Bai-Shuan Liu

Evaluation of a Non-Woven Fabric Coated with a Chitosan Bi-Layer Composite for Wound Dressing

This study presents a novel design of an easily stripped bi-layer composite that consists of an upper layer of a soybean protein non-woven fabric coated with a lower layer, a genipin-crosslinked chitosan film, as a wound dressing material. This study examines the in vitro properties of the genipin-crosslinked chitosan film and the bi-layer composite. Furthermore, in vivo experiments are conducted to study wounds treated with the composite in a rat model. Experimental results show that the degree of crosslinking and the in vitro degradation rate of the genipin-crosslinked chitosan films can be controlled by varying the genipin contents. In addition, the genipin contents should exceed 0.025 wt.-% of the chitosan-based material if complete crosslinking reactions between genipin and chitosan molecules are required. Water contact angle analysis shows that the genipin-crosslinked chitosan film is not highly hydrophilic; therefore, the genipin-crosslinked chitosan layer is not entangled with the soybean protein non-woven fabric, which forms an easily stripped interface layer between them. Furthermore, this new wound dressing material provides adequate moisture, thereby minimizing the risk of wound dehydration, and exhibits good mechanical properties. The in vivo histological assessment results reveal that epithelialization and reconstruction of the wound are achieved by covering the wound with the composite, and the composite is easily stripped from the wound surface without damaging newly regenerated tissue.

Peripheral nerve repair of transplanted undifferentiated adipose tissue-derived stem cells in a biodegradable reinforced nerve conduit †

This study proposes a biodegradable nerve conduit containing genipin-cross-linked gelatin annexed with tricalcium phosphate ceramic particles (genipin-gelatin-tricalcium phosphate, GGT) in peripheral nerve regeneration. Firstly, cytotoxicity tests revealed that the GGT-extracts were not toxic, and promoted the proliferation and neuronal differentiation of adipose tissue-derived stem cells (ADSCs). Secondly, the GGT composite film effectively supported ADSCs attachment and growth. Additionally, the GGT substrate was biocompatible with the neonatal rat sciatic nerve and produced a beneficial effect on peripheral nerve repair through in vitro tissue culture. Finally, the experiments in this study confirmed the effectiveness of a GGT/ADSCs nerve conduit as a guidance channel for repairing a 10-mm gap in a rat sciatic nerve. Eight weeks after implantation, the mean recovery index of compound muscle action potentials (CMAPs) was significantly different between the GGT/ADSCs and autografts groups (p < 0.05), both of which were significantly superior to the GGT group (p < 0.05). Furthermore, walking track analysis also showed a significantly higher sciatic function index (SFI) score (p < 0.05) and better toe spreading development in the GGT/ADSCs group than in the autograft group. Histological observations and immunohistochemistry revealed that the morphology and distribution patterns of nerve fibers in the GGT/ADSCs nerve conduits were similar to those of the autografts. The GGT nerve conduit offers a better scaffold for the incorporation of seeding undifferentiated ADSCs, and opens a new avenue to replace autologous nerve grafts for the rapid regeneration of damaged peripheral nerve tissues and an improved approach to patient care.

Nanocomposites of genipin-crosslinked chitosan/silver nanoparticles--structural reinforcement and antimicrobial properties.

This study investigates the feasibility of a novel nanocomposite (GC/Ag) of a genipin-crosslinked chitosan (GC) film in which was embedded various amounts of Ag nanoparticles for wound-dressing applications. In situ UV-vis results revealed that adding chitosan solution did not affect the characteristics of Ag nanoparticles. The water uptake ratios and surface hydrophilicity of the GC/Ag nanocomposite were better and the degradation rates slightly lower than those of the pure GC film. The presence of Ag nanoparticles enhanced L929 cell attachment and growth. Its function as an anti-microbial agent in a GC/Ag nanocomposite was assessed for Ag contents of over 100 ppm. In conclusion, silver ions had dual functions--structural reinforcement and provision of antimicrobial properties to a biocompatible polymer.

Transplantation of Adipose Tissue-Derived Stem Cells for Treatment of Focal Cerebral Ischemia

The neurological functional disabilities caused by cerebral infarction significantly deteriorate life quality and increase the medical and socio-economic costs. Although some molecular agents show potential in acting against the pathological mechanisms in animal studies, none has been proven effective for cerebral ischemia treatment in human patients. New treatment strategy needs to be developed. Stem cell therapy is promising for neural regeneration and thus become one of the current trends. More evidence has shown stem cells, such as embryonic stem cells (ESCs), skeletal muscle satellite cells and mesenchymal stem cells, to be useful in tissue repair and regeneration. However all these stem cells mentioned above have limitations. Adipose tissue-derived stem cells (ADSCs) are an alternative autologous stem cell source for the characters as abundant, easy to obtain, immunological and ethic problem free. So far, this treatment strategy has been rarely adopted on ischemic brain injury. In this study, we investigated the transplantation effects of rat ADSCs for the treatment of cerebral ischemia in rats. ADSCs were isolated from rat adipose tissue and then induced to initiate neural differentiation. Following neural induction, ADSCs developed neural morphology and displayed molecular expression of Nestin, MAP2 and GFAP. We evaluate the neurobehavioral function, infarct volume and cell properties as apoptosis, survival, migration, proliferation, differentiation and immunogenicity. Treatment with i-ADSCs (induction from ADSCs) results in better functional recovery and more reduction in hemispheric atrophy then without i-ADSCs in other groups. Our study demonstrates that i-ADSCs therapy is promising in stroke treatment and finally leads to an efficacious therapeutic modalities for much better outcome in clinical patients.

Fabrication and evaluation of a biodegradable proanthocyanidin-crosslinked gelatin conduit in peripheral nerve repair.

This study proposed a novel and biodegradable nerve guide conduit in its applicability to peripheral nerve regeneration. A naturally occurring proanthocyanidin (PA) was selected as a cross-linking reagent in preparing the PA-crosslinked gelatin (PCG) conduit. Experimental results indicate that 5 wt % of PA was optimal in the complete cross-linking reaction in the PCG conduit. The PCG conduit was brownish and round with a rough outer surface whereas its inner lumen was smooth. The cross-linked networks of the PCG conduit resisted enzymatic hydrolysis under in vitro degradation studies. PA and gelatin were released from the soaked PCG conduit. During the release phase, the concentrations of PA, gelatin, and PCG-soaking solutions were not only nontoxic but also promoted the viability and growth of Schwann cells. The PCG conduit more effectively supported cell attachment and growth. The effectiveness of the PCG conduit as a guidance channel was studied when it was used to repair a 10 mm gap in the rat sciatic nerve. Throughout the 8-week experimental period, the peak amplitude and area under the muscle action potential curve both increased with the recovery period. Histological observations revealed that various regenerated nerve fibers crossed through and beyond the gap region. These results suggest that the PCG conduit can be a candidate for peripheral nerve repair.

Large-area irradiated low-level laser effect in a biodegradable nerve guide conduit on neural regeneration of peripheral nerve injury in rats.

This study used a biodegradable composite containing genipin-cross-linked gelatin annexed with β-tricalcium phosphate ceramic particles (genipin-gelatin-tricalcium phosphate, GGT), developed in a previous study, as a nerve guide conduit. The aim of this study was to analyse the influence of a large-area irradiated aluminium-gallium-indium phosphide (AlGaInP) diode laser (660 nm) on the neural regeneration of the transected sciatic nerve after bridging the GGT nerve guide conduit in rats. The animals were divided into two groups: group 1 comprised sham-irradiated controls and group 2 rats underwent low-level laser (LLL) therapy. A compact multi-cluster laser system with 20 AlGaInP laser diodes (output power, 50mW) was applied transcutaneously to the injured peripheral nerve immediately after closing the wound, which was repeated daily for 5 min for 21 consecutive days. Eight weeks after implantation, walking track analysis showed a significantly higher sciatic function index (SFI) score (P<0.05) and better toe spreading development in the laser-treated group than in the sham-irradiated control group. For electrophysiological measurement, both the mean peak amplitude and nerve conduction velocity of compound muscle action potentials (CMAPs) were higher in the laser-treated group than in the sham-irradiated group. The two groups were found to be significantly different during the experimental period (P<0.005). Histomorphometric assessments revealed that the qualitative observation and quantitative analysis of the regenerated nerve tissue in the laser-treated group were superior to those of the sham-irradiated group. Thus, the motor functional, electrophysiologic and histomorphometric assessments demonstrate that LLL therapy can accelerate neural repair of the corresponding transected peripheral nerve after bridging the GGT nerve guide conduit in rats.

Regenerative effect of adipose tissue-derived stem cells transplantation using nerve conduit therapy on sciatic nerve injury in rats.

This study proposed a biodegradable GGT nerve conduit containing genipin crosslinked gelatin annexed with tricalcium phosphate (TCP) ceramic particles for the regeneration of peripheral nerves. Cytotoxicity tests revealed that GGT‐extracts were non‐toxic and promoted proliferation and neuronal differentiation in the induction of stem cells (i‐ASCs) derived from adipose tissue. Furthermore, the study confirmed the effectiveness of a GGT/i‐ASCs nerve conduit as a guidance channel in the repair of a 10‐mm gap in the sciatic nerve of rats. At eight weeks post‐implantation, walking track analysis showed a significantly higher sciatic function index (SFI) (P < 0.05) in the GGT/i‐ASC group than in the autograft group. Furthermore, the mean recovery index of compound muscle action potential (CMAP) differed significantly between GGT/i‐ASCs and autograft groups (P < 0.05), both of which were significantly superior to the GGT group (P < 0.05). No severe inflammatory reaction in the peripheral nerve tissue at the site of implantation was observed in either group. Histological observation and immunohistochemistry revealed that the morphology and distribution patterns of nerve fibers in the GGT/i‐ASCs nerve conduits were similar to those of the autografts. These promising results achieved through a combination of regenerative cells and GGT nerve conduits suggest the potential value in the future development of clinical applications for the treatment of peripheral nerve injury. Copyright

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.

Characteristics and biocompatibility of a biodegradable genipin-cross-linked gelatin/β-tricalcium phosphate reinforced nerve guide conduit

To modulate the mechanical properties of nerve guide conduit for surgical manipulation, this study develops a biodegradable composite containing genipin cross-linked gelatin annexed with β-tricalcium phosphate ceramic particles as a nerve guide material. The conduit was dark bluish and round with a rough and compact outer surface compared to the genipin cross-linked gelatin conduit (without β-tricalcium phosphate). Water uptake and swelling tests indicate that the conduit noticeably increases the stability in water, and the hydrated conduit does not collapse and stenose. The conduit has a sufficiently high level of mechanical properties to serve as a nerve guide. After subcutaneous implantation on the dorsal side of a rat, the degraded conduit only evokes a mild tissue response, and the formation of a very thin fibrous capsule surrounds the conduit. This paper assesses the effectiveness of the conduit as a guidance channel when we use it to repair a 10 mm gap in the rats sciatic nerve. The experimental results show no gross inflammatory reactions of the peripheral nerve tissues at the implantation site in either group. In overall gross examination, the diameter of the intratubular and newly formed nerve fibers in the conduits exceeds that of the silicone tubes during the implantation period. The quantitative results indicate the superiority of the conduits over the silicone tubes. This study microscopically observes the nerve regeneration in the tissue section at the middle region of all implanted conduits. Therefore, the histomorphometric assessment demonstrates that the conduit could be a candidate for peripheral nerve repair.

Neural regeneration in a novel nerve conduit across a large gap of the transected sciatic nerve in rats with low-level laser phototherapy

This study proposes a biodegradable nerve conduit comprising 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) cross-linked gelatin annexed with β-tricalcium phosphate (β-TCP) ceramic particles (EDC-gelatin-TCP, EGT). For this study, the EGT-implant site in rats was irradiated using 660-nm GaAlAsP laser diodes (50 mW) for trigger point therapy to investigate the use of low-level laser (LLL) stimulation in the regeneration of a 15-mm transected sciatic nerve. Animals were divided into three groups: a control group undergoing autologous nerve graft (autograft); a sham-irradiated group (EGT), and an experimental group undergoing laser stimulation (EGT/LS). Two trigger points on the surgical incision along the sciatic nerve were irradiated transcutaneously for 2 min daily for 10 consecutive days. Twelve weeks after implantation, walking track analysis showed a significantly higher sciatic functional index (SFI; p < 0.05) and improved toe spreading development in the autograft and EGT/LS groups, compared to the EGT group. In the electrophysiological measurement, the mean recovery index (peak amplitude and area) of the compound muscle action potential curves in the autograft and EGT/LS groups showed significantly improved functional recovery than in the EGT group (p < 0.05). Compared with the EGT group, the autograft and EGT/LS groups showed a reduction in muscular atrophy. Histomorphometric assessments showed that the EGT/LS group had undergone more rapid nerve regeneration than the EGT group. Therefore, motor function, electrophysiological reaction, muscular reinnervation, and histomorphometric assessments demonstrate that LLL therapy can accelerate the repair of a 15-mm transected peripheral nerve in rats after being bridged with the EGT nerve conduit.