Introduction:
IVD degeneration and aging involve decreased NP cell quantity and activity. Tie2-expressing NP progenitor cells hold promise for cell therapy due to their regenerative capacity. Challenges in expanding these cells arise from age and disease, prompting studies to optimize culture methods to enhance Tie2-expressing cell retention. Recently, we identified an optimized culture condition that facilitates cell rejuvenation, leading to increased positivity of Tie2-expressing cells. This discovery prompted us to investigate whether Tie2-expressing cells derived from older tissue sources exhibit comparable functionality and regenerative potential to Tie2+ NP cells obtained from younger patients, to broaden the sources of cell materials.
Methods:
Surgical tissues (Tokai University IRB approval: 17R173) and with patient consent, were obtained. Tissues from patients under 25 years (Young) and over 60 years (Older) were washed, fragmented, and cultured as tissue fragments and subsequent mono-layer cultures following Sako et al. Resulting cells were used for TrypLE-detached cells were re-seeded and cultured for an additional 4 weeks to assess proliferation rates. Flow cytometry determined cell surface marker expression (Tie2, GD2, CD24), and IntraPrep examined type I and type II collagen and proteoglycan positivity. NP cells (1,000 cells/mL) were seeded in MethocultTM media for 14 days to assess colony formation rate. Finally, obtained cell products were applied in coccygeal induced disc degeneration rat models; 18G needle disc puncture at Co5/6-Co7/8. Directly after, each rat received 25 µL of saline solution (Sham), 25 µL of 1x105 Old-, and 25 µL of 1x105 Young NP cells in one of each level. Rats were followed for 3 months, with monthly radiographic assessment followed by sacrifice for histological assessment. Histological score was performed through ORS Spine rat specific classification scheme.
Results:
Tie2-positivity rates were high and comparable in cells from older (17.5%) and young (20.8%) sources. GD2-positivity was significantly higher in older NP cells. Despite similar Tie2-rates, Young NP cells exhibited superior proliferative capacity, colony-forming units, and matrix production. Transplantation of both cell products enhanced disc height index outcomes, with young cells outperforming old cells. Histological assessment revealed responder and non-responder discs, emphasizing the impact of donor age on Tie2-expressing NP cells functionality.
Discussion :
The study underscores the importance of Tie2-expressing NP cells in IVD regeneration, highlighting the influence of donor age on cell functionality. Tie2 emerges as a key marker for regenerative potential, suggesting epigenetic changes in older NP progenitor cells due to age and chronic inflammation. This warrants further investigation as a potential therapeutic target.