• Journal of the Korean Association of Oral and Maxillofacial Surgeons
• /
• v.30 no.1
• /
• pp.1-16
• /
• 2004

The use of artificial nerve conduit containing viable Schwann cells is one of the most promising strategies to repair the peripheral nerve injury. To fabricate an effective nerve conduit whose microstructure and internal environment are more favorable in the nerve regeneration than existing ones, a new three-dimensional Schwann cell culture technique using $Matrigel^{(R)}$. and dorsal root ganglion (DRG) was developed. Nerve conduit of three-dimensionally arranged Schwann cells was fabricated using direct seeding of freshly harvested DRG into a $Matrigel^{(R)}$ filled silicone tube (I.D. 1.98 mm, 14 mm length) and in vitro rafting culture for 2 weeks. The nerve regeneration efficacy of three-dimensionally cultured Schwann cell conduit (3D conduit group, n=6) was assessed using SD rat sciatic nerve defect of 10 mm, and compared with that of silicone conduit filled with $Matrigel^{(R)}$ and Schwann cells prepared from the conventional plain culture method (2D conduit group, n=6). After 12 weeks, sciatic function was evaluated with sciatic function index (SFI) and gait analysis, and histomorphology of nerve conduit and the innervated tissues of sciatic nerve were examined using image analyzer and electromicroscopic methods. The SFI and ankle stance angle (ASA) in the functional evaluation were $-60.1{\pm}13.9$, $37.9^{\circ}{\pm}5.4^{\circ}$ in 3D conduit group (n=5) and $-87.0{\pm}12.9$, $32.2^{\circ}{\pm}4.8^{\circ}$ in 2D conduit group (n=4), respectively. And the myelinated axon was $44.91%{\pm}0.13%$ in 3D conduit group and $13.05%{\pm}1.95%$ in 2D conduit group to the sham group. In the TEM study, 3D conduit group showed more abundant myelinated nerve fibers with well organized and thickened extracellular collagen than 2D conduit group, and gastrocnemius muscle and biceps femoris tendon in 3D conduit group were less atrophied and showed decreased fibrosis with less fatty infiltration than 2D conduit group. In conclusion, new three-dimensional Schwann cell culture technique was established, and nerve conduit fabricated using this technique showed much improved nerve regeneration capacity than the silicone tube filled with $Matrigel^{(R)}$ and Schwann cells prepared from the conventional plain culture method.

• Macromolecular Research
• /
• v.14 no.1
• /
• pp.94-100
• /
• 2006

Although the peripheral nerve system has a relatively good regenerating capacity compared to the central nerve system, peripheral nerve repair remains a clinical challenge as restoration of normal nerve function is highly variable. Synthetic tubular nerve conduits were designed as an alternative repair method in order to replace the need for an isograft. These nerve conduits guide regenerating axons from the proximal toward the distal end, maintain within growth-promoting molecules released by the nerve stumps, and protect regenerating axons from infiltrating scar tissue. In this work, we prepared cinnamoylated alginate (CA)-collagen-chitosan nerve conduit using the lyophilization method to generate a controllable parallel channel in the center and then investigated its influence on peripheral nerve regeneration in an animal study. At 12 weeks after implantation, histological study showed that tissue cable was continuously bridging the gap of the sciatic nerve in all rats. Our newly developed nerve conduit is a promising tool for use in peripheral nerve regeneration and provides a suitable experimental model for future clinical application.

• Journal of Dental Anesthesia and Pain Medicine
• /
• v.17 no.3
• /
• pp.191-198
• /
• 2017

Background: For peripheral nerve regeneration, recent attentions have been paid to the nerve conduits made by tissue-engineering technique. Three major elements of tissue-engineering are cells, molecules, and scaffolds. Method: In this study, the attachments of nerve cells, including Schwann cells, on the nerve conduit and the effects of both growth factor and adhesion molecule on these attachments were investigated. Results: The attachment of rapidly-proliferating cells, C6 cells and HS683 cells, on nerve conduit was better than that of slowly-proliferating cells, PC12 cells and Schwann cells, however, the treatment of nerve growth factor improved the attachment of slowly-proliferating cells. In addition, the attachment of Schwann cells on nerve conduit coated with fibronectin was as good as that of Schwann cells treated with glial cell line-derived neurotrophic factor (GDNF). Conclusion: Growth factor changes nerve cell morphology and affects cell cycle time. And nerve growth factor or fibronectin treatment is indispensable for Schwann cell to be used for implantation in artificial nerve conduits.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
• /
• v.32 no.4
• /
• pp.295-307
• /
• 2006

Styela clava, called non-native tunicate or sea squirt, is habitat which include bays and harbors in Korea and several sites in the sea faced world. We fabricate cellulose membrane nerve conduit (CMNC) from this native sea squirt skin, and evaluate the capacity of promoting peripheral nerve regeneration in the rat sciatic nerve defect model. After processing the pure cellulose membrane from the sea squirt skin as we already published before, CMNC was designed as a non-tubular sheet with 14 mm length and 4 mm width. Total eleven male Spraque-Dawley rats (12 weeks, weighing 250 to 300g) were divided into sham group (n=2), silicone tube grafted control group (n=3) and experimental group (n=6). Each CMNC grafted nerve was evaluated after 4, 8 and 12 weeks in the experimental group, and after 12 weeks, sciatic function was evaluated with sciatic function index (SFI) and gait analysis, and histomorphology of nerve conduit and the innervated tissues of sciatic nerve were all examined using image analyzer and electromicroscopic methods in the all groups. The regenerated axon and nerve sheath were found only in the inner surface of the CMNC after 4 weeks and became more thicker after 8 and 12 weeks. In the TEM study, CMNC grafted group showed more abundant organized myelinated nerve fibers with thickened extracellular matrix than silicone conduit grafted group after 12 weeks. The sciatic function index (SFI) and ankle stance angle (ASA) in the functional evaluation were $-47.2{\pm}3.9$, $35.5^{\circ}{\pm}4.9^{\circ}$ in CMNC grafted group (n=2) and $-80.4{\pm}7.4$, $29.2^{\circ}{\pm}5.3^{\circ}$ in silicone conduit grafted group (n=3), respectively. And the myelinated axon was 41.59% in CMNC group and 9.51% in silicone conduit group to the sham group. The development of a bioactive CMNC to replace autogenous nerve grafts offers a potential and available approach to improved peripheral nerve regeneration. As we already published before, small peptide fragment derived from the basement membrane matrix proteins of squirt skin, which is a kind of anchoring protein composed of glycocalyx, induced the effective axonal regeneration with rapid growth of Schwann cells beneath the inner surface of CMNC. So the possibilities of clinical application as a peripheral nerve regeneration will be able to be suggested.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
• /
• v.29 no.3
• /
• pp.151-156
• /
• 2003

This study was performed to develop a useful nerve conduit which provides favorable environment for Schwann cell viability and proliferation. Milipore membrane of $0.45{\mu}m$ pore size was selected because it permits nutritional inflow from the outside of the conduit and prevents from invading the fibrotic tissue into the conduit. The membrane was rolled and sealed to form a conduit of 2mm diameter and 20mm length. To improve the axonal regeneration and to render better environment for endogenous and exogenous Schwann cell behaviour, the microgeometry and surface of conduit was modified by coating with thin film of calcium phosphate. Cellular viability within the conduit and attachment to its wall were assessed with MTT assay and SEM study. Milipore filter conduit showed significantly higher rate of Schwann cell attachment and viability than the culture dish. However, the reverse was true in case of fibroblast. Coating with thin film of low crystalline calcium phosphate made more favorable environment for both cells with minimal change of pore size. These findings means the porous calcium phosphate coated milipore nerve conduit can provide much favorable environment for endogenous Schwann cell proliferation and exogenous ones, which are filled within the conduit for the more advanced strategy of peripheral nerve regeneration, with potential of reducing fibrotic tissue production.

• Archives of Plastic Surgery
• /
• v.32 no.5
• /
• pp.613-618
• /
• 2005

When a large peripheral nerve defect occurs, an autologous nerve graft is the most ideal method of recinstruction. But an autologous nerve graft has many limitations due to donor site morbidities. Many previous focused on finding the ideal nerve conduit. Among them, $Gore-Tex^{(R)}$ has several advantages over other conduits. It can be manipulated to a suitable size, does not collapse easily, and it is a semi- permeable material that contain pores. A round shaped nerve can be newly formed because of its smooth inner surface. The purpose of this study was to evaluate the availability of $Gore-Tex^{(R)}$ tube as a nerve conduit at the peripheral nerve defect in the rat sciatic nerve. The 10 mm nerve gap was made in each group. A $Gore-Tex^{(R)}$ tube filled with skeletal muscle was inserted and autologous nerve graft was harvested, respectively. In the experimental group, we placed a 0.5 mm thickness, $30{\mu}m$ pored, 1.8 mm in diameter and 14 mm length tube with skeletal muscle inserted inside. In the control group, the nerve gap was inserted with a rat sciatic nerve. We estimated the results electrophysiologically and histologically to 16 weeks postoperatively. Results in the nerve conduction velocity, total myelinated axon count, myelin sheath thickness and mean nerve fiber diameter, the experimental group was substantially lower than that of the control group, but the statistic difference was not significant (p<0.05). The morphology was very similar in both groups, microscopically. From the above results, We conclude that $Gore-Tex^{(R)}$ qualifies as an ideal nerve conduit. It is suggested that $Gore-Tex^{(R)}$ tube filled with skeletal muscle may, substitute for an autologous nerve graft.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
• /
• v.31 no.3
• /
• pp.199-218
• /
• 2005

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$(1{\times}10^6)$ or BDNF-Ad infected Schwann cells$(1{\times}10^6)$ 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 $1.54{\pm}4.0{\times}10^6$ and $9.66{\pm}9.6{\times}10^6$. 3. The mRNA of BDNF in BDNF-Ad infected Schwann cells was detected using RT-PCR. In Schwann cell $0.69\;{\mu}g/{\mu}l$ of DNA was detected and in BDNF-Adenovirus transfected Schwann cell $0.795\;{\mu}g/{\mu}l$ 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.

• Proceedings of the Polymer Society of Korea Conference
• /
• 2006.10a
• /
• pp.181-181
• /
• 2006

We developed a novel method to fabricate a nerve guide conduit (NGC) with the porosity of submicron pore sizes (to prevent fibrous tissue infiltration) and hydrophilicity (for effective oxygen and nutrient permeation) using poly(lactic-co-glycolic acid) (PLGA) and Pluronic F127 by a modified immersion precipitation method designed by our laboratory. It was recognized that the hydrophilized PLGA/F127 (3 wt%) tube can be a good candidate as a NGC from the analyses of its morphology, mechanical strength, hydrophilicity, model nutrient permeability and in vivo nerve regeneration behavior using a rat model.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
• /
• v.26 no.3
• /
• pp.270-278
• /
• 2000

This study was conducted to compare the vein graft with the nerve graft, and evaluated the availability of the vein graft on the reconstruction of the inferior alveolar nerve defect. The experimental animals were 12 rabbits weighing $1.5{\sim}2.0kg$, divided into 3 groups : sham operation group, vein conduit group and nerve graft group. All nerves were excised and histomorphometric analysis was performed at 2, 4, 6, 12, 16 weeks after operation. The obtained results were as follows. 1. Histologic examination revealed the regenerated nerve fibers within the lumen of the vein graft and nerve graft at 6 weeks after repair. 2. Axon diameter was significantly larger in nerve graft group(p<0.05) than in vein graft group at 6weeks, and larger in nerve graft group than in vein graft group at 16weeks. 3. Axon density was higher in the vein graft group at 16 weeks. 4. The myelin of the regenerated nerve fibers in distal segment of the vein graft group was thick, approaching the proximal segment at 16weeks. This means remyelination in distal segment in the vein graft group. These results suggested that autogenous vein graft may be used as an alternative to autogenous nerve graft.

• Archives of Plastic Surgery
• /
• v.47 no.2
• /
• pp.187-193
• /
• 2020

Upper limb nerve damage is a common condition, and evidence suggests that functional recovery may be limited following peripheral nerve repair in cases of delayed reconstruction or reconstruction of long nerve defects. A 26-year-old man presented with traumatic injury from a wide, blunt wound of the right forearm caused by broken glass, with soft tissue loss, complete transection of the radial and ulnar arteries, and a large median nerve gap. The patient underwent debridement and subsequent surgery with a microsurgical free radial fasciocutaneous flap to provide a direct blood supply to the hand; the cephalic vein within the flap was employed as a venous vascularized chamber to wrap the sural nerve graft and to repair the wide gap (14 cm) in the median nerve. During the postoperative period, the patient followed an intensive rehabilitation program and was monitored for functional performance over 5 years of follow-up. Our assessment demonstrated skin tropism and sufficient muscle power to act against strong resistance (M5) in the muscles previously affected by paralysis, as well as a good localization of stimuli in the median nerve region and an imperfect recovery of two-point discrimination (S3+). We propose a novel and efficient procedure to repair >10-cm peripheral nerve gap injuries related to upper limb trauma.