Growth and Stem Cell Characteristics of Tendon-Derived Cells with Different Initial Seeding Densities: An In Vitro Study in Mouse Flexor Tendon Cells

Tendon stem/progenitor cells (TSPCs) are considered promising seed cells for tendon regeneration. Previous studies reported that a low seeding density favors TSPC growth, whereas a high seeding density favors tenocyte growth. We aimed to distinguish TSPCs from tenocytes by seeding tendon-derived cells at a density gradient. In this study, tendon-derived cells were isolated from flexor digitorum profundus tendons of mice and seeded at the initial densities of 50, 500, 5,000, and 50,000/cm2. We found that distinct cell colonies were formed from cells with initial seeding densities of 50 and 500/cm2, but colonies were not discernible for cells seeded at 5,000 and 50,000/cm2. There was a positive correlation between cell proliferation rate and seeding density, but a negative correlation between cell senescence and seeding density. The cell proliferation rate decreased gradually during serial passages. All cells exhibited restricted differentiation potentials, and expressed stem cell markers and relatively high levels of tenogenic markers without notable differences among cells seeded at different densities. We concluded that a pure population of TSPCs could not be isolated from mouse digital flexor tendons through culturing cells at a density gradient. Cells seeded at low densities had very limited proliferative ability and did not show more prominent stem cell characteristics when compared with cells seeded at high densities.