J Korean Assoc Oral Maxillofac Surg.  2005 Jun;31(3):199-218.

Sciatic nerve regeneration using calcium phosphate coated conduit and brain-derived neurotrophic factor gene-transfected schwann cell in rat

• Choi WJ
• Ahn KM
• Hwang SJ
• Choung PH
• Kim MJ
• Kim NY
• Yoo SB
• Jahng JW
• Kim HM
• Kim JS
• Kim YH
• Kim SM
• Oh SJ
• Kim SJ
• Yang BE
• Lee JH

Abstract

PURPOSE OF STUDY: Peripheral nerve regeneration depends on neurotrophism of distal nerve stump, recovery potential of neuron, supporting cell like Schwann cell and neurotrophic factors such as BDNF. Peripheral nerve regeneration can be enhanced by the conduit which connects the both sides of transected nerve. The conduit maintains the effects of neurotrophism and BDNF produced by Schwann cells which can be made by gene therapy. In this study, we tried to enhance the peripheral nerve regeneration by using calcium phosphate coated porous conduit and BDNF-Adenovirus infected Schwann cells in sciatic nerve of rats.
MATERIALS AND METHODS
Microporous filter which permits the tissue fluid essential for nerve regeneration and does not permit infiltration of fibroblasts, was made into 2mm diameter and 17mm length conduit. Then it was coated with calcium phosphate to improve the Schwann cell adhesion and survival. The coated filter was evaluated by SEM examination and MTT assay. For effective allogenic Schwann cell culture, dorsal root ganglia of 1-day old rat were extracted and treated with enzyme and antimitotic Ara-C. Human BDNF cDNA was obtained from cDNA library and amplified using PCR. BDNF gene was inserted into adenovirus shuttle vector pAACCMVpARS in which E1 was deleted. We infected the BDNF-Ad into 293 human mammary kidney cell-line and obtained the virus plaque 2 days later. RT-PCR was performed to evaluate the secretion of BDNF in infected Schwann cells. To determine the most optimal m.o.i of BDNF-Ad, we infected the Schwann cells with LacZ adenovirus in 1, 20, 50, 75, 100, 250 m.o.i for 2 hours and stained with beta-galactosidase. Rats(n=24) weighing around 300g were used. Total 14mm sciatic nerve defect was made and connected with calcium phosphate coated conduits. Schwann cells(1x10(6)) or BDNF-Ad infected Schwann cells(1x10(6)) were injected in conduit and only media(MEM) was injected in control group. Twelve weeks after surgery, degree of nerve regeneration was evaluated with gait analysis, electrophysiologic measurements and histomorphometric analysis.
RESULTS
1. Microporous Millipore filter was effective conduit which permitted the adhesion of Schwann cells and inhibited the adhesion of fibroblast. We could enhance the Schwann cell adhesion and survival by coating Millipore filter with calcium phosphate. 2. Schwann cell culture technique using repeated treatment of Ara-C and GDNF was established. The mean number of Schwann cells obtained 1 and 2 weeks after the culture were 1.54+/-4.0*10(6) and 9.66+/-9.6*10(6). 3. The mRNA of BDNF in BDNF-Ad infected Schwann cells was detected using RT-PCR. In Schwann cell 0.69 microgram/microliter of DNA was detected and in BDNF-Adenovirus transfected Schwann cell 0.795 microgram/microliter of DNA was detected. The most effective infection concentration was determined by LacZ Adenovirus and 75 m.o.i was found the most optimal.
CONCLUSION
BDNF-Ad transfected Schwann cells successfully regenerated the 14mm nerve gap which was connected with calcium phosphate coated Millipore filter. The BDNF-Ad group showed better results compared with Schwann cells only group and control group in aspect to sciatic function index, electrophysiologic measurements and histomorphometric analysis.

Keyword

Peripheral nerve regeneration; Calcium phosphate coating; Nerve conduit; Schwann cell; BDNF(Brain-derived Neurotrophic factor); Adenovirus

MeSH Terms

Adenoviridae
Animals
beta-Galactosidase
Brain-Derived Neurotrophic Factor*
Calcium*
Cell Adhesion
Cell Culture Techniques
Cytarabine
DNA
DNA, Complementary
Fibroblasts
Gait
Ganglia, Spinal
Gene Library
Genetic Therapy
Genetic Vectors
Glial Cell Line-Derived Neurotrophic Factor
Humans
Kidney
Micropore Filters
Nerve Growth Factors
Nerve Regeneration
Neurons
Peripheral Nerves
Polymerase Chain Reaction
Rats*
Regeneration*
RNA, Messenger
Schwann Cells
Sciatic Nerve*
Brain-Derived Neurotrophic Factor
Calcium
Cytarabine
DNA
DNA, Complementary
Glial Cell Line-Derived Neurotrophic Factor
Nerve Growth Factors
RNA, Messenger
beta-Galactosidase