Preparation of acellular nerve matrix using Trito X-100 and sodium deoxycholate as extracting agent: Is there an optimal time? / 中国组织工程研究

ABSTRACT

BACKGROUND: Compared to other preparation method, chemical extraction can almost removed all cellular components,reduce the possibility of immunological rejection, and remain the integrality of nerve graft. However, there are still problems need to be explored.OBJECTIVE: To investigate the optimal condition of acellular nerve graft using Trito X-100 and sodium deoxycholate as extracting agent.DESIGN, TIME AND SETTING: Randomized grouping, controlled cytology observation. The experiment was performed at the Department of Stomatology, Affiliated Hospital of Binzhou Medical University, from February to June 2009.MATERIALS Fifteen New Zealand white rabbits, aged 3-4 months, weighing 2.5-3.0 kg. Triton X-100 and sodium deoxycholate were provided by Sigma Company, USA.METHODS: The bilateral facial nerve were obtained from rabbits, and removed the adipose tissue and epineurium of the nerve surface under the surgery microscope, then divided these nerves into 66 segments, with each length of 10 mm. The 66 neurons were randomly divided into 11 groups, with 6 neurons in each group. Except the control group, all neurons were placed into Petri dish for 12 hours bathing using distilled water at room temperature, then 5 groups of which were cultured with Triton X-100 for 12,24, 36, 48, and 60 hours, oscillation at room temperature; the remained 5 groups were cultured with 3% Triton X-100 for 12 hours,followed by 4% sodium deoxycholate for 12 hours, repeated for 1-5 cycles.MAIN OUTCOME MEASURES: Haematoxylin-eosin staining; degrees of decellularization and integrality of fiber pipe.RESULTS: Only use Triton X-100 to deal with the nerve of New Zealand white rabbits, even if 60 hours, could not to remove all the cellular components, and the basement membrane of Schwann cells were greatly destroyed. After 2 cycles treatment of Trito X-100 combined with sodium deoxycholate, cellular components and myelin sheath of nerve fibers and axons were removed effectively, and basement membrane of Schwann cell was remained, with epineurium and perineurium could be seen.CONCLUSION: Oscillation accompanied by 2 cycles treatment of Trito X-100 and sodium deoxycholate can obtain acellular nerve graft by removing cellular components completely, and reserving integrated basement membrane of Schwann cells.