Differential phenotypic behaviors of human degenerative nucleus pulposus cells under normoxic and hypoxic conditions: influence of oxygen concentration during isolation, expansion, and cultivation.

BACKGROUND CONTEXT

Intervertebral discs (IVDs) are the largest avascular structures in the body; therefore, cells within these discs might be adapted to low-oxygen conditions. Although it has been demonstrated that a low oxygen concentration could promote synthesis of the extracellular matrix by IVD cells in the in vitro culture, isolation, expansion, and cultivation of IVD cells under classical tissue culture O2 saturation could still be detrimental.

PURPOSE

To investigate the phenotypic differences between human degenerative nucleus pulposus (NP) cells during isolation and expansion under normoxic (Nx: 21% O2) or hypoxic (Hx: 3.5% O2) conditions.

STUDY DESIGN

We investigated in vitro isolation, expansion, and cultivation of human NP cells.

METHODS

Human NP tissue samples were obtained from patients who underwent lumbar disc surgeries. Nucleus pulposus cells were then isolated, expanded, and cultivated under normoxic or hypoxic conditions. To determine whether the effects of normoxic expansion are reversible, another group of cells was isolated and expanded in normoxic conditions and then cultivated under hypoxic conditions (Nx→Hx group). Cellular proliferation, RNA expression of selected genes, and immunohistochemical staining were performed to evaluate the phenotypic behaviors of human NP cells under different conditions.

RESULTS

Expressions of Type II collagen and aggrecan in the Nx→Hx group were significantly higher than those in the normoxic group but were significantly lower than those in the hypoxic group. The normoxic group showed higher expression of matrix metalloproteinase (MMP)-2 and MMP-13 than did the other groups. Expression levels of hypoxia-inducible factors (HIFs) were significantly higher in the normoxic groups; however, a greater degree of HIF-1α staining was found in the hypoxic group, whereas a greater degree of HIF-2α staining was found in the normoxic group.

CONCLUSIONS

Human degenerative NP cells isolated, expanded, and cultivated in hypoxic conditions could better preserve the cells' regenerative potential. Compromised properties that were observed during isolation and expansion under normoxic conditions could only be partially rescued by later hypoxic cultivation. The superior phenotypic behaviors of human NP cells under hypoxia may be related to higher HIF-1α production and lower HIF-2α production. Cells that are isolated, expanded, and cultivated under hypoxic conditions may show better regenerative results when transplanted; therefore, the isolation and expansion processes of human degenerative NP cells should be managed in a hypoxic environment.