Chen-Jung Chang

Effects of nerve growth factor from genipin-crosslinked gelatin in polycaprolactone conduit on peripheral nerve regeneration—In vitro and in vivo

The gelatin solution crosslinked by genipin (0, 0.1, 0.5, 1.0, and 1.5% w/w) was studied as a nerve growth factor (NGF) carrier (GGp0, GGp0.1, GGp0.5, GGp1.0, and GGp1.5) in a polycaprolactone conduit in large-gap nerve regeneration. The GGp0 and GGp0.1 displayed the highest activity of PC12 cells and inhibited the reduction of 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl-tetrazolium bromide (MTT). No cytotoxicity was found in all groups by lactate dehydrogenase (LDH) release. The NGF-releasing characters were obtained by ELISA tests. A relatively fast release rate appeared during the first 10 days and then a subsequent slower release profile followed. NGF was higher in GGp0.1 than in GGp0 and GGp0.1 after 10 days. The bioactivity of the released NGF remains the same when measuring the neurite outgrowth of PC 12 cells. Finally, the controlled-release conduits were implanted into 12-mm long sciatic nerve gaps of rats. In addition, the best site of NGF carrier was determined either by filling carrier into the conduit lumen or by sucking carrier to the conduit wall. Four and 8 weeks after implantation, morphological analysis revealed that GGp0.1 conduits had markedly larger and more number of myelin axons in the midconduit and distal nerve. Further, sucking the carrier into the conduit wall was an efficient and convenient way to prevent the regeneration of axons and vessels from being impaired by the lumens carrier. The genipin-crosslinked gelatin is a promising carrier in producing a high release concentration and a long release period of NGF to promote the regeneration over a large-gap nerve injury.

In Vitro and In Vivo Effects of Ginkgo biloba Extract EGb 761 on Seeded Schwann Cells within Poly(DL-lactic acid-co-glycolic acid) Conduits for Peripheral Nerve Regeneration

This study investigated the effects of Ginkgo biloba (EGb 761) extract on seeded Schwann cells within poly(DL-lactic acid-co-glycolic acid) (PLGA) conduits by in vitro and in vivo trials for peripheral nerve regeneration. The seeding efficiency of Schwann cells in serum-deprived culture medium, which simulated the environment of mechanical trauma on an injured nerve site, was improved by adding different dosages of EGb 761 (0, 1, 10, 20, 50, 100, 200 mg/mL). The analytical results showed enhanced cell attachment and survival, reduced LDH release and increased MTT values, particularly in the range 10-100 mg/mL. The PLGA nerve conduits seeded with Schwann cells (6 103 cells) and filled with gelatin containing EGb 761 (0, 10, 50, 100 mg/mL) were implanted to 10-mm right sciatic nerve defects in rats. Autograft was performed as another control. Electromyography was assessed based on the motor unit action potential (MUAP) and fibrillation potential (Fib) at 2, 4, and 6 weeks during all periods. The specimens of the experimental and control groups were harvested for histological analysis at 6 weeks after surgery. The Fib was found to gradually decay, and the MUAP was found not to be present until 4 weeks after surgery. Meanwhile, the experimental groups were all statically better than the control group (without EGb 761) and autografts were observed at 6 weeks, especially at the concentration of 10 mg/mL, where there was higher amplitude of MUAP and a significantly larger number of myelinated axons. This study concluded that a proper concentration of EGb 761 (10-50 mg/mL) promoted seeding efficiency of Schwann cells in a tissue-engineered PLGA conduit. Addition of EGb 761 in Schwann cells-seeded conduit could increase the total number of myelinated axons in nerve regeneration and improve peripheral nerve functional recovery.

Comparison between two different methods of immobilizing NGF in poly(DL-lactic acid-co-glycolic acid) conduit for peripheral nerve regeneration by EDC/NHS/MES and genipin.

For surface modification and nerve regeneration, chitosan, followed by nerve growth factor (NGF), was immobilized onto the interior surface of poly (lactic acit-co-glycolic) conduits, using EDC/NHS/MES system (EDCs) and genipin (GP). Four new conduits were, therefore, obtained and named by immobilizing order-EDCs/EDCs, GP/EDCs, EDCs/GP, and GP/GP groups. The immobilized methods used were evaluated and compared, respectively. The researchers found that the EDCs- and GP-cross-linked chitosan displayed higher hydrophilic than pure poly (DL-lactic acid-co-glycolic acid) (PLGA) in water contact angle experiment, which meant the cell compatibility was improved by the modification. Scanning electron microscopic observations revealed that the GP-cross-linking of chitosan greatly improved cell compatibility while cultured rat PC12 cells were flatter and more spindle-shaped than EDCs-cross-linked chitosan. The results concerning the GP-cross-linked chitosan revealed significant proliferation of the seeded cells relative to pure PLGA films, as determined by counting cells and MTT assay. The NGF was released from the modified conduits in two separate periods--an initial burst in 5 days and then slow release from day 10 to day 40. The GP/EDCs group had the highest NGF value among all groups after the 5th day. Finally, the controlled-release conduits were used to bridge a 10 mm rat sciatic nerve defect. Six weeks following implantation, morphological analysis revealed the highest numbers of myelinated axons in the midconduit and distal regenerated nerve in GP/EDCs group. Therefore, the results confirm that GP/EDCs groups with good cell compatibility and effective release of NGF can considerably improve peripheral nerve regeneration.

Effects of Near-Field Ultrasound Stimulation on New Bone Formation and Osseointegration of Dental Titanium Implants In Vitro and In Vivo

A near-field ultrasound stimulation system was designed for use in in vitro and in vivo trials. The intensity of ultrasound was studied to optimize the osseointegration of the dental titanium implant into the adjacent bone. MG63 osteoblast-like cells were seeded on commercial purity titanium (CP-Ti) plate, and then sonicated for 3 min/day at a frequency of 1 MHz and intensities of 0.05, 0.15 and 0.30 W/cm(2), using either pulsed or continuous ultrasound. Cells were analyzed to determine viability (inhibition of (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction) and alkaline phosphatase (ALP). Tissue culture was performed in vitro by placing a CP-Ti plate in a cultured rat neonatal calvarial defect in response to ultrasound stimulation. In the in vivo trial, screw-shaped CP-Ti implants were inserted into the metaphysis of rabbit tibia, and then stimulated by ultrasound for 10 min daily for 30 d. All samples were processed for histomorphometric evaluation and analyzed by image system. Color Doppler ultrasonography was inspected to evaluate the supply of blood flow. Pulsed ultrasound groups had higher MTT and ALP than control. Tissue culture indicated that pulsed ultrasound groups promoted cell migration and new bone regeneration more effectively than in the control. In animal study, blood flow and mature type I collagen fibers were more prevalent around titanium implants, and bone formation was accelerated in pulsed ultrasound groups. In conclusion, low-intensity pulsed ultrasound at 0.05-0.3 W/cm(2) may accelerate cell proliferation and promote the maturation of collagen fibers and support osteointegration.

Effect of an Epineurial-Like Biohybrid Nerve Conduit on Nerve Regeneration.

A novel approach of making a biomimetic nerve conduit was established by seeding adipose-derived adult stem cells (ADSCs) on the external wall of porous poly(D, L-lactic acid) (PLA) nerve conduits. The PLA conduits were fabricated using gas foaming salt and solvent–nonsolvent phase conversion. We examined the effect of two different porous structures (GS and GL) on ADSC growth and proliferation. The GS conduits had better structural stability, permeability, and porosity, as well as better cell viability at 4, 7, and 10 days. The epineuriallike tissue was grown from ADSC-seeded conduits cultured for 7 days in vitro and then implanted into 10-mm rat sciatic nerve defects for evaluation. The regeneration capacity and functional recovery were evaluated by histological staining, electrophysiology, walking track, and functional gait analysis after 6 weeks of implantation. Experimental data indicated that the autograft and ADSC-seeded GS conduits had better functional recovery than the blank conduits and ADSC-seeded GL conduits. The area of regenerated nerve and number of myelinated axons quantified based on the histology also indicated that the autograft and AGS groups performed better than the other two groups. We suggested that ADSCs may interact with endogenous Schwann cells and release neurotrophic factors to promote peripheral nerve regeneration. The design of the conduit may be critical for producing a biohybrid nerve conduit and to provide an epineurial-like support.