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Tissue repair engineering supported by nanoparticles and stem cells has been demonstrated as being an efficient strategy for promoting the healing potential during the regeneration of damaged tissues. In the current study, we prepared various nanomaterials including pure Pul, pure Col, Pul–Col, Pul–Au, Pul–Col–Au, and Col–Au to investigate their physicochemical properties, biocompatibility, biological functions, differentiation capacities, and anti-inflammatory abilities through in vitro and in vivo assessments. The physicochemical properties were characterized by SEM, DLS assay, contact angle measurements, UV-Vis spectra, FTIR spectra, SERS, and XPS analysis. The biocompatibility results demonstrated Pul–Col–Au enhanced cell viability, promoted anti-oxidative ability for MSCs and HSFs, and inhibited monocyte and platelet activation. Pul–Col–Au also induced the lowest cell apoptosis and facilitated the MMP activities. Moreover, we evaluated the efficacy of Pul–Col–Au in the enhancement of neuronal differentiation capacities for MSCs. Our animal models elucidated better biocompatibility, as well as the promotion of endothelialization after implanting Pul–Col–Au for a period of one month. The above evidence indicates the excellent biocompatibility, enhancement of neuronal differentiation, and anti-inflammatory capacities, suggesting that the combination of pullulan, collagen, and Au nanoparticles can be potential nanocomposites for neuronal repair, as well as skin tissue regeneration in any further clinical treatments.

Background It has previously been shown that bevacizumab, when added to chemotherapy, improved overall survival in several cancers. In glioblastoma multiforme (GBM), bevacizumab increased progression-free survival and it is widely used for tumor recurrence, though it has failed to improve overall survival (OS) in controlled trials. However, an effective biomarker for predicting the prognosis of bevacizumab treatment has yet to be identified. This study, therefore, aimed to retrospectively analyze the polymorphisms of p53 codon 72 and the clinical characteristics of GBM specimens from Taiwanese patients. Methods The polymorphisms of p53 codon 72 in 99 patients with GBM treated at Taichung Veterans General Hospital in Taiwan from 2007 to 2017 were analyzed using direct DNA sequencing and PCR-RFLP analysis. Results We found that among these GBM patients, the distribution of codon 72 polymorphisms was 28.3% for proline homozygotes (Pro/Pro), 38.4% for arginine homozygotes (Arg/Arg), and 33.3% for proline/arginine heterozygotes (Pro/Arg). Although the polymorphisms of p53 codon 72 were not directly associated with the overall survival of GBM, both the Arg/Arg and Arg/Pro genotypes were associated with significant benefits in terms of overall survival in patients treated with CCRT plus bevacizumab compared to patients treated with CCRT alone. Conclusions This pilot study suggests that both the Arg/Arg and Arg/Pro genotypes of p53 codon 72 polymorphism may have value as independent prognostic or predictive parameters for bevacizumab treatment response and failure. Relatedly, the results of the study further demonstrate the utility of stratifying GBM patients according to bevacizumab sensitivity.

Additional file 1.

A previous study confirmed that a novel splicing variant of large vascular endothelial growth factor (L-VEGF) termed L-VEGF144, a nucleolus protein, is found in glioblastoma cells and specimens, but the actual biological function and clinical significance of L-VEGF144 remain unclear. In this study, we analyzed the expression of L-VEGF144 in 68 glioblastoma multiforme specimens using reverse transcriptase-polymerase chain reaction analysis. The results showed that the high expression of L-VEGF144 was associated with a poor prognosis in the bevacizumab plus concurrent chemoradiotherapy with temozolomide treatment. In addition, we constructed a series truncated and mutant form of L-VEGF144 to confirm that exon 6a of L-VEGF144 is able to engage in the nuclear importation and found that 8 lysines within exon 6a play a critical role in the nucleolus aggregation of L-VEGF144. Also, the transfection of the L-VEGF144 increased the number of nucleoli. Furthermore, the recombinant protein Flag-L-VEGF144 and commercial VEGF protein have similar growth stimulatory activities in terms of inducing glioblastoma cell proliferation in vitro. Taken together, these results indicated that the expression of L-VEGF144 could potentially serve as an independent indicator of poor prognosis in bevacizumab treatment.

Recent studies have shown the potential of artificially synthesized conduits in the repair of peripheral nerve injury. Natural biopolymers have received much attention because of their biocompatibility. To investigate the effects of novel electrospun absorbable poly(ε-caprolactone)/type I collagen nanofiber conduits (biopolymer nanofiber conduits) on the repair of peripheral nerve injury, we bridged 10-mm-long sciatic nerve defects with electrospun absorbable biopolymer nanofiber conduits, poly(ε-caprolactone) or silicone conduits in Sprague-Dawley rats. Rat neurologica1 function was weekly evaluated using sciatic function index within 8 weeks after repair. Eight weeks after repair, sciatic nerve myelin sheaths and axon morphology were observed by osmium tetroxide staining, hematoxylin-eosin staining, and transmission electron microscopy. S-100 (Schwann cell marker) and CD4 (inflammatory marker) immunoreactivities in sciatic nerve were detected by immunohistochemistry. In rats subjected to repair with electrospun absorbable biopolymer nanofiber conduits, no serious inflammatory reactions were observed in rat hind limbs, the morphology of myelin sheaths in the injured sciatic nerve was close to normal. CD4 immunoreactivity was obviously weaker in rats subjected to repair with electrospun absorbable biopolymer nanofiber conduits than in those subjected to repair with poly(ε-caprolactone) or silicone. Rats subjected to repair with electrospun absorbable biopolymer nanofiber conduits tended to have greater sciatic nerve function recovery than those receiving poly(ε-caprolactone) or silicone repair. These results suggest that electrospun absorbable poly(ε-caprolactone)/type I collagen nanofiber conduits have the potential of repairing sciatic nerve defects and exhibit good biocompatibility. All experimental procedures were approved by Institutional Animal Care and Use Committee of Taichung Veteran General Hospital, Taiwan, China (La-1031218) on October 2, 2014.

The expression levels of different vascular endothelial growth factor A (VEGF) isoforms are associated with the angiogenesis and the patient's prognoses in human cancers. Ribosomes specifically scan from 5' to 3' CUG initiation codon in the long 5'-untranslated region (5'-UTR) of the VEGF mRNA, resulting in the generation of high mol wt VEGF isoform [call large VEGF (L-VEGF)]. Alternative splicing of VEGF mRNA transcripts results in several isoforms with distinct properties that are dependent up their exon compositions. In this study, we observed two novel kinds of splicing VEGF isoforms that transcripted at the first upstream CUG codon, and which we have named large-VEGF144 (LVEGF144), and large-VEGF138 (L-VEGF138). The expression levels of messenger RNA for the different VEGF splice forms were analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR). After DNA sequencing, the genetic structure of L-VEGF144 involved not only a partial exon 1, exon 6a, and exons 7-8, but also an unique 108- nucleotides insertion of VEGF intron 5 interposed between exon 1 and exon 6. At the same time, L-VEGF144 lacked most of the Nterminal fragments (exons 1-5). We further found that a specific detection model could easily and rapidly confirm the presence of L-VEGF144 mRNA fragments in the biopsies or cell lines via RT-PCR assay. In addition, we used visible fluorescent fusion proteins to prove that both L-VEGF144 and L-VEGF138 have nuclear localization ability. Taken together, the findings of this study indicate that, unlike previously identified isoforms, these novel VEGF isoforms are likely to suggest a further level of complexity in the angiogenic process.

This paper proposes a novel biodegradable nerve conduit comprising 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) cross-linked gelatin, annexed with β-tricalcium phosphate (TCP) ceramic particles (EDC-Gelatin-TCP, EGT). In this study, the EGT-implant site in rats was irradiated using a large-area 660 nm AlGaInP diode laser (50 mW) to investigate the feasibility of laser stimulation in the regeneration of a 15-mm transected sciatic nerve. The animals were divided into three groups: a sham-irradiated group (EGT/sham); an experimental group undergoing low-level laser (LLL) therapy (EGT/laser); a control group undergoing autologous nerve grafts (autografts). Twelve weeks after implantation, walking track analysis showed a significantly higher sciatic functional index (p < 0.05) and improved toe spreading development in the EGT/laser and autograft groups than in the EGT/sham group. In electrophysiological measurement, both the mean peak amplitude and the area under the compound muscle action potential curves in the EGT/laser and autograft groups showed significantly improved functional recovery than the EGT/sham group (p < 0.05). Compared with the EGT/sham group, the EGT/laser and autograft groups displayed a reduction in muscular atrophy. Histomorphometric assessments revealed that the EGT/laser group had undergone more rapid nerve regeneration than the EGT/sham group. The laser-treated group also presented greater neural tissue area as well as larger axon diameter and thicker myelin sheath than the tube group without the laser treatment, indicating improved nerve regeneration. Thus, these assessments demonstrate that LLL therapy can accelerate the repair of a transected peripheral nerve in rats after being bridged with EGT conduit. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 101A:239–252, 2013.

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

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

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.

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.

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.

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.

This study employed a rat model of sciatic nerve injury to investigate the effects of postoperative low-power far-infrared (FIR) radiation therapy on nerve repair following end-to-end neurorrhaphy. The rat models were divided into the following 3 groups: (1) nerve injury without FIR biostimulation (NI/sham group); (2) nerve injury with FIR biostimulation (NI/FIR group); and (3) noninjured controls (normal group). Walking-track analysis results showed that the NI/FIR group exhibited significantly higher sciatic functional indices at 8 weeks after surgery (P<0.05) compared with the NI/sham group. The decreased expression of CD4 and CD8 in the NI/FIR group indicated that FIR irradiation modulated the inflammatory process during recovery. Compared with the NI/sham group, the NI/FIR group exhibited a significant reduction in muscle atrophy (P<0.05). Furthermore, histomorphometric assessment indicated that the nerves regenerated more rapidly in the NI/FIR group than in the NI/sham group; furthermore, the NI/FIR group regenerated neural tissue over a larger area, as well as nerve fibers of greater diameter and with thicker myelin sheaths. Functional recovery, inflammatory response, muscular reinnervation, and histomorphometric assessment all indicated that FIR radiation therapy can accelerate nerve repair following end-to-end neurorrhaphy of the sciatic nerve.

The aim of this study was to evaluate peripheral nerve regeneration across a 15-mm gap in the sciatic nerve of the rat, using a silicone rubber nerve guide filled with different concentrations of astragaloside (0, 50, 100, and 200 μM). Collagen was also filled in the chambers to prevent the astragaloside from leakage. At the end of 8 weeks, animals from the group treated with astragaloside, especially at the concentration of 50 μM, had a higher rate of successful regeneration across the wide gap, a significantly larger number of myelinated axons, and a greater evoked action potential than the control group. However, the high-dose astragaloside (200 μM) completely reversed this positive effect of growth-promoting capability and inhibited nerve regeneration. Thus, astragaloside plays a dual role in anastomosis, being salutary in aiding the growth of axons in peripheral nerve but also detrimental, terminating the nerve regenerative processes if improperly applied. © 2005 Wiley Periodicals, Inc. J Biomed Mater Res, 2006

A biodegradable composite which was composed of genipin cross-linked gelatin mixing with tricalcium phosphate ceramic particles (GGT) was developed as a bone substitute. This study was evaluated by the biological response of rabbit calvarial bone to assess the potential of the GGT composite as a biodegradable and osteoconductive bone substitute. Eighteen New Zealand white rabbits were used for cranial implantation. Bone defects (15×15 mm) of nine rabbits were filled with the GGT composites, while the others were filled with the de-proteinized bovine bones as controls. Three rabbits were examined for each group in every time period at 4, 8 and 12 weeks post-surgery. The assessment included serial post-operative gross examinations, radiographic analyses and histological evaluations. This study demonstrated that this composite is: (1) malleable, with easily molded to the calvarial bone defect without fracture; (2) biocompatible, with no evidence of adverse tissue reaction; (3) osteoconductive, with progressive growth of new bone into the calvarial bone defect; (4) biodegradable, with progressive replacement of the composite by new bone. Additionally, results of both radiographic analyses and histological evaluations revealed obviously greater new bone ingrowth in the GGT composite compared with the de-proteinized bovine bone at the same implantation time. Therefore, the GGT composite could serve as a useful bone substitute for repairing bone defects.

The aim of this study is to prepare networks of Bloom number 300 gelatin and genipin as degradable biomaterials. The cross-linking ability of genipin on gelatin was studied and discs were made from the genipin-fixed gelatin. The swelling ratio, the degree of cross-linking, and the rate of degradation of the discs were measured using ninhydrin assay and differential scanning calorimetry (DSC) experiments. The results indicated that genipin is a good cross-linker for the gelatin. Additionally, varying the concentration of genipin can control the rate of degradation and the degree of cross-linking of the genipin-fixed gelatin. Finally, the concentration of genipin should exceed 0.5% of the overall weight of the gelatin-based material if a complete cross-linking reaction between gelatin and genipin molecules is required.

A biodegradable composite (GGT) containing tricalcium phosphate ceramic particles and genipin crosslinked gelatin was developed for use as a bone substitute. The objective of this study was to assess the biocompatibility and the osteoconductivity of the GGT composite on new bone formation in vitro. Additionally, biodegradation and biocompatibility of the GGT composite in animals were investigated. Results of the GGT composites cocultured with osteoblasts showed that the concentration of genipin used as a crosslinking agent should be

The purpose of this study was to prepare and evaluate in vitro the feasibility and cytocompatibility of a novel composite (GGT) as a large defect bone substitute. The composite is tricalcium phosphate ceramic particles combined with genipin crosslinked gelatin. After soaking the GGT composites in Ringer solutions at 37°C for 7, 14, 28, 42, 56, and 84 days, the in vitro biologic degradation rate and biocompatibility were determined. Substances released from soaked GGT composites were analyzed with an ultraviolet visible light spectrophotometer. In addition, the solution soaking the GGT was co-cultured with osteoblasts to determine whether or not the released substances from GGT could facilitate the growth of bone cells. After they had been cultured for 2 days, the osteoblasts were tested for differentiation and proliferation by alkaline phosphatase (ALP) activity and a MTT assay. Results indicate that the concentration of the genipin solution is a critical factor in deciding the crosslinking degree of the GGT composite. Complete crosslinking reaction in the GGT composite occurred when 0.5 wt % of genipin had been added. Cytotoxic testing revealed that 80 ppm of the genipin in the culture medium served as the level over which cytotoxicity to osteoblasts could be produced. In addition, we found that gelatin and calcium continuously were released from the GGT composite in the soaking solution, which promoted differentiation and proliferation of the osteoblasts. © 2003 Wiley Periodicals, Inc. J Biomed Mater Res 67A: 1163–1169, 2003

The purpose of this study was to prepare and evaluate the feasibility and biocompatibility of a new composite as a bone substitute. The new composite (GTGC) was mainly composed of tricalcium phosphate ceramics and gelatin to which Chi-Li-Saan, a Chinese medicinal remedy was added. The GTGC composite was manually packed into cylindrical Teflon molds, dried overnight in an oven and sterilized by γ-ray prior to use. Mature New Zealand rabbits, weighting 3–3.5 kg, underwent full-thickness excision of the parietal bone. In the experimental group, bone defects of 12 animals were filled with the GTGC composites and another 12 unreconstructed rabbits were considered as controls. Three rabbits were examined for each group in every time period at 2, 4, 8 and 12 weeks after operation. There was no evidence of adverse tissue reaction to the GTGC composite. In addition, examination with light and fluorescent microscopy revealed a significantly greater amount of new bone ingrowth in the GTGC group at the same implantion time as compared with the controls. Therefore, the GTGC composite could serve as a useful substitute when repairing bone defects.

This study proposed a novel combination of neural regeneration techniques for the repair of damaged peripheral nerves. A biodegradable nerve conduit containing genipin-cross-linked gelatin was annexed using beta-tricalcium phosphate (TCP) ceramic particles (genipin-gelatin-TCP, GGT) to bridge the transection of a 15 mm sciatic nerve in rats. Two trigger points were irradiated transcutaneously using 660 nm of gallium-aluminum arsenide phosphide (GaAlAsP) via laser diodes for 2 min daily over 10 consecutive days. Walking track analysis showed a significant improvement in sciatic functional index (SFI)(P<0.01)and pronounced improvement in the toe spreading ability of rats undergoing laser stimulation. Electrophysiological measurements (peak amplitude and area) illustrated by compound muscle action potential (CMAP) curves demonstrated that laser stimulation significantly improved nerve function and reduced muscular atrophy. Histomorphometric assessments revealed that laser stimulation accelerated nerve regeneration over a larger area of neural tissue, resulting in axons of greater diameter and myelin sheaths of greater thickness than that observed in rats treated with nerve conduits alone. Motor function, electrophysiological reactions, muscular reinnervation, and histomorphometric assessments all demonstrate that the proposed therapy accelerated the repair of transected peripheral nerves bridged using a GGT nerve conduit.

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; p0.05) 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 (p0.05). 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.

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.

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. © 2011 Wiley Periodicals, Inc. J Biomed Mater Res Part A:, 2012.

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.

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 rat's 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.

This study proposes a biodegradable GGT composite nerve guide conduit containing genipin-cross-linked gelatin and tricalcium phosphate (TCP) ceramic particles in peripheral nerve regeneration. The proposed genipin-cross-linked gelatin annexed with TCP ceramic particles (GGT) conduit was dark bluish and round with a rough and compact surface. Water uptake and swelling tests indicated that the hydrated GGT conduit exhibited increased stability with not collapsing or stenosis. The GGT conduit had higher mechanical properties than the genipin-cross-linked gelatin without TCP ceramic particles (GG) conduit and served as a better nerve guide conduit. Cytotoxicity tests revealed that the GGT conduit was not toxic and that it promoted the viability and growth of neural stem cells. The experiments in this study confirmed the effectiveness of the GGT conduit as a guidance channel for repairing a 10-mm gap in rat sciatic nerve. Walking track analysis showed a significantly higher sciatic function index score and better toe spreading development in the GGT group than in the silicone group 8 weeks after implantation. Gross examination revealed that the diameter of the intratubular newly formed nerve fibers in GGT conduits exceeded those in silicone tubes after the implantation period. Histological observations revealed that the morphology and distribution patterns of nerve fibers in the GGT conduits at 8 weeks after implantation were similar to those of normal nerves. The quantitative results indicated the superiority of the conduits over the silicone tubes. Motor functional and histomorphometric assessments demonstrate that the proposed GGT conduit is a suitable candidate for peripheral nerve repair.

This study presents a novel design of an easily stripped bilayer composite that consists of an upper layer of soybean protein nonwoven fabric coated with a lower layer, genipin-crosslinked chitosan and Bletilla striata herbal extract (GCB) film as a wound dressing material (GCB/NWF). Preliminary tests investigated the in vitro characteristics of the genipin-crosslinked chitosan (GC) film. Experimental results showed that the dark bluish color of the GC film may have resulted from the reaction of genipin with the amino acid group in the chitosan solution. Additionally, adding more wt% genipin caused the bluish color of the GC films to be darker. This new wound dressing material also exhibited good mechanical properties. Genipin and chitosan were released from the soaked GC film. Cytotoxic test revealed that 20 ppm of the genipin in the culture medium could be criteria, over which cytotoxicity to L929 fibroblasts could be produced. However, the chitosan and B. striata herbal extract not only were not toxic but also promoted the viability and growth of L929 fibroblasts. Additionally, the GCB film more effectively supported cell attachment and growth. The in vivo histological assessment results revealed that epithelialization and reconstruction of the wound were achieved by covering the wound with the GCB/NWF wound dressing material, and it would be easily stripped from the wound surface without damaging newly regenerated tissue. POLYM. COMPOS., 2010. © 2009 Society of Plastics Engineers

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.

The biological properties of commercial pure titanium (cp-Ti) dental implants can be improved by surface treatment. In this study, the cp-Ti surfaces were prepared to enable machined surfaces (TM) to be compared to the machined, sandblasted, laser irradiated and dual-acid etched surfaces (TA). The surface elements and roughness were characterized. The biocompatibility was evaluated by cell and organ culture in vitro. The removal torque was measured in rabbit implantation. Surface characterization revealed that TA surface was more oxidized than TM surface. The TA surface had micrometric, beehive-like coarse concaves. The average roughness (2.28 mum) was larger than that typical of acid-etched surfaces. Extracts of both materials were not cytotoxic to bone cells. The morphology of cells attached on the TA surface was superior to that on the TM surface. TA promoted cell migration and repaired damaged bones more effectively in organ culture. The formation of bone-like nodules on TA disk exceeded that on TM disk. Rabbit tibia implantation also proved that TA implant had greater removal torque value. These results suggested that TA had good osteoconductivity and was a potential material for dental implantation.

We have a clarification to make in our paper “Low-Level Laser Stimulation on Adipose-Tissue-Derived Stem Cell Treatments for Focal Cerebral Ischemia in Rats” [1]. Because the GAPDH gene is often stably and constitutively expressed at high levels in most cells and tissues, it is considered a housekeeping gene. For this reason, GAPDH is commonly used by biological researchers as a loading control for western blot. GAPDH was just used as internal control for gene expression analysis as it was supposed to have a uniform expression in both Figures 5(a) and 9(a).

The purpose of this investigation was to prepare and evaluate the feasibility and biocompatibility of a new composite as a large defect bone substitute. The new GTGG was mainly composed of tricalcium phosphate ceramic particles and glutaraldehyde crosslinked gelatin in which Gui-Lu-Jiao was added (a mixture of Cervi Colla Cornus and Colla Plastri Testudinis). In the in vitro study, rat's calvaria osteoblasts were used to study bone characteristics upon exposure to different concentrations of the Gui-Lu-Jiao solution. In the in vivo study, GTGG composites were implanted into the defects of calvarial bones in mature New Zealand rabbits to test their osteogenerative characteristics. As a result, we found that Gui-Lu-Jiao added to the culture could promote the proliferation of osteoblasts. In addition, GTGG could induce a large amount of new bone growth in the rabbit's calvarial bone defect. Therefore, the GTGG composite might be a potential bone substitute.

This study investigates the biological effects of traditional Chinese medicines on the activities of bone cells using rat bone cells. Then, a mixture of a GGT composite, that is, a novel biodegradable composite containing genipin crosslinked gelatin and tricalcium phosphate, and traditional Chinese medicine (TCM) was prepared as a GGT-TCM composite. A cultured neonatal rat calvarias organ was used to measure the potential of GGT-TCM composite for use in promoting the regeneration of defective bone tissue. The mitochondria activity of the bone cells following exposure to various concentrations of crude extracts of five herbal Chinese medicines was measured by colorimetric assay. Biochemical markers, such as alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP) titers were analyzed to evaluate the activities of bone cells. Finally, we examined the organ culture units, which were maintained in cultured medium for 5 weeks. Morphology of tissue was observed, and the quantitative evaluation of the regenerated bone was determined. In a bone cells culture experiment, adding Cuscuta chinensis Lam. (TCM-5) to the bone cells culture clearly promoted the proliferation and differentiation of the osteoblasts from their precursor cells; but the reduced amount of TRAP indicated that the medicine significantly inhibited the osteoclasts activities. Opposite bone cell responses were observed when Loranthus parasiticus Merr. (TCM-3) and Achyranthes bidentata Bl. (TCM-4) were added to the bone cells culture. Eucommia ulmoides Oliv. (TCM-1) and Dipsacus asper Wall. (TCM-2) potentially influence the proliferation and differentiation of the osteoblasts from their precursor cells, but they did not affect the osteoclasts activities. The finding from the organ culture indicated that Chinese medicine effectively increased the rate of tissue regeneration of damaged bones.

Application of combining herbal medicine and biomedical material science to nerve regeneration is a new approach. In this study, we describe a novel use of purified genipin, which can be extracted from Gardenia jasminoides Ellis, fixing the gelatin to be an extracellular matrix for peripheral nerve regeneration. A 10-mm gap of rat sciatic nerve was created between the proximal and distal nerve stumps, which were sutured into silicone rubber tubes filled with either the genipin-fixed gelatin or collagen gel. Silicone rubber tubes filled with saline were used as controls. Six weeks after implantation, regeneration across the nerve gaps occurred in 80 and 90% of the animals from the groups of genipin-fixed gelatin and collagen, respectively, whereas only 30% in the control group. Large numbers of myelinated axons were also seen in the genipin-fixed gelatin (5104±3278) and the collagen groups (8063±1807). These findings indicated that the genipin-fixed gelatin could be an acceptable extracellular matrix for nerve regeneration.

Peripheral nerve injuries are common in clinical practice because of traumas such as crushing and sectioning. Lesions of the nerve structure result in lost or diminished sensitivity and/or motor activity in the innervated territory. The degree of lesion depends on the specific nerve involved, the magnitude and type of pressure exerted, and the duration of the compression. The results of such injuries commonly include axonal degeneration and retrograde degeneration of the corresponding neurons in the spinal medulla, followed by very slow regeneration (Rochkind et al., 2001). The adverse effects on the daily activities of patients with a peripheral nerve injury are a determining factor in establishing the goals of early recovery (Rodriguez et al., 2004). The most severe form of nerve damage involves complete transection of the nerve, which results in the loss of sensory and motor function at the site of injury. Although a degree of recovery can be expected in most untreated nerve injuries, the process is slow and often incomplete. Moreover, despite considerable advances in microsurgical techniques, the functional results of peripheral nerve repair remain largely unsatisfactory. The regrowth of nerves across large gaps is particularly challenging, usually requiring a nerve graft to correctly bridge the proximal and distal nerve stumps. At present, nerve autografting is the most common treatment used to repair peripheral nerve defects. However, this recognized “gold standard” technique has a number of inherent disadvantages, such as limited availability of donor tissue (IJkema-Paassen et al., 2004), secondary deformities, potential differences in tissue structure and size (Nichols et al., 2004), and numbness at donor sites (Bini et al., 2004). Although xenografts and allografts have been proposed as alternatives to autografts, the success rate of these techniques remains poor, often resulting in immune rejection. Thus, researchers have invested considerable effort in developing synthetic nerve conduits for the repair of peripheral nerve defects.