Effect of optimized collagenase digestion on isolated and cultured nucleus pulposus cells in degenerated intervertebral discs

This study aims to explore the optimized digestive method of collagenase to nucleus pulposus (NP) cells by observing the digestive effects of type I and II collagenase in different concentrations to NP in degenerated intervetebral discs. NP were collected from 18 human herniated intervertebral disc samples, and digested by type I and II collagenase, which were separated or combined in different concentrations. NP cells were counted using an inverted microscope, and the activities were determined by trypan blue staining at 4, 8, 16, and 24 hours after digestion. The growth of NP cells was also observed. The amount of NP cells with combined collagenases was greater than that separated in an identical concentration. With the combined collagenases at 4 and 8 hours, the higher concentration, the greater the amount of NP cells became. The amount of cells in extremely low concentrations of collagenase increased after 16 and 24 hours, and its activities remained at a higher level. The optimized digestion of extremely low concentrations of type I and II collagenase combined could save enzymes, was less harmful to NP cells, and was more adapted to separated and cultured NP cells.