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[In vivo study on tissue engineered skeletal muscle with hypoglossal nerve implantation]
Xiufa Tang1, Fugui Zhang, Yang Feng
1Department of Head & Neck Oncology, West China School of Stomatology, Sichuan University, Chengdu Sichuan 610041, PR China. tangxf1963@163.com
Objective:
To construct tissue engineered skeletal muscle in vivo using glial cell derived neurotrophic factor (GDNF) genetically modified myoblast (Mb) on acellular collagen sponge with hypoglossal nerve implantation, and to observe whether structural or functional connection could be established between engineered tissue and motor nerve or not.
Methods:
Mbs were isolated from 7 male Lewis rats at age of 2 days, cultured and genetically modified by recombinant adenovirus carrying GDNF cDNA (Mb(GDNF)). Calf skin-derived acellular collagen sponge was used as scaffold; cell adhesion was detected by scanning electron microscope after 24 hours. Hypoglossal nerve was implanted into Mb-scaffold complex (Mb group, n = 27) or Mb(GDNF)-scaffold complex (Mb(GDNF) group, n = 27) in 54 female Lewis rats at age of 8 weeks. HE staining was performed at 1, 6, and 12 weeks postoperatively, and immunohistochemistry staining and fluorescence in situ hybridization were used.
Results:
Mb(GDNF) could highly expressed GDNF gene. Mb and Mb(GDNF) could adhere to the scaffold and grew well. HE staining showed tight junctions between implant and peripheral tissue with new muscle fiber and no distinguished line at 12 weeks in 2 groups. Immunohistochemistry staining showed that positive cells of myogenin and slow skeletal myosin were detected, as well as positive cells of acetylcholine receptor alpha1 at 1, 6, and 12 weeks. The positive cells of Y chromosome decreased with time. At 1, 6, and 12 weeks, the positive neurons were 261.0 +/- 6.6, 227.3 +/- 8.5, and 173.3 +/- 9.1, respectively in Mb(GDNF) group, and were 234.7 +/- 5.5, 196.0 +/- 13.5, and 166.7 +/- 11.7, respectively in Mb group; significant differences were found between 2 groups at 1 and 6 weeks (P < 0.05), no significant difference at 12 weeks (P > 0.05).
Conclusion:
Connection can be established between engineered tissue and implanted hypoglossal nerve. Recombinant GDNF produced by Mb(GDNF) might play a critical role in protecting central motor neurons from apoptosis by means of retrograde transportation.
