929:Cell Culture Techniques / Q000379:methods; D015687:Cell Hypoxia / Q000502:physiology; D048429:Cell Size; D002478:Cells, Cultured; D024043:Collagen Type II / Q000378:metabolism; D004285:Dogs; D004548:Elasticity; D005109:Extracellular Matrix / Q000378:metabolism / Q000502:physiology / Q000648:ultrastructure; D020723:Glucuronic Acid; D006603:Hexuronic Acids; D007403:Intervertebral Disc / Q000166:cytology* / Q000502:physiology*; D008855:Microscopy, Electron, Scanning; D009672:Notochord / Q000166:cytology*; D010100:Oxygen / Q000494:pharmacology*; D023822:Tissue Engineering / Q000379:methods*
D053674:Aggrecans; D000464:Alginates; D024043:Collagen Type II; D006603:Hexuronic Acids; D020723:Glucuronic Acid; D010100:Oxygen