Carboxylated Poly-L-lysine Potentially Reduces Human Sperm DNA Fragmentation after Freeze-Thawing, and Its Function Is Enhanced by Low-Dose Resveratrol.

Sperm DNA fragmentation (SDF) that occurs during the freezing-thawing of sperm may negatively impact the treatment outcomes of assisted reproductive technologies (ART). In a previous study, we developed a human sperm cryopreservation reagent containing carboxylated poly-L-lysine (CPLL) that reduced SDF after freeze-thawing compared with clinically popular cryopreservation reagents containing human serum albumin. However, it is unclear whether CPLL reduces SDF, as it differed from the constituents of the commercial cryopreservation reagents used for comparison. Therefore, here, we examined whether CPLL reduces the SDF of human sperm and evaluated reactive oxygen species (ROS) levels and lipid peroxidation (LPO), which are the causes of SDF; mitochondrial injury, ROS production; and impaired sperm motility. Furthermore, optimal antioxidants and their concentrations that could further enhance the reduction in SDF were determined for future clinical application in ART and underwent the same functional evaluations. CPLL can reduce SDF via inhibition of intracytoplasmic ROS and LPO. Furthermore, the addition of 0.1 mM resveratrol avoided the enhancement of SDF, which potentially affects mitochondrial and cytoplasmic ROS and LPO. This novel human sperm cryopreservation reagent containing CPLL and resveratrol has the potential to improve treatment outcomes in ART using frozen sperm.

Spontaneous Osteogenic Differentiation of Human Mesenchymal Stem Cells by Tuna-Bone-Derived Hydroxyapatite Composites with Green Tea Polyphenol-Reduced Graphene Oxide.

In recent years, bone tissue engineering (BTE) has made significant progress in promoting the direct and functional connection between bone and graft, including osseointegration and osteoconduction, to facilitate the healing of damaged bone tissues. Herein, we introduce a new, environmentally friendly, and cost-effective method for synthesizing reduced graphene oxide (rGO) and hydroxyapatite (HAp). The method uses epigallocatechin-3-O-gallate (EGCG) as a reducing agent to synthesize rGO (E-rGO), and HAp powder is obtained from Atlantic bluefin tuna (Thunnus thynnus). The physicochemical analysis indicated that the E-rGO/HAp composites had exceptional properties for use as BTE scaffolds, as well as high purity. Moreover, we discovered that E-rGO/HAp composites facilitated not only the proliferation, but also early and late osteogenic differentiation of human mesenchymal stem cells (hMSCs). Our work suggests that E-rGO/HAp composites may play a significant role in promoting the spontaneous osteogenic differentiation of hMSCs, and we envision that E-rGO/HAp composites could serve as promising candidates for BTE scaffolds, stem-cell differentiation stimulators, and implantable device components because of their biocompatible and bioactive properties. Overall, we suggest a new approach for developing cost-effective and environmentally friendly E-rGO/HAp composite materials for BTE application.

Novel external reinforcement device for gastrointestinal anastomosis in an experimental study.

BACKGROUND: Anastomotic leakage has been reported to occur when the load on the anastomotic site exceeds the resistance created by sutures, staples, and early scars. It may be possible to avoid anastomotic leakage by covering and reinforcing the anastomotic site with a biocompatible material. The aim of this study was to evaluate the safety and feasibility of a novel external reinforcement device for gastrointestinal anastomosis in an experimental model. METHODS: A single pig was used in this non-survival study, and end-to-end anastomoses were created in six small bowel loops by a single-stapling technique using a circular stapler. Three of the six anastomoses were covered with a novel external reinforcement device. Air was injected, a pressure test of each anastomosis was performed, and the bursting pressure was measured. RESULTS: Reinforcement of the anastomotic site with the device was successfully performed in all anastomoses. The bursting pressure was 76.1 ± 5.7 mmHg in the control group, and 126.8 ± 6.8 mmHg in the device group, respectively. The bursting pressure in the device group was significantly higher than that in the control group (p = 0.0006). CONCLUSIONS: The novel external reinforcement device was safe and feasible for reinforcing the anastomoses in the experimental model.

Improved fertility of frozen-thawed porcine spermatozoa with 3,3-dimethylglutaric anhydride poly-L-lysine as a novel cryoprotectant.

In this study, we determined the efficacy of 3,3-dimethylglutaric anhydride poly-L-lysine (DMGA-PLL) as a cryoprotectant for porcine spermatozoa. Porcine spermatozoa were cryopreserved in a freezing extender containing 3% (v/v) glycerol and various concentrations of DMGA-PLL. At 12 h after thawing, the motility index of spermatozoa cryopreserved with 0.25% (v/v) DMGA-PLL (25.9) was significantly (P < 0.01) higher than that of spermatozoa cryopreserved with 0%, 0.125%, or 0.5% DMGA-PLL (10.0-16.3). In addition, the blastocyst formation rate of embryos derived from spermatozoa cryopreserved with 0.25% DMGA-PLL (22.8%) was significantly (P < 0.01) higher than that of embryos derived from spermatozoa cryopreserved with 0%, 0.125%, or 0.5% DMGA-PLL (7.9%-10.9%). The mean number of total piglets born to sows inseminated with spermatozoa cryopreserved without DMGA-PLL (9.0) was significantly (P < 0.05) lower than that of total piglets born to sows inseminated with spermatozoa stored at 17°C (13.8). However, when spermatozoa cryopreserved with 0.25% DMGA-PLL were used for artificial insemination, the mean number of total piglets (11.7) was not significantly different from that obtained following artificial insemination using spermatozoa stored at 17°C. The results showed the usefulness of DMGA-PLL as a cryoprotectant in the cryopreservation of porcine spermatozoa.

Successful production of offspring derived from mouse zygotes vitrified with carboxylated ε-poly-L-lysine and polyvinyl alcohol without serum.

The vitrification of zygotes is important for their use as donors for generating genome-edited mice. We previously reported the successful vitrification of mouse zygotes using carboxylated ε-poly-L-lysine (COOH-PLL). However, this vitrification solution contains fetal calf serum (FCS), which contains unknown factors and presents risks of pathogenic viral and microbial contamination. In this study, we examined whether polyvinyl alcohol (PVA) can be used as an alternative to FCS in vitrification solutions for mouse zygotes. When COOH-PLL was added to the vitrification solutions, zygotes vitrified with solutions containing 0.01% PVA (PV0.01) and those vitrified in a control solution containing FCS (75.6%) developed into blastocysts (78.4%). In addition, there were no significant differences in the ability to develop to term between the control solution (46.6%) and PV0.01 (44.1%) groups. In conclusion, we clearly demonstrated that PVA can replace FCS in our vitrification solution supplemented with COOH-PLL for mouse zygotes.

Cryopreservation of pig spermatozoa using carboxylated poly-L-lysine as cryoprotectant.

In this study, we cryopreserved pig spermatozoa using carboxylated poly-L-lysine (CPLL) as the cryoprotectant to determine its efficacy. Pig spermatozoa were placed in a freezing extender containing 3% (v/v) glycerol and different CPLL concentrations. The motility indices of the spermatozoa cryopreserved with 0.25% (v/v) CPLL at 6 (59.3), 9 (53.7), and 12 (26.2) h after thawing were significantly higher (P < 0.01 or P < 0.05) than those of the spermatozoa cryopreserved without CPLL (53.7, 40.1, and 17.5 at 6, 9, and 12 h after thawing, respectively). The concentration of CPLL in the freezing extender did not affect the ability of frozen-thawed spermatozoa to fertilize oocytes in vitro. However, the blastocyst formation rate of embryos derived from spermatozoa cryopreserved with 0.25% CPLL (24.6%) was significantly higher (P < 0.01) than that of embryos derived from spermatozoa cryopreserved without CPLL (11.2%). The conception rate of the sows inseminated with spermatozoa cryopreserved with 0.25% CPLL (72.2%) was not significantly different from that of the sows inseminated with spermatozoa stored at 17°C (81.3%). However, the mean number of total piglets born to the former (10.0) was significantly lower (P < 0.05) than that of total piglets born to the latter (13.4). The results showed that CPLL in the freezing extender maintained the motility of frozen-thawed pig spermatozoa and improved the in vitro development of embryos produced by in vitro fertilization. In addition, we have demonstrated that piglets could be obtained with artificial insemination using spermatozoa cryopreserved with CPLL.

Elucidating the degradation mechanism of a self-degradable dextran-based medical adhesive.

We developed a self-degradable medical adhesive, LYDEX, consisting of periodate-oxidized aldehyde-functionalized dextran (AD) and succinic anhydride-treated ε-poly-l-lysine (SAPL). After gelation and adhesion of LYDEX by Schiff base bond formation between the AD aldehyde groups and SAPL amino groups, molecular degradation associated with the Maillard reaction is initiated, but the detailed degradation mechanism remains unknown. Herein, we elucidated the degradation mechanism of LYDEX by analyzing the main degradation products under typical solution conditions in vitro. The degradation of the LYDEX gel with a sodium periodate/dextran content of 2.5/20 was observed using gel permeation chromatography and infrared and 1H NMR spectroscopy. The AD ratio in the AD-SAPL mixture increased as the molecular weight decreased with the degradation time. This discovery of LYDEX self-degradability is useful for clarifying other polysaccharide hydrogel degradation mechanisms, and valuable for the use of LYDEX in medical applications, such as hemostatic or sealant materials.

Gene expression analysis of human induced pluripotent stem cells cryopreserved by vitrification using StemCell Keep.

In recent years, regenerative medicine research using human somatic and induced pluripotent stem cells has advanced considerably, promoting clinical applications. However, it is essential that these cells are cryopreserved safely and effectively. Most cryopreservation solution agents contain dimethyl sulfoxide (DMSO), which exhibits strong toxicity and can potentially promote cell differentiation. Hence, it is important to explore substitutes for DMSO in cryoprotectant solutions. One such alternative is StemCell Keep (SCK), a DMSO-free solution that has been reported to effectively cryopreserve human induced pluripotent stem cells (hiPS cells). To clarify the effect of cryopreservation agents on cells, DNA microarray analysis is useful, as it can identify a large number of gene expression differences in cryopreserved cells, as well as functional increases in gene groups. In this study, we performed gene expression analysis of SCK-cryopreserved hiPS cells using a DNA microarray gene chip. The hiPS cells vitrified with SCK or DMSO-based vitrification solutions were thawed and cultured on Matrigel under feeder-free conditions, and RNA was extracted for DNA microarray analysis. Genes obtained from DNA microarray data were classified by the keywords of Gene Ontology Biological Process Term, and their relationships were analyzed using DAVID or the GeneMANIA database. SCK-cryopreserved hiPS cells expressed several anti-apoptotic genes, as well as genes related to cell adhesion or proliferation at levels that were nearly equivalent to those of non-frozen hiPS cells. Gene enrichment analysis with selected genes of SCK-cryopreserved hiPS cells whose expression differences were superior to those of DAP-cryopreserved showed strong interactions of negative regulation of apoptotic process, cell adhesion and positive regulation of cell proliferation in DAVID analysis. We demonstrated that SCK successfully maintained the key functions of hiPS cells, including anti-apoptosis, cell adhesion, and cell proliferation, during cryopreservation.

Polyphenols-loaded electrospun nanofibers in bone tissue engineering and regeneration.

Bone is a complex structure with unique cellular and molecular process in its formation. Bone tissue regeneration is a well-organized and routine process at the cellular and molecular level in humans through the activation of biochemical pathways and protein expression. Though many forms of biomaterials have been applied for bone tissue regeneration, electrospun nanofibrous scaffolds have attracted more attention among researchers with their physicochemical properties such as tensile strength, porosity, and biocompatibility. When drugs, antibiotics, or functional nanoparticles are taken as additives to the nanofiber, its efficacy towards the application gets increased. Polyphenol is a versatile green/phytochemical small molecule playing a vital role in several biomedical applications, including bone tissue regeneration. When polyphenols are incorporated as additives to the nanofibrous scaffold, their combined properties enhance cell attachment, proliferation, and differentiation in bone tissue defect. The present review describes bone biology encompassing the composition and function of bone tissue cells and exemplifies the series of biological processes associated with bone tissue regeneration. We have highlighted the molecular mechanism of bioactive polyphenols involved in bone tissue regeneration and specified the advantage of electrospun nanofiber as a wound healing scaffold. As the polyphenols contribute to wound healing with their antioxidant and antimicrobial properties, we have compiled a list of polyphenols studied, thus far, for bone tissue regeneration along with their in vitro and in vivo experimental biological results and salient observations. Finally, we have elaborated on the importance of polyphenol-loaded electrospun nanofiber in bone tissue regeneration and discussed the possible challenges and future directions in this field.

Carboxylated Poly-l-Lysine as a Macromolecular Cryoprotective Agent Enables the Development of Defined and Xeno-Free Human Sperm Cryopreservation Reagents.

In human sperm cryopreservation, test yolk buffer and human serum albumin have been used as permeating macromolecular-weight cryoprotectants. In clinical reproductive medicine, human serum albumin is frequently used because of low risks of zoonoses and allergic reactions. However, the risk of allogeneic infectious diseases exists, and the supply may be unstable because human serum albumin is derived from human blood. Therefore, the development of xeno-free human sperm cryopreservative reagents that could overcome the aforementioned problems is warranted. We succeeded in developing a new xeno-free and defined sperm cryopreservation reagent containing glycerol, carboxylated poly-l-lysine, and raffinose. The cryopreservation reagent was not significantly different in terms of sperm motility, viability, and DNA fragmentation and was comparable in performance to a commercial cryopreservation reagent containing human serum albumin. Moreover, the addition of saccharides was essential for its long-term storage. These results may help elucidate the unknown function of macromolecular-weight permeating cryoprotective agents.

Carboxylated ε-poly-L-lysine, a cryoprotective agent, is an effective partner of ethylene glycol for the vitrification of embryos at various preimplantation stages.

It has been known that different protocols are used for embryo preservation at different stages due to different sensitivity to the physical and physiological stress caused by vitrification. In this study, we developed a common vitrification protocol using carboxlated ε-poly-l-lysine (COOH-PLL), a new cryoprotective agent for the vitrification of mouse embryos at different stages. The IVF-derived Crl:CD1(ICR) x B6D2F1/Crl pronuclear, 2-cell, 4-cell, and 8-cell, morula and blastocyst stage embryos were vitrified with 15% (v/v) ethylene glycol (EG) and 10% (w/v) COOH-PLL (E15P15) or 15% (v/v) EG and 15% (v/v) dimethyl sulfoxide (E15D15) using the minimal volume cooling method. The survival of vitrified embryos from pronuclear to blastocyst stages was equivalent between E15P15 and E15D15 groups. However, the rate of development to blastocysts was significantly lower in E15P15 than E15D15. The rates of survival and development to blastocysts were dramatically improved by a slight modification of EG and COOH-PLL concentrations (E20P10). After transferring 17 (E20P10) and 15 (E15D15) vitrified/warmed blastocysts, 8 and 7 pups were obtained (47.1% and 46.7%, respectively). Taken together, these results indicate that our vitrification protocol is appropriate for the vitrification of mouse embryos at different stages.

Molecular Design of Polyampholytes for Vitrification-Induced Preservation of Three-Dimensional Cell Constructs without Using Liquid Nitrogen.

Current slow-freezing methods are too inefficient for cryopreservation of three-dimensional (3D) tissue constructs. Additionally, conventional vitrification methods use liquid nitrogen, which is inconvenient and increases the chance of cross-contamination. Herein, we have developed polyampholytes with various degrees of hydrophobicity and showed that they could successfully vitrify cell constructs including spheroids and cell monolayers without using liquid nitrogen. The polyampholytes prevented ice crystallization during both cooling and warming, demonstrating their potential to prevent freezing-induced damage. Monolayers and spheroids vitrified in the presence of polyampholytes yielded high viabilities post-thawing with monolayers vitrified with PLL-DMGA exhibiting more than 90% viability. Moreover, spheroids vitrified in the presence of polyampholytes retained their fusibilities, thus revealing the propensity of these polyampholytes to stabilize 3D cell constructs. This study is expected to open new avenues for the development of off-the-shelf tissue engineering constructs that can be prepared and preserved until needed.

Development of an efficient vitrification method for chondrocyte sheets for clinical application.

INTRODUCTION: Regenerative therapy using chondrocyte sheets is effective for osteoarthritis. The clinical application of chondrocyte sheet therapy is expected to be further advanced by the use of a feasible cryopreservation technique. Previously, we developed a chondrocyte sheet vitrification method; however, it was too complex to be used for routine clinical application. Here, we aimed to develop a prototype method for vitrifying chondrocyte sheets for clinical practice. METHODS: We developed a "circulating vitrification bag" as a container to process cell sheets for vitrification in an efficient and sanitary fashion. Moreover, we invented the "vitrification storage box", which is useful for the vitrification of cell sheets, long-term preservation, and transportation. These devices were used to vitrify rabbit chondrocyte sheets, which were then assessed for their structural characteristics and the viability of the component cells after rewarming. RESULTS: In all cell sheet samples (n = 7) vitrified by the circulating vitrification bag method, the integrity of the sheet structure was maintained, and the cell survival rate was similar to that of non-vitrified samples (91.0 ± 2.9% vs. 90.0 ± 3.0%). Proteoglycan and type II collagen, which are major components of cartilage, were densely and evenly distributed throughout the chondrocyte sheet subjected to vitrification similarly to that observed in the non-vitrified sheet. After long-term storage using the vitrification storage box, the cell sheets maintained normal structure and cell viability (survival rate: 81.2 ± 1.0% vs. 84.3 ± 1.8%) compared to the non-vitrified sheet. CONCLUSION: Our results indicate that the circulating vitrification bag method is an effective approach for realizing the clinical application of vitrified chondrocyte sheets. The vitrification storage box is also useful for the long-term preservation of vitrified cell sheets, further enhancing the feasibility of the clinical application of cryopreserved chondrocyte sheets.

Management of retrosternal adhesion after median sternotomy by controlling degradation speed of a dextran and ε-poly (L-lysine)-based biocompatible glue.

OBJECTIVE: Retrosternal adhesion after median sternotomy possibly raises the risk of cardiac injury at resternotomy. A biodegradable glue "Lydex" is composed of food additives, dextran and ε-poly (L-lysine), and the degradation speed can be controlled by the composition. In the present study, we evaluated the preventative effect of Lydex on retrosternal adhesion and the relationship between degradation speed and the progression of retrosternal fibrosis. METHODS: Japanese white rabbits are subjected to median sternotomy. Lydex 1, 2 and 3 were loaded at the retrosternal space of rabbits in allocated groups before sternal closure, respectively (n = 11 for each group). Retrosternal adhesion was macroscopically evaluated after surgery. Retainment of Lydex, retrosternal fibrosis and the infiltration of macrophages are histologically evaluated, respectively. RESULTS: All Lydex groups exhibited less retrosternal adhesion at 4 weeks after loading compared to unloaded control. The degradation speed of Lydex varied according to the compositions. Lydex with faster degradation (Lydex 2 or Lydex 3) showed lower progression of retrosternal fibrosis compared to that with slower degradation (Lydex 1) [fibrosis ratio: control vs Lydex 1 vs Lydex 2 vs Lydex 3: 0.60 ± 0.15 vs 0.18 ± 0.17 vs 0.00 ± 0.00 vs 0.00 ± 0.00, P = 0.0005 (Lydex 1 vs Lydex 2), P = 0.0005 (Lydex 1 vs Lydex 3)]. Retrosternal infiltrations of macrophages in Lydex 1 and Lydex 3 groups are not higher compared to that in unloaded control. CONCLUSIONS: The degradation speed of Lydex could be controlled according to the compositions. The degradation speed affected the progression of retrosternal fibrosis.

Virus-Incorporated Biomimetic Nanocomposites for Tissue Regeneration.

Owing to the astonishing properties of non-harmful viruses, tissue regeneration using virus-based biomimetic materials has been an emerging trend recently. The selective peptide expression and enrichment of the desired peptide on the surface, monodispersion, self-assembly, and ease of genetic and chemical modification properties have allowed viruses to take a long stride in biomedical applications. Researchers have published many reviews so far describing unusual properties of virus-based nanoparticles, phage display, modification, and possible biomedical applications, including biosensors, bioimaging, tissue regeneration, and drug delivery, however the integration of the virus into different biomaterials for the application of tissue regeneration is not yet discussed in detail. This review will focus on various morphologies of virus-incorporated biomimetic nanocomposites in tissue regeneration and highlight the progress, challenges, and future directions in this area.

Efficacy of a novel endoscopically deliverable muco-adhesive hemostatic powder in an acute gastric bleeding porcine model.

This study investigated the effectiveness of new hemostatic adhesive powder (UI-EWD) in a swine mode of acute gastric bleeding. Gastric ulcer bleeding was induced endoscopically at two locations in each of eight heparinized mini-pigs. UI-EWD and saline were sprayed endoscopically in the experimental (n = 5) and control groups (n = 3), respectively. The hemostatic effect and hydrogel persistence on ulcers were periodically evaluated endoscopically. Initial hemostasis was achieved successfully in all lesions in the experimental group. Follow-up endoscopy showed minor delayed bleeding in 10% at 6 hours in the experimental group, whereas re-bleeding was observed in 50% at 6 hours in the control group. UI-EWD gel persisted at 90%, 80%, and 50% of ulcer bases at 6, 18, and 42 hours post-application, respectively. This study suggests that muco-adhesive UI-EWD may be effective in the endoscopic treatment of active ulcer bleeding.

Controlling the degradation of an oxidized dextran-based hydrogel independent of the mechanical properties.

The objective of this study is to control and elucidate the mechanism of molecular degradation in a polysaccharide hydrogel. Glycidyl methacrylate (GMA) immobilized dextran (Dex-GMA) was oxidized by periodate to introduce aldehyde groups (oxidized Dex-GMA). The hydrogel was formed by the addition of dithiothreitol to the oxidized Dex-GMA solution through thiol Michael addition with the preservation of the aldehyde group for degradation points. It was experimentally determined that the degradation of this hydrogel can be controlled by the addition of amino groups and the speed of degradation can be controlled independently of mechanical properties because crosslinking and degradation points are different. In addition, the molecular mechanism of the crosslinking between the thiol and aldehyde groups was found to control the degradation of dextran derivatives. It is expected that these results will be beneficial in the design of polymer materials in which the speed of degradation can be precisely controlled. In addition, the cytotoxicity of oxidized Dex-GMA was approximately 3000 times lower than that of glutaraldehyde. The low cytotoxicity of the aldehyde in oxidized Dex-GMA was the likely reason for the harmless functionalized polysaccharide material. Possible future clinical applications include cell scaffolds in regenerative medicine and carriers for drug delivery systems.

Aligned laminin core-polydioxanone/collagen shell fiber matrices effective for neuritogenesis.

Neural tissue regeneration is a significant challenge, because severe nerve injury is quite difficult to regenerate spontaneously. Although, many studies have been devoted to promote nerve regeneration, there are still many technical challenges to achieve satisfactory results. In this study, we designed biomimetic matrices composed of aligned laminin core-polydioxanone/collagen shell (Lam-PDO/Col) fibers, which can provide both topographical and biochemical cues for promoting neuritogenesis. The aligned Lam-PDO/Col core-shell fiber matrices were fabricated by magnetic field-assisted electrospinning with the coaxial system, and their potential as biofunctional scaffolds for promoting neuritogenesis was explored. It was demonstrated that the aligned Lam-PDO/Col core-shell fibers were successfully fabricated, and the laminin in the core of fibers was steadily and continuously released from fibers. In addition, the cellular behaviors of hippocampal neuronal cells on the matrices were significantly enhanced. Moreover, the aligned Lam-PDO/Col fiber matrices effectively improved and guided neurite outgrowth as well as the neurogenic differentiation by providing both topographical and biochemical cues through aligned fiber structure and sustained release of laminin. Collectively, it is suggested that the aligned Lam-PDO/Col core-shell fiber matrices are one of the most promising approaches for promoting neuritogenesis and neural tissue regeneration.

Ternary Aligned Nanofibers of RGD Peptide-Displaying M13 Bacteriophage/PLGA/Graphene Oxide for Facilitated Myogenesis.

Recently, there have been tremendous efforts to develop the biofunctional scaffolds by incorporating various biochemical factors. In the present study, we fabricated poly(lactic-co-glycolic acid) (PLGA) nanofiber sheets decorated with graphene oxide (GO) and RGD peptide. The decoration of GO and RGD peptide was readily achieved by using RGD peptide-displaying M13 bacteriophage (RGD-M13 phage) and electrospinning. Furthermore, the aligned GO-decorated PLGA/RGD peptide (GO-PLGA/RGD) ternary nanofiber sheets were prepared by magnetic field-assisted electrospinning, and their potentials as bifunctional scaffolds for facilitating myogenesis were explored. We characterized the physicochemical and mechanical properties of the sheets by scanning electron microscopy, Raman spectroscopy, contact angle measurement, and tensile test. In addition, the C2C12 skeletal myoblasts were cultured on the aligned GO-PLGA/RGD nanofiber sheets, and their cellular behaviors, including initial attachment, proliferation and myogenic differentiation, were evaluated. Our results revealed that the GO-PLGA/RGD nanofiber sheets had suitable physicochemical and mechanical properties for supporting cell growth, and could significantly promote the spontaneous myogenic differentiation of C2C12 skeletal myoblasts. Moreover, it was revealed that the myogenic differentiation was further accelerated on the aligned GO-PLGA/RGD nanofiber sheets due to the synergistic effects of RGD peptide, GO and aligned nanofiber structure. Therefore, , it is suggested that the aligned GO-PLGA/RGD ternary nanofiber sheets are one of the most promising approaches for facilitating myogenesis and promoting skeletal tissue regeneration.

Cryoprotective effect of antifreeze polyamino-acid (Carboxylated Poly-l-Lysine) on bovine sperm: A technical note.

In this study, investigating Carboxylated Poly-l-Lysine (CPLL) for its effectiveness as a new cryoprotectant for bovine sperm is aimed. CPLL is an ampholytic polymer compound, has cryoprotective properties similar to those of anti-freeze protein. The cryopreservation medium used for control group consisted of 6.5% (v/v) glycerol, the cryopreservation medium used for experimental group consisted of 3.25% (v/v) glycerol + 0.5% (w/v) CPLL. There was no consequential difference in sperm motility parameter after thawing whereas there was huge distinction for sperm membrane integrity rate (control vs experimental; 49.6 vs 60.7%, P < 0.01). Conception rate of artificial insemination of experimental group was significantly higher than that of control group (79.0% vs 53.1%, P < 0.01). These results suggest CPLL has protected sperm membrane and leads to improve fertility. This is the first report using CPLL for bovine sperm cryopreservation, it is also expected CPLL can be applied to other animal species.