Porous Nano-Fiber Structure of Modified Electrospun Chitosan GBR Membranes Improve Osteoblast Calcium Phosphate Deposition in Osteoblast-Fibroblast Co-Cultures.

Desirable characteristics of electrospun chitosan membranes (ESCM) for guided bone regeneration are their nanofiber structure that mimics the extracellular fiber matrix and porosity for the exchange of signals between bone and soft tissue compartments. However, ESCM are susceptible to swelling and loss of nanofiber and porous structure in physiological environments. A novel post-electrospinning method using di-tert-butyl dicarbonate (tBOC) prevents swelling and loss of nanofibrous structure better than sodium carbonate treatments. This study aimed to evaluate the hypothesis that retention of nanofiber morphology and high porosity of tBOC-modified ESCM (tBOC-ESCM) would support more bone mineralization in osteoblast-fibroblast co-cultures compared to Na2CO3 treated membranes (Na2CO3-ESCM) and solution-cast chitosan solid films (CM-film). The results showed that only the tBOC-ESCM retained the nanofibrous structure and had approximately 14 times more pore volume than Na2CO3-ESCM and thousands of times more pore volume than CM-films, respectively. In co-cultures, the tBOC-ESCM resulted in a significantly greater calcium-phosphate deposition by osteoblasts than either the Na2CO3-ESCM or CM-film (p < 0.05). This work supports the study hypothesis that tBOC-ESCM with nanofiber structure and high porosity promotes the exchange of signals between osteoblasts and fibroblasts, leading to improved mineralization in vitro and thus potentially improved bone healing and regeneration in guided bone regeneration applications.

Rev1 overexpression accelerates N-methyl-N-nitrosourea (MNU)-induced thymic lymphoma by increasing mutagenesis.

Rev1 has two important functions in the translesion synthesis pathway, including dCMP transferase activity, and acts as a scaffolding protein for other polymerases involved in translesion synthesis. However, the role of Rev1 in mutagenesis and tumorigenesis in vivo remains unclear. We previously generated Rev1-overexpressing (Rev1-Tg) mice and reported that they exhibited a significantly increased incidence of intestinal adenoma and thymic lymphoma (TL) after N-methyl-N-nitrosourea (MNU) treatment. In this study, we investigated mutagenesis of MNU-induced TL tumorigenesis in wild-type (WT) and Rev1-Tg mice using diverse approaches, including whole-exome sequencing (WES). In Rev1-Tg TLs, the mutation frequency was higher than that in WT TL in most cases. However, no difference in the number of nonsynonymous mutations in the Catalogue of Somatic Mutations in Cancer (COSMIC) genes was observed, and mutations involved in Notch1 and MAPK signaling were similarly detected in both TLs. Mutational signature analysis of WT and Rev1-Tg TLs revealed cosine similarity with COSMIC mutational SBS5 (aging-related) and SBS11 (alkylation-related). Interestingly, the total number of mutations, but not the genotypes of WT and Rev1-Tg, was positively correlated with the relative contribution of SBS5 in individual TLs, suggesting that genetic instability could be accelerated in Rev1-Tg TLs. Finally, we demonstrated that preleukemic cells could be detected earlier in Rev1-Tg mice than in WT mice, following MNU treatment. In conclusion, Rev1 overexpression accelerates mutagenesis and increases the incidence of MNU-induced TL by shortening the latency period, which may be associated with more frequent DNA damage-induced genetic instability.

Chitosan Membranes Stabilized with Varying Acyl Lengths Release Cis-2-Decenoic Acid and Bupivacaine at Controlled Rates and Inhibit Pathogenic Biofilm.

BACKGROUND: Adherence of complex bacterial biofilm communities to burned tissue creates a challenge for treatment, with infection causing 51% of burn victim deaths. This study evaluated the release of therapeutics from wound care biomaterials and their antimicrobial activity against pathogens Staphylococcus aureus, Acinetobacter baumannii, and Pseudomonas aeruginosa. METHODS: Electrospun chitosan membranes (ESCMs) were fabricated and acylated with chain lengths ranging from 6-10 carbons then loaded with 0.15 mg of anti-biofilm agent, cis-2-decenoic acid (C2DA), and 0.5 mg of local anesthetic, bupivacaine. RESULTS: Combinations of therapeutics released from modified ESCMs at a cumulative amount of 45-70% of bupivacaine and less than 20% of C2DA. Results from bacterial studies suggest that this combination reduced biofilm 10-fold for S. aureus, 2-fold for Acinetobacter baumannii, and 2-3-fold for Pseudomonas aeruginosa by 24 hours. Additionally, dual loaded groups reduced planktonic Staphylococcus aureus ~4-fold by 24 hours as well as Acinetobacter baumannii ~3-fold by 48 hours. CONCLUSIONS: The combination of therapeutics used has a significant role in biofilm prevention for selected strains via direct contact or diffusion in aqueous solutions.

BMAL1 positively correlates with genes regulating steroidogenesis in human luteinized granulosa cells.

In brief: In this study, we examined the relationship between BMAL1 expression and the genes regulating steroid biosynthesis in human luteinized granulosa cells. BMAL1 function is crucial for steroid production and proper ovarian function, highlighting the importance of circadian clock regulation in female reproductive health. Abstract: Human luteinized granulosa cells were collected to analyze circadian clock gene expression and its effect on the genes regulating steroid biosynthesis. We used siRNA to knock down the expression of BMAL1 in KGN cells. We measured the expression levels of genes regulating steroid biosynthesis and circadian clock RT-qPCR. We demonstrated that BMAL1 expression positively correlates with genes regulating steroid biosynthesis (CYP11A1, CYP19A1, STAR, and ESR2). The knockdown of BMAL1 in KGN cells revealed a significant decrease in steroid synthase expression. In contrast, when BMAL1 was overexpressed in KGN and HGL5 cells, we observed a significant increase in the expression of steroid synthases, such as CYP11A1 and CYP19A1. These results indicated that BMAL1 positively controls 17ß-estradiol (E2) secretion in granulosa cells. We also demonstrated that dexamethasone synchronization in KGN cells enhanced the rhythmic alterations in circadian clock genes. Our study suggests that BMAL1 plays a critical role in steroid biosynthesis in human luteinized granulosa cells, thereby emphasizing the importance of BMAL1 in the regulation of reproductive physiology.

Imeglimin profoundly affects the circadian clock in mouse embryonic fibroblasts.

OBJECTIVE: Imeglimin is a novel antidiabetic drug structurally related to metformin. Metformin has been shown to modulate the circadian clock in rat fibroblasts. Accordingly, in the present study, we aimed to determine whether imeglimin can impact the circadian oscillator in mouse embryonic fibroblasts (MEFs). METHODS: MEFs carrying a Bmal1-Emerald luciferase (Bmal1-ELuc) reporter were exposed to imeglimin (0.1 or 1 mM), metformin (0.1 or 1 mM), a nicotinamide phosphoribosyltransferase inhibitor FK866, and/or vehicle. Subsequently, Bmal1-ELuc expression and clock gene mRNA expression levels were measured at 10-min intervals for 55 h and 4-h intervals for 32 h, respectively. RESULTS: Imeglimin significantly prolonged the period (from 26.3 to 30.0 h at 0.1 mM) and dose-dependently increased the amplitude (9.6-fold at 1 mM) of the Bmal1-ELuc expression rhythm; however, metformin exhibited minimal effects on these parameters. Moreover, imeglimin notably impacted the rhythmic mRNA expression of clock genes (Bmal1, Per1, and Cry1). The concurrent addition of FK866 partly inhibited the effects of imeglimin on both Bmal1-ELuc expression and clock gene mRNA expression. CONCLUSION: Collectively, these results reveal that imeglimin profoundly affects the circadian clock in MEFs. Further studies are needed to evaluate whether imeglimin treatment could exert similar effects in vivo.

Cis-2-Decenoic Acid and Bupivacaine Delivered from Electrospun Chitosan Membranes Increase Cytokine Production in Dermal and Inflammatory Cell Lines.

Wound dressings serve to protect tissue from contamination, alleviate pain, and facilitate wound healing. The biopolymer chitosan is an exemplary choice in wound dressing material as it is biocompatible and has intrinsic antibacterial properties. Infection can be further prevented by loading dressings with cis-2-decenoic acid (C2DA), a non-antibiotic antimicrobial agent, as well as bupivacaine (BUP), a local anesthetic that also has antibacterial capabilities. This study utilized a series of assays to elucidate the responses of dermal cells to decanoic anhydride-modified electrospun chitosan membranes (DA-ESCMs) loaded with C2DA and/or BUP. Cytocompatibility studies determined the toxic loading ranges for C2DA, BUP, and combinations, revealing that higher concentrations (0.3 mg of C2DA and 1.0 mg of BUP) significantly decreased the viability of fibroblasts and keratinocytes. These high concentrations also inhibited collagen production by fibroblasts, with lower loading concentrations promoting collagen deposition. These findings provide insight into preliminary cellular responses to DA-ESCMs and can guide future research on their clinical application as wound dressings.

Titanium coated with 2-decenoic analogs reduces bacterial and fungal biofilms.

AIMS: Due to antibiotic tolerance of microbes within biofilm, non-antibiotic methods for prevention and treatment of implant-related infections are preferable. The goal of this work is to evaluate a facile loading strategy for medium-chain fatty-acid signaling molecules 2-heptycyclopropane-1-carboxylic acid (2CP), cis-2-decenoic acid (C2DA), and trans-2-decenoic acid, which all act as diffusible signaling factors (DSFs), onto titanium surfaces for comparison of their antimicrobial efficacy. METHODS AND RESULTS: Titanium coupons were drop-coated with 0.75 mg of DSF in ethanol and dried. Surface characteristics and the presence of DSF were confirmed with Fourier Transform infrared spectroscopy, x-ray photoelectron spectroscopy, and water contact angle. Antimicrobial assays analyzing biofilm and planktonic Staphylococcus aureus, Escherichia coli, or Candida albicans viability showed that planktonic growth was reduced after 24-h incubation but only sustained through 72 h for S. aureus and C. albicans. Biofilm formation on the titanium coupons was also reduced for all strains at the 24-h time point, but not through 72 h for E. coli. Although ∼60% of the loaded DSF was released within the first 2 days, enough remained on the surface after 4 days of elution to significantly inhibit E. coli and C. albicans biofilm. Cytocompatibility evaluations with a fibroblast cell line showed that none of the DSF-loaded groups decreased viability, while C2DA and 2CP increased viability by up to 50%. CONCLUSIONS: In this study, we found that DSF-loaded titanium coupons can inhibit planktonic microbes and prevent biofilm attachment, without toxicity to mammalian cells.

Desynchronization between Food Intake and Light Stimulations Induces Uterine Clock Quiescence in Female Mice.

BACKGROUND: Dysmenorrhea is associated with breakfast skipping in young women, suggesting that fasting in the early active phase disrupts uterine functions. OBJECTIVES: To investigate the possible involvement of the uterine clock system in fasting-induced uterine dysfunction, we examined core clock gene expressions in the uterus using a 28-h interval-fed mouse model. METHODS: Young female mice (8 wk of age) were divided into 3 groups: group I (ad libitum feeding), group II (time-restricted feeding, initial 4 h of the active period every day), and group III (time-restricted feeding for 8 h with a 28-h cycle). Groups II and III have the same fasting interval of 20 h. After analyzing feeding and wheel running behaviors during 2 wk of dietary restriction, mice were sacrificed at 4-h intervals, and the expression profiles of clock genes in the uterus and liver were examined by qPCR. RESULTS: The mice in group I took food mainly during the dark phase and those in group II during the initial 4 h of the dark phase, whereas those in group III delayed feeding time by 4 h per cycle. In all groups, spontaneous wheel running was observed during the dark phase. There was no difference in the quantity of feeding and the amount of running exercise among the 3 groups during the second week. The mRNA expressions of peripheral clock genes, Bmal1, Clock, Per1, Per2, Cry1, Nr1d1, and Dbp and a clock-controlled gene, Fabp1, in the uterus showed rhythmic oscillations with normal sequential expression cascade in groups I and II, whereas their expressions decreased and circadian cycles disappeared in group III. In contrast, liver core clock genes in group III showed clear circadian cycles. CONCLUSIONS: Fluctuations in the timing of the first food intake impair the uterine clock oscillator system to reduce clock gene expressions and abolish their circadian rhythms.

The Circadian Clock, Nutritional Signals and Reproduction: A Close Relationship.

The circadian rhythm, which is necessary for reproduction, is controlled by clock genes. In the mouse uterus, the oscillation of the circadian clock gene has been observed. The transcription of the core clock gene period (Per) and cryptochrome (Cry) is activated by the heterodimer of the transcription factor circadian locomotor output cycles kaput (Clock) and brain and muscle Arnt-like protein-1 (Bmal1). By binding to E-box sequences in the promoters of Per1/2 and Cry1/2 genes, the CLOCK-BMAL1 heterodimer promotes the transcription of these genes. Per1/2 and Cry1/2 form a complex with the Clock/Bmal1 heterodimer and inactivate its transcriptional activities. Endometrial BMAL1 expression levels are lower in human recurrent-miscarriage sufferers. Additionally, it was shown that the presence of BMAL1-depleted decidual cells prevents trophoblast invasion, highlighting the importance of the endometrial clock throughout pregnancy. It is widely known that hormone synthesis is disturbed and sterility develops in Bmal1-deficient mice. Recently, we discovered that animals with uterus-specific Bmal1 loss also had poor placental development, and these mice also had intrauterine fetal death. Furthermore, it was shown that time-restricted feeding controlled the uterine clock's circadian rhythm. The uterine clock system may be a possibility for pregnancy complications, according to these results. We summarize the most recent research on the close connection between the circadian clock and reproduction in this review.

Isolation and characterization of Zygosaccharomyces sp. yeast strains from miso.

There is currently great interest in the salt-tolerant yeast strains used to produce miso and soy sauce. Since the isolation of Zygosaccharomyces sp. strain from Japanese miso more than 60 years, several hybrid strains have been identified in fermented foods. Studies have shown that the active mating-type locus of the original Zygosaccharomyces sp. yeast strain is located between the T-subgenome sequence and the P-subgenome sequence. In this study, 32 salt-tolerant Zygosaccharomyces sp. yeast strains were isolated from five miso factories in Hiroshima Prefecture, Japan. Analysis by flow cytometry revealed that 27 strains were diploid and five strains were haploid. PCR analysis indicated that the 27 diploid strains had the same chromosomal structure of the active mating-type (MAT) locus as the original yeast strain isolated from miso 60 years ago. In addition, the 27 diploid strains were allodiploid, namely, natural hybrids of Z. rouxii and a related species, while the five haploid strains were all Z. rouxii. We found that cells of yeast strains isolated from miso changed morphologically when co-cultured with a yeast strain of opposite mating-type under nitrogen starvation conditions. The DNA sequence of the active mating-type locus and the results of cell morphology changes by co-culture were consistent with the mating type of each strain shown in the mating experiments. These findings will be useful for the future production of miso and soy sauce.

Evaluation of Magnesium-Phosphate Particle Incorporation into Co-Electrospun Chitosan-Elastin Membranes for Skin Wound Healing.

Major challenges facing clinicians treating burn wounds are the lack of integration of treatment to wound, inadequate mechanical properties of treatments, and high infection rates which ultimately lead to poor wound resolution. Electrospun chitosan membranes (ESCM) are gaining popularity for use in tissue engineering applications due to their drug loading ability, biocompatibility, biomimetic fibrous structure, and antimicrobial characteristics. This work aims to modify ESCMs for improved performance in burn wound applications by incorporating elastin and magnesium-phosphate particles (MgP) to improve mechanical and bioactive properties. The following ESCMs were made to evaluate the individual components' effects; (C: chitosan, CE: chitosan-elastin, CMg: chitosan-MgP, and CEMg: chitosan-elastin-MgP). Membrane properties analyzed were fiber size and structure, hydrophilic properties, elastin incorporation, MgP incorporation and in vitro release, mechanical properties, degradation profiles, and in vitro cytocompatibility with NIH3T3 fibroblasts. The addition of both elastin and MgP increased the average fiber diameter of CE (~400 nm), CMg (~360 nm), and CEMg (565 nm) compared to C (255 nm). Water contact angle analysis showed elastin incorporated membranes (CE and CEMg) had increased hydrophilicity (~50°) compared to the other groups (C and CMg, ~110°). The results from the degradation study showed mass retention of ~50% for C and CMg groups, compared to ~ 30% seen in CE and CEMg after 4 weeks in a lysozyme/PBS solution. CMg and CEMg exhibited burst-release behavior of ~6 µg/ml or 0.25 mM magnesium within 72 h. In vitro analysis with NIH3T3 fibroblasts showed CE and CEMg groups had superior cytocompatibility compared to C and CMg. This work has demonstrated the successful incorporation of elastin and MgP into ESCMs and allows for future studies on burn wound applications.

Sustained effect of habitual feeding time on daily rhythm of core body temperature in mice.

Background and aim: Circadian clocks in most peripheral tissues are entrained mainly by feeding. Therefore, this study aimed to investigate whether the daily rhythm of core body temperature (CBT), including the effect of diet-induced thermogenesis, varies according to habitual feeding time. Methods: Wild-type and uncoupling protein 1 (UCP1) knockout mice were fed only during the first 4 h (Breakfast group) or the last 4 h of the dark period (Dinner group) for 17 days. On day 18, both groups were fed twice for 2 h, at the same starting times. Locomotor activity and CBT were measured continuously during the experiment. Results: On day 18, CBT increased at the beginning of each feeding period, regardless of the group and strain. However, the CBT increase induced by the first meal decreased sharply in the Breakfast group and mildly in the Dinner group; the opposite was observed after the second meal. In UCP1 knockout, but not wild-type, mice, the total amount of CBT was significantly lower in the Dinner group than in the Breakfast group. These effects were mostly independent of the locomotor activity and food intake. Conclusion: These results reveal that the effect of habitual feeding time on the daily rhythm of CBT is sustained at least until the following day. These effects may be mediated by both UCP1-dependent and -independent mechanisms.

Uterine Deletion of Bmal1 Impairs Placental Vascularization and Induces Intrauterine Fetal Death in Mice.

Recently, it was demonstrated that the expression of BMAL1 was decreased in the endometrium of women suffering from recurrent spontaneous abortion. To investigate the pathological roles of uterine clock genes during pregnancy, we produced conditional deletion of uterine Bmal1 (cKO) mice and found that cKO mice could receive embryo implantation but not sustain pregnancy. Gene ontology analysis of microarray suggested that uterine NK (uNK) cell function was suppressed in cKO mice. Histological examination revealed the poor formation of maternal vascular spaces in the placenta. In contrast to WT mice, uNK cells in the spongiotrophoblast layer, where maternal uNK cells are directly in contact with fetal trophoblast, hardly expressed an immunosuppressive NK marker, CD161, in cKO mice. By progesterone supplementation, pregnancy could be sustained until the end of pregnancy in some cKO mice. Although this treatment did not improve the structural abnormalities of the placenta, it recruited CD161-positive NK cells into the spongiotrophoblast layer in cKO mice. These findings indicate that the uterine clock system may be critical for pregnancy maintenance after embryo implantation.

Plastic litter fate and contaminant transport within the urban environment, photodegradation, fragmentation, and heavy metal uptake from storm runoff.

A significant portion of urban litter is plastic which contaminates the environment and threatens ecological safety. The conversion of plastic litter into small fragments called microplastics (MPs) intensifies their critical risks by facilitating their transport and altering their physicochemical features. This study focuses on low density polyethylene (LDPE) and polyethylene terephthalate (PET) as the main components of urban litter. The photodegradation of LDPE and PET MPs due to the accelerated weathering experiments is investigated through surface chemistry and morphology analysis. The influence of MPs' photodegradation on their fragmentation behavior is evaluated through the innovative accelerated mechanical weathering experiments that simulated the abrasion of MPs with the road deposits. Furthermore, the role of MPs as the vehicles to transport the heavy metals from the urban environment to the water resources is evaluated by studying the kinetics of lead (Pb) uptake by new and weathered MPs in synthetic stormwater. The surface morphology investigation revealed the formation of crazes and the crack networks onto the MPs due to the weathering experiments. The surface chemistry analysis revealed the generation of several oxidized carbon surface functional groups onto the photodegraded MPs and their increased susceptibility to fragmentation due to the abrasion with the road deposits. The photodegradation increased the Pb accumulation onto the LDPE and PET MPs from 467 µg/m2 and 21 µg/m2 to 2290 µg/m2 and 725 µg/m2, after five days of metal exposure. The fundamental knowledge developed in this research provides a better conceptual understanding of the mechanisms controlling MPs persistence and contaminant transport within the urban environment, which is crucial to estimate their negative impacts on the ecosystem.

3D reconstructed brain images reveal the possibility of the ogg1 gene to suppress the irradiation-induced apoptosis in embryonic brain in medaka (Oryzias latipes).

The accumulation of oxidative DNA lesions in neurons is associated with neurodegenerative disorders and diseases. Ogg1 (8-oxoG DNA glycosylase-1) is a primary repair enzyme to excise 7,8-dihydro-8-oxoguanine (8-oxoG), the most frequent mutagenic base lesion produced by oxidative DNA damage. We have developed ogg1-deficient medaka by screening with a high resolution melting (HRM) assay in Targeting-Induced Local Lesions In Genomes (TILLING) library. In this study, we identified that ogg1-deficient embryos have smaller brains than wild-type during the period of embryogenesis and larvae under normal conditions. To reveal the function of ogg1 when brain injury occurs during embryogenesis, we examined the induction of apoptosis in brains after exposure to gamma-rays with 10 Gy (137Cs, 7.3 Gy/min.) at 24 h post-irradiation both in wild-type and ogg1-deficient embryos. By acridine orange (AO) assay, clustered apoptosis in irradiated ogg1-deficient embryonic brains were distributed in a similar manner to those of irradiated wild-type embryos. To evaluate possible differences of gamma-ray induced apoptosis in both types of embryonic brains, we constructed 3D images of the whole brain based on serial histological sections. This analysis identified that the clustered apoptotic volume was about 3 times higher in brain of irradiated ogg1-deficient embryos (n = 3) compared to wild-type embryos (n = 3) (P = 0.04), suggesting that irradiation-induced apoptosis in medaka embryonic brain can be suppressed in the presence of functional ogg1. Collectively, reconstruction of 3D images can be a powerful approach to reveal slight differences in apoptosis induction post-irradiation.

Synthesis and Characterization of 2-Decenoic Acid Modified Chitosan for Infection Prevention and Tissue Engineering.

Chitosan nanofiber membranes are recognized as functional antimicrobial materials, as they can effectively provide a barrier that guides tissue growth and supports healing. Methods to stabilize nanofibers in aqueous solutions include acylation with fatty acids. Modification with fatty acids that also have antimicrobial and biofilm-resistant properties may be particularly beneficial in tissue regeneration applications. This study investigated the ability to customize the fatty acid attachment by acyl chlorides to include antimicrobial 2-decenoic acid. Synthesis of 2-decenoyl chloride was followed by acylation of electrospun chitosan membranes in pyridine. Physicochemical properties were characterized through scanning electron microscopy, FTIR, contact angle, and thermogravimetric analysis. The ability of membranes to resist biofilm formation by S. aureus and P. aeruginosa was evaluated by direct inoculation. Cytocompatibility was evaluated by adding membranes to cultures of NIH3T3 fibroblast cells. Acylation with chlorides stabilized nanofibers in aqueous media without significant swelling of fibers and increased hydrophobicity of the membranes. Acyl-modified membranes reduced both S. aureus and P.aeruginosa bacterial biofilm formation on membrane while also supporting fibroblast growth. Acylated chitosan membranes may be useful as wound dressings, guided regeneration scaffolds, local drug delivery, or filtration.

Long-Term Controlled Release of Simvastatin from Photoprinted Triple-Networked Hydrogels Composed of Modified Chitosan and PLA-PEG Micelles.

Local delivery of active agents using injectable or implantable hydrogels for tissue and bone regeneration is a promising therapy, but it remains challenging for controlling dose and duration of release. Simvastatin (SMV), a hydrophobic drug, has shown potential for osteogenic stimulation. Secure loading of hydrophobic drugs by physical interactions is particularly difficult to establish in hydrophilic polymer matrices, and their sustained release over several months for long-term regeneration has rarely been reported. Additionally, mechanical properties of hydrogels must be improved for a sufficient support while maintaining eventual biodegradability. This study assesses the effect of controlled SMV release from 3D-printed triple-network hydrogels for osteogenic stimulation and characterizes their mechanical and biological properties as an implant. SMV is loaded into polymeric micelles of polylactide/poly(ethylene glycol) triblock copolymers (PLA-PEG-PLA) and mixed with N-methacryloyl chitosan and PEG dimethacrylate to fabricate hydrogels by photo-cross-linked 3D printing. The hydrogel properties and drug release profiles have shown significant dependance on the polymer compositions. The SMV release from the triple-polymer-network hydrogel has continued for 17 weeks of observation. Cytocompatibility of hydrogels with various formulations is confirmed. The tunable triple-network hydrogels loaded with SMV provide a potential therapeutic value for bone regeneration.

Time-Restricted Feeding Regulates Circadian Rhythm of Murine Uterine Clock.

BACKGROUND: Skipping breakfast is associated with dysmenorrhea in young women. This suggests that the delay of food intake in the active phase impairs uterine functions by interfering with circadian rhythms. OBJECTIVES: To examine the relation between the delay of feeding and uterine circadian rhythms, we investigated the effects of the first meal occasion in the active phase on the uterine clock. METHODS: Zeitgeber time (ZT) was defined as ZT0 (08:45) with lights on and ZT12 (20:45) with lights off. Young female mice (8 wk of age) were divided into 3 groups: group I (ad libitum consumption), group II (time-restricted feeding during ZT12-16, initial 4 h of the active period), and group III (time-restricted feeding during ZT20-24, last 4 h of the active period, a breakfast-skipping model). After 2 wk of dietary restriction, mice in each group were killed at 4-h intervals and the expression profiles of uterine clock genes, Bmal1 (brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein 1), Per1 (period circadian clock 1), Per2, and Cry1 (cryptochrome 1), were examined. RESULTS: qPCR and western blot analyses demonstrated synchronized circadian clock gene expression within the uterus. Immunohistochemical analysis confirmed that BMAL1 protein expression was synchronized among the endometrium and myometrium. In groups I and II, mRNA expression of Bmal1 was elevated after ZT12 at the start of the active phase. In contrast, Bmal1 expression was elevated just after ZT20 in group III, showing that the uterine clock rhythm had shifted 8 h backward. The changes in BMAL1 protein expression were confirmed by western blot analysis. CONCLUSIONS: This study is the first to indicate that time-restricted feeding regulates a circadian rhythm of the uterine clock that is synchronized throughout the uterine body. These findings suggest that the uterine clock system is a new candidate to explain the etiology of breakfast skipping-induced uterine dysfunction.

In vitro evaluation of loaded chitosan membranes for pain relief and infection prevention.

Wounds resulting from surgeries, implantation of medical devices, and musculoskeletal trauma result in pain and can also result in infection of damaged tissue. Up to 80% of these infections are due to biofilm formation either on the surface of implanted devices or on surrounding wounded tissue. Bacteria within a biofilm have intrinsic growth and development characteristics that allow them to withstand up to 1,000 times the minimum inhibitory concentration of antibiotics, demonstrating the need for new therapeutics to prevent and treat these infections. Cis-2-decenoic acid (C2DA) is known to disperse preformed biofilms and can prevent biofilm formation entirely for some strains of bacteria. Additionally, local anesthetics like bupivacaine have been shown to have antimicrobial effects against multiple bacterial strains. This study sought to evaluate hexanoic acid-treated electrospun chitosan membranes (HA-ESCM) as wound dressings that release C2DA and bupivacaine to simultaneously prevent infection and alleviate pain associated with musculoskeletal trauma. Release profiles of both therapeutics were evaluated, and membranes were tested in vitro against Methicillin-resistant Staphylococcus aureus (MRSA) to determine efficacy in preventing biofilm infection and bacterial growth. Results indicate that membranes release both therapeutics for 72 hr, and release profile can be tailored by loading concentration. Membranes were effective in preventing biofilm growth but were toxic to fibroblasts when loaded with 2.5 or 5 mg of bupivacaine.

A Study of Combining Elastin in the Chitosan Electrospinning to Increase the Mechanical Strength and Bioactivity.

While electrospun chitosan membranes modified to retain nanofibrous morphology have shown promise for use in guided bone regeneration applications in in vitro and in vivo studies, their mechanical tear strengths are lower than commercial collagen membranes. Elastin, a natural component of the extracellular matrix, is a protein with extensive elastic property. This work examined the incorporation of elastin into electrospun chitosan membranes to improve their mechanical tear strengths and to further mimic the native extracellular composition for guided bone regeneration (GBR) applications. In this work, hydrolyzed elastin (ES12, Elastin Products Company, USA) was added to a chitosan spinning solution from 0 to 4 wt% of chitosan. The chitosan-elastin (CE) membranes were examined for fiber morphology using SEM, hydrophobicity using water contact angle measurements, the mechanical tear strength under simulated surgical tacking, and compositions using Fourier-transform infrared spectroscopy (FTIR) and post-spinning protein extraction. In vitro experiments were conducted to evaluate the degradation in a lysozyme solution based on the mass loss and growth of fibroblastic cells. Chitosan membranes with elastin showed significantly thicker fiber diameters, lower water contact angles, up to 33% faster degradation rates, and up to seven times higher mechanical strengths than the chitosan membrane. The FTIR spectra showed stronger amide peaks at 1535 cm-1 and 1655 cm-1 in membranes with higher concentrated elastin, indicating the incorporation of elastin into electrospun fibers. The bicinchoninic acid (BCA) assay demonstrated an increase in protein concentration in proportion to the amount of elastin added to the CE membranes. In addition, all the CE membranes showed in vitro biocompatibility with the fibroblasts.